Abstracts from the 50th International Musculoskeletal Biology Workshop, July 22 – 27, 2023, Midway, Utah

Chemokines are a family of small cytokines or signaling proteins secreted by cells. CXCL12 is upregulated in injury sites, where it acts as a chemotactic agent, attracting CXCR4-expressing bone marrow stromal cells, which play a pivotal role in the healing and regeneration of tissue throughout the body. Furthermore, CXCL12 expression has been observed in regions experiencing inflammation-induced bone destruction and fracture sites. In this study, we identified a novel peptide derived from CXCL12, which can promote the osteogenesis of bone marrow stromal cells as well as bone formation and healing. Multipotent bone marrow stromal cells treated with peptide showed enhanced alizarin red S staining and higher alkaline phosphatase (ALP) activity. Moreover, ALP and osterix proteins were more abundantly expressed when cells were treated with peptide than CXCL12. Histology and microcomputed tomography data at 4, 8, 12 weeks demonstrated that both rabbit and goat models transplanted with polycaprolactone (PCL) scaffolds coated with peptide showed significantly greater bone formation than animals transplanted with PCL scaffolds alone. The results of our study prove that peptide can notably stimulate the osteogenic differentiation of multipotent bone marrow stromal cells, and the transplantation of PCL + peptide scaffolds into rabbit and goat animal models can result in greater bone tissue formation and repair. The peptide biomaterial holds substantial promise for bone tissue engineering. Figure 1. Surgical procedures involved in bone defect creation in rabbit and goat. Rabbit Step 1. Tibia exposition, Step 2. Bone defect, Step 3. From a point 1.5 cm away a rectangular cortical bone 5 mm wide and 1.5 cm long was formed, Step 4, Step 5. The defect was treated using the prepared scaffolds. Goat Step 1. The goat was laid in a right lateral recumbency position, Step 2. The femoral shaft was exposed using a retractor and a longitudinal incision was made on the left posterior thigh, Step 3. Cortical bone defects, Step 4, Step 5. Scaffolds were implanted into the bone defect area. Figure 2. BFP-5 increases osteogenic differentiation markers in cells. RT-PCR analysis and relative mRNA expression level for RUNX2, ALP, Osteopontin, Collagen type I, DLX-5, and Osterix. The above data represent the mean ± SD from triplicate measurements (# p < 0.05 ODM vs negative control; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 peptide vs ODM). Figure 3. Histological and micro-CT analysis of PCL and PCL+Peptide scaffold in rabbits after 4, 8, 12 weeks postimplantation and in goats after 12 week post-implantation. Quantification of bone regeneration in rabbit and goat bone volume (BV), bone volume per total volume (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), and bone mineral density (BMD), as measured by micro-CT. Acknowledgement: This work was supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (Project Number: 9991007120, KMDF_PR_20200901_0038). 40 A novel periosteal cell type, identified by single cell RNA sequencing, senses mechanical loading and in response to it differentiates into osteoblasts Xuehua Li, PhD, Intawat Nookaew, PhD, Jinhu Xiong, PhD University of Arkansas for Medical Sciences, Little Rock, AR, United States Mechanical loading stimulates periosteal bone formation and osteocytes are considered to be the major cells sensing and transducing mechanical signals  in bone. However, a complete understanding of the  identity of mechanosensitive cells in periosteum is lacking. To determine which cell types are responding to mechanical stimulation in periosteum, we isolated periosteal cells from loaded tibia at different time points and performed 10X‐based single cell RNA sequencing (scRNA‐seq). The left tibiae of 7‐month‐old female C57BL/6J mice were loaded with 9N at 1 bout per day for 1, 2, and 5 days.  Periosteal cells were then isolated 12 hours after loading for 12‐hour time point and 24 hours after the last set of loading for the 2‐ and 5‐day  time points.  scRNA‐seq  identified 8  clusters:  Fibro‐1,  Fibro‐2, Osteo‐X, Osteoblasts, Chondrocytes, Articular Chondrocytes, Tenocytes, and a new cluster we refer to as Fibro‐L. Comparing clusters between the control and 12‐hour groups, we found that the number of Fibro‐L is greatly increased within 12 hours after loading. The dramatic change of the cell number  in this cluster within 12 hours cannot be explained by cell proliferation but  the change  in gene expression profile induced by mechanical loading. Differential gene expression analysis of this cluster showed increased expression of Ptgs2, Lif, and Il‐6 which are classic early response genes to mechanical loading.  To confirm the increased expression of these genes in periosteum, we localized their mRNA using RNAscope based in‐situ hybridization. Tibiae collected 4 hours after loading were used for RNAscope. Consistent with scRNA‐seq analysis, we found increased Ptgs2, Lif, and Il‐6 expression in osteocytes as well as cells at the periosteal surface, suggesting that periosteal cells are also capable of sensing mechanical stimulation. Differential gene expression analysis of this cluster at later time points showed that proliferation related genes such as Ki67 and Top2a were significantly elevated. In addition, RNAscope showed a dramatic increase of Top2a expression in the tibial periosteum 5 days after loading, demonstrating increased cell proliferation in periosteal cells.  More importantly, cells in the Fibro‐L cluster became Sp7 and Spp1 positive at day 5 consistent with their differentiation towards osteoblasts. Taken  together, our  results provide evidence  that a subset of periosteal cells may directly sense mechanical  loads and differentiate into osteoblasts. 41 Systemic Administration of Mes-1022 Enhances Bone Formation in a Critical-Size Segmental Femoral Defect Rat Model Jack Chapman, Catherine Julien, MSc, Michal Kulasek, Taylor deVet, MASc, Mayumi Umebayashi, DMD, Frank Rauch, MD, Bettina M. Willie, PhD Research Centre, Shriners Hospital for Children-Canada, Montreal, QC, Canada, Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada, Departments of Bioengineering and Biomedical Engineering, McGill University, Montreal, QC, Canada, Faculté de medicine, Université de Montréal, Montreal, QC, Canada Introduction: Up to 5% of bone fractures exhibit delayed healing or non-union (1). Long bone critical size defects (CSD) model several aspects of nonunion, but most current therapies show suboptimal efficacy in such models (2). Mes-1022, a novel hydroxyapatite-targeted prostaglandin E2 receptor 4 agonist prodrug, has been shown to promote bone healing when applied locally with bone graft material (3), but it is unclear whether systemic administration enhances healing. The aim of this study was to evaluate the efficacy of systemically-administered MES-1022 on the healing of a femoral segmental CSD in the absence of bone grafting and on the mass and microstructure of the intact contralateral femur in a rat model. Methods: Ten-week-old female Sprague-Dawley rats (7/group) were randomized to receive 12 weekly subcutaneous injections of MES-1022 at 5 mg/kg (high dose), MES-1022 at 1.7 mg/kg (low dose), or Vehicle (PBS + 25 nM EDTA). Rats underwent a 5 mm osteotomy of the left femoral midshaft, stabilized by a unilateral external fixator. Bone markers (P1NP, TRAcP-5b) and open field activity were measured at baseline and throughout the study. Rats were sacrificed on day 84 and the osteotomized and unoperated femora were imaged via micro-CT at 8 μm voxel size. Volumes of interest (VOI) for the unoperated femur were 5% of bone length at mid-diaphysis and 10% at the distal metaphysis. The osteotomized femur VOI was 7 mm in length. A threshold of 819 mg HA/cm was determined by Otsu in the unoperated femur. For osteotomy region analyses, a range of 35% – 70% of this density was used to distinguish newly mineralized tissue (i.e., new bone and calcified cartilage), referred to as non-quiescent bone (NQ), and a range of 70-100% of the density identified existing mineralized bone (quiescent bone; Q). Defect site healing parameters for Q, NQ, and total (T) included tissue volume (TV, mm), mineralized callus volume (BV, mm), mineralized callus volume fraction (BV/TV, mm/mm), callus tissue mineral density (TMD, mg HA/cm), and callus tissue mineral content (TMC, mg). Standard cortical and trabecular microCT parameters were calculated for the unoperated femur (n = 5). All MicroCT analyses were performed using Xamflow software. Three-point bending (day 84) was used to assess bone strength of the unoperated femora. Data were analyzed using an ANOVA followed by post-hoc Tukey testing, with significance at p ≤ 0.05. Results: Full bony bridging of the osteotomy occurred in 1 of 7 rats in the high dose MES-1022 group, versus 0 of 7 rats in both the vehicle and low dose groups. ANOVA indicated MES-1022 treatment had a significant effect on NQ BV/TV and NQ TMD and a trending effect on NQ TMC and NQ BV. Tukey’s tests showed high dose treated rats (433 ± 19) had greater NQ TMD compared with vehicle treated rats (412 ± 12, p = 0.0221). The high dose group also had greater NQ BV/TV (0.21 ± 0.09) compared to the low dose group (0.08 ± 0.05, p = 0.0264) and a trend for greater NQ TMC versus low dose animals (2.34 ± 2.03, p = 0.0541). There was also a trend of greater NQ BV in the high dose (20.91 ± 17.34) compared to low dose treated rats (5.50 ± 4.72, p = 0.0689). For unoperated femurs, increased trabecular number and bone volume fraction, as well as decreased trabecular separation were present at the metaphysis of high dose animals compared to both low dose and vehicle. There was no difference in any whole bone mechanical property (maximum load, stiffness, post-yield displacement, or work-to-fracture) or in open field physical activity among treatment groups. The high dose cohort had higher levels of P1NP than the vehicle group at all post-baseline timepoints (p < 0.02) except day 69 (p = 0.0667). The serum bone resorption marker TRAcP-5b was lower in the high dose group versus vehicle rats at days 41 and 55 (p < 0.0006) and also versus the low dose group at days 27, 55, and 69 (p < 0.04). Conclusion: Systemic administration of MES-1022 (at 5 mg/kg) increased bone mass in the unoperated femur and had favorable effects on the healing of a segmental femoral CSD, including complete bony bridging in one animal, greater callus NQ TMD, and increased bone formation markers. Histological analysis of callus tissue composition is ongoing to provide additional insights on healing outcomes. Future studies may determine whether including bone graft material in the defect site enables systemically-administered MES-1022 to further improve bridging and healing. Conflicts of Interest: BW and FR received institutional funding from Mesentech. References: (1) Wildemann, et. al. (2021). Nature reviews. 7(1), 57. (2) Schmidt-Bleek et. al. (2016); Cytokine & growth factor reviews, 27, 141–148. (3) Sheikh, Z., et al. (2020); J Biomed Mater Res B Appl Biomater, 108(6): p. 2670-2680. 42 Healing non-union fractures using bone-targeted polymeric nanoparticles Vignesh K. Rangasami1, Baixue Xiao2, and Danielle S.W. Benoit1,2 1 Department of Bioengineering, Knight Campus, University of Oregon, OR, United States, 2 Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States IntroductionNon-unions arise when there is an impaired or complete cessation of the regenerative capacity of bone.1 Among the many risk factors for non-unions, age is most significant.2 Current non-union management typically involves surgery, often with autograft and fixation,3 are costly with varied success rates.4 As such, there is a need to develop non-surgical options. To this end, we have pioneered the development of a nanoparticle (NP) drug delivery system functionalized with a targeting peptide to promote non-union healing. NPs composed of poly(styrene-alt-maleic anhydride-b-poly(styrene) (PSMA-b-PS). NPs are then functionalized with a peptide (TBP) that specifically binds to tartrate resistant acid phosphatase (TRAP), a protein deposited by osteoclasts during fracture healing and non-union.5 The NPs have been designed to deliver a GSK-3b inhibitor (AR28) to improve fracture healing by activating the Wnt/b-catenin pathway.6 Drug loaded nanoparticles (TBP-NPAR28) enhanced fracture healing. These results motivate future studies of the effect of TBP-NPAR28 in impaired fracture healing models using aged mice. MethodsThe PSMA-b-PS diblock polymers were synthesized and self-assembled as previously described.5 Briefly, excess styrene was added along with maleic anhydride (MA) (4:1 [STY]/[MA]) in the presence of the chain transfer agent (CTA) 4-Cyano-4-dodecylsulfanyltrithiocarbonylsulfanylpentanoic 18 acid (DCT) (100:1 [MA]/[CTA]). The radical initiator 2,2′-Azo-bis(isobutylnitrile) (AIBN) was used (10:1 [CTA]/[initiator]) in dioxane. The reaction was purged with nitrogen for 45 minutes before being placed in a 60 °C oil bath for 72 hours. The reaction mixture was then dissolved in acetone before precipitation in petroleum ether after which they were dried under vacuum and taken for further characterization. The TBP was conjugated to the surface of the TBPNPs through maleic anhydride ring opening, as previously described.7 These NPs were evaluated in a middiaphyseal femur fracture Balb/c mice models. ResultsCharacterization of the polymer revealed the molecular weight (MW) of 1st block (PSMA) as 21 kDa and MW of 2nd block (PS) as 24 kDa with a PDI of 1.0. The synthesized TBP-NPAR28 have a hydrodynamic diameter of 67 ± 7 nm and a zeta potential of -24 ± 1 mV. TBP-NPAR28 were injected three days post-fracture into mice. Enhanced bone healing was observed 4 weeks post-injection through microcomputed tomography (μCT) in the TBP-NPAR28. Bone volume was 1.8and 1.5-fold higher in the TBP-NPAR28 group compared to untreated control and free drug. Immunofluorescence of the fracture site after 10 days revealed an increased number of M2 macrophages in the TBP-NPAR28 group compared to the control group, which was confirmed by flow cytometry. Further investigation by bulk RNAseq revealed that the TBP-NPAR28 enhanced fracture healing by down-regulating pro-inflammatory pathways, including TNF, NFkB, and IL17 and enriched genes that were involved in angiogeneisis and arteriogenesis, which are essential for fracture healing. DiscussionIn sum, data suggest that TBP-NPAR28 modulates and rebalances M2/M1 ratio resulting from upregulation in Wnt/B-catenin signaling, which is associated with enhanced fracture healing in both bone formation volume and mechanical properties after TBP-NPAR28 treatment. With these results, we believe this TBP-NPAR28 can be used to restart the healing process in aged mice with impaired fracture healing conditions that lead to nonunions, including age. Figure A,BFractured mouse femurs were analyzed via microcomputed tomography (μCT) at 4 weeks post-treatment to evaluate in vivo bone callus formation. Quantification of μCT demonstrated that TBP-NPAR28 significantly increased both bone volume (B(i)) and bone volume fraction (B(ii)). CImmunofluorescence staining of F4/80, iNOS, and CD206, where M1 macrophages were determined as F4/80+iNOS+ and F4/80+CD206+ for M2 subtype. DFlow cytometry analysis of M1 and M2 macrophage phenotypes, gated as double positive for MHCII/CD38 (M1) and CD206/CD163 (M2), at day 10 post-fracture ESignaling pathway analysis of differential genes in TBP-NPAR28 and free drug AR28 at day 10 post-injection. ReferencesStewart, S. K. Fracture Non-Union: A Review of Clinical Challenges and Future Research Needs. Malays Orthop J 13, 1 (2019). 2. Zura, R., Mehta, S., Della Rocca, G. J. & Steen, R. G. Biological Risk Factors for Nonunion of Bone Fracture. JBJS Rev 4, (2016). 3. Giannotti, S. et al. Current medical treatment strategies concerning fracture healing. Clinical Cases in Mineral and Bone Metabolism 10, 116 (2013). 4. Stewart, S. K. Fracture Non-Union: A Review of Clinical Challenges and Future Research Needs. Malays Orthop J 13, 1 (2019). 5. Wang, Y. et al. Fracture-Targeted Delivery of β-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing. ACS Nano 11, 9445–9458 (2017). 6. Sisask, G. et al. Rats treated with AZD2858, a GSK3 inhibitor, heal fractures rapidly without endochondral bone formation. Bone 54, 126–132 (2013). 7. Chandrasiri, I., Liu, Y., Adjei-Sowah, E., Xiao, B. & Benoit, D. S. W. Reproducible and controlled peptide functionalization of polymeric nanoparticles. Frontiers in Biomaterials Science 1, 9 (2022) 43 Skeletal Consequences of Preterm Birth in a Pig Model Brittany M. Wilson, PhD1, Frank C. Ko, PhD1, Amal Adra, MD1*, Meghan M. Moran, PhD1, Martin B. Rasmussen, BSc2, Thomas Thymann, DVM, PhD2, Per T. Sangild, PhD, DMSc, DVSc2, and D. Rick Sumner, PhD1 1Rush University Medical Center, Chicago, IL, USA. 2University of Copenhagen, Frederiksberg, Denmark, *now at St. Louis University, St. Louis, MO, USA Premature, or preterm, birth occurs in about 10% of all  live births and leads to various short‐ and long‐term health consequences. Since about 80% of skeletal mineral accrual occurs during the final  trimester of gestation, preterm birth disrupts  a  critical  period  of  skeletal  development  and  leaves  preterm  neonates  at  increased  risk  for  osteopenia  and metabolic bone disease of prematurity (MBDP). Preterm birth has also been associated with reduced peak bone mass and increased risk for osteoporosis later in life. While many studies have measured bone mineral content or density in preterm neonates, mechanistic insight into how preterm birth affects the mechanical competence of bone requires a large animal model  that  is  compatible with human  skeletal development. The pig model of preterm birth has already been used  to examine multiple  developing  tissues  and may  provide  a  resource  to  study  how  prematurity  affects  postnatal  skeletal development. Thus, the purpose of this study was to evaluate the pig as a model for postnatal skeletal development after premature birth. Preterm pigs  (Landrace  x  Yorkshire  x Duroc;  all  raised  in Denmark) were born on day 106 of  ~117 of  gestation by cesarean section and were reared artificially. Term control pigs were born vaginally on day 117 ± 2 of gestation and reared naturally by their mother sow on a farm. Groups of term and preterm pigs, respectively, were sacrificed on postnatal days 1 (n=6 and 7), 5 (n=7 and 17), and 19 (n=7 and 13). Pigs were weighed, blood and urine were collected (only on days 5 and 19), and hindlimbs were dissected. Plasma was used to determine levels of alkaline phosphatase, calcium, phosphate, and creatinine. Calcium, phosphate, and creatinine were also measured in urine to calculate the rates of calcium excretion and tubular  reabsorption  of  phosphate.  Serum  was  used  to  measure  parathyroid  hormone  (PTH),  vitamin  D  (25‐OHD), osteocalcin, C‐terminal crosslinks of type I collagen (CTX‐I), and procollagen type I N‐terminal propeptide (PINP). Micro‐ computed tomography was used to assess femoral mid‐diaphysis cortical bone geometry and bone mineral density (BMD), distal metaphysis integral BMD and trabecular bone BMD. Femurs were tested in three‐point bending to determine whole‐ bone stiffness and maximum load to failure. Data were collapsed across sex since initial statistical tests indicated that sex was  not  significant.  Statistical  analyses  included  multivariate  two‐way  analyses  of  variance  (ANOVAs),  allometry  to determine the relationship between bone cross‐sectional properties and length, and multivariate one‐way ANOVAs in pigs of similar post‐conceptual age. Preterm pigs were smaller and gained less body mass over time than term pigs (p<0.001). Femur length was reduced in preterm compared to term pigs (p<0.001). Calcium and phosphate were reduced in preterm pigs (p=0.013 and p<0.001, respectively). Alkaline phosphatase levels decreased between day 5 and 19 in term pigs but remained constant in preterm pigs (p=0.005), leading to elevated levels at day 19 in preterm compared to term pigs. Calcium excretion was elevated in preterm pigs (p<0.001). Tubular reabsorption of phosphate was depressed in preterm compared to term pigs on day 5. PTH remained  low in preterm pigs while  there was a  large  increase  in term pigs between days 5 and 19 (p=0.029). 25‐OHD increased from day 5 to 19 in preterm pigs while levels remained stable in term pigs over time (p=0.001). Preterm pigs had higher  osteocalcin  levels  (p=0.004),  lower  PINP  (p=0.008),  and  higher  CTX‐I  levels  than  term  pigs.  Cortical  bone  area, medullary area, and total area were each reduced in preterm compared to term pigs at each time point (all p<0.001) as was cortical  thickness (p=0.022). By day 19  in preterm pigs compared to term pigs, metaphyseal  integral BMD was reduced (p=0.021). Whole‐bone stiffness(p=0.009) and maximum load to failure (p<0.001) were reduced in preterm pigs compared to term pigs at all 3 time points. Preterm pig femurs were slender for their length. When pigs of a similar post‐conceptual age were compared many of the skeletal phenotypes persisted. A major barrier to understanding the skeletal consequences of preterm birth is the lack of a translational animal model. We report here that the pig model recapitulated many commonly observed features of preterm birth, including altered mineral metabolism,  reduced  cortical  bone  area,  and  increased  bone  resorption.  Clinical  features  of MBDP were  also recapitulated, including reduced phosphate and increased alkaline phosphatase. We also show here, for the first time, that additional  properties  of  bone  structure  and  turnover,  as  well  as  whole‐bone mechanical  properties  were  affected  by preterm birth in pigs. While additional long‐term studies are needed, the pig model of preterm birth has utility for studying skeletal consequences and interventions after preterm birth. Acknowledgements Funding provided by NIH T32AR073157, NIH R21HD102026, and the University of Copenhagen. 44 Contribution of Proliferating 3.6Col1a1 Expressing Cells to Early Fracture Callus Andre F. Coello, Jennifer A. McKenzie, PhD, Katherine R. Hixon, PhD, Nicole R. Gould, PhD, Leyi Chen, MS, Anna N. Miller, MD, Matthew J. Silva, PhD Department of Orthopaedic Surgery, Department of Biomedical Engineering, Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, St. Louis, MO, Dartmouth Engineering, Dartmouth College, Hanover, NH Atrophic nonunion occurs when an osteochondral callus fails to form after fracture. Proliferation of periosteal cells is a key feature of fracture healing. We have previously shown that proliferation of periosteal cells during the first 2 weeks of fracture healing is essential for normal healing. This was determined using mice expressing a thymidine kinase (TK+) ‘suicide gene’ driven by the 3.6Col1a1 promoter, which is active in osteoblast lineage periosteal cells. In this mouse model, proliferating TK+ cells die when dosed with ganciclovir (GCV), without affecting non-dividing cells. Here, we sought to investigate the role of proliferating 3.6Col1a1 expressing cells in early stages of fracture healing and determine their critical proliferation window. This was done using the 3.6Col1a1-TK mouse model to study how fracture healing is affected when GCV is administered for different durations following a full femur fracture in 12-week-old TK+ mice and their wild type (WT) littermates. This study was IACUC approved. First, mice were dosed with GCV daily for 3, 7, and 14 days starting on the day of fracture and healing was evaluated at 21 days post fracture (DPF). Radiographs and histology at 21DPF showed that GCV dosing in TK+ mice for 3 days led to a slightly smaller, yet fully bridged mineralized callus. In contrast, GCV dosing for 7 and 14 days led to a significant impairment in callus formation and failure to bridge by 21DPF, compared to the control group (Fig. 1, right). Mice in the TK+ group dosed with GCV for 7 days developed calluses composed mostly of woven bone, yet still had 20% less woven bone, 10% more cartilage, and 10% more fibrous tissue than the control group (Fig. 2C). The greatest effect on healing was observed in the TK+ group dosed with GCV for 14 days, where calluses had 70% less woven bone, 15% more cartilage, and 45% more fibrous tissue than controls (Fig. 2C). We then focused on earlier time points to further understand the role of proliferating osteoblast lineage cells in the first 10 days after fracture. Histology at 5DPF (Fig. 1, left) showed that GCV dosing for 3 and 5 days resulted in TK+ mice with slightly smaller fracture calluses that had ~15% less woven bone compared to control mice (Fig. 2A). μCT analysis at 10DPF showed a trend where TK+ mice developed smaller calluses with less mineralization as GCV dosing duration increased. Histology at 10DPF (Fig. 1, middle) showed TK+ mice dosed with GCV formed calluses composed largely of cartilage (30%) and fibrous (40%) tissues, with only 10%-15% being woven bone (Fig. 2B). In contrast, control mice formed larger calluses composed mostly of woven bone (>40%) with less cartilage (25%) and fibrous (15%) tissues. We conclude that proliferation of osteoblast lineage cells between 3 and 10 days post fracture is required for normal fracture healing in mice. References: [1] Hixon, K.R., McKenzie, J.A., Sykes, D.A.W., Yoneda, S., Hensley, A., Buettmann, E.G., Zheng, H., Skouteris, D., McAlinden, A., Miller, A.N. and Silva, M.J. (2021), Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model. J Bone Miner Res, 36: 2243-2257. https://doi.org/10.1002/jbmr.4424 Figure 1: Representative Picrosirius Red and Alcian Blue stained histology images at 5 days post fracture (left), 10 days post fracture (middle), and 21 days post fracture (right). (Top row) Control slides show normal healing, (Bottom row) TK+ samples administered GCV for the stated duration show abnormal healing from ablation of proliferating 3.6Col1a1 expressing cells. Figure 2: Average callus composition by tissue type for each group at (A) 5 days post fracture, (B) 10 days post fracture, and (C) 21 days post fracture. Percentages for each tissue type calculated by quantification of Picrosirius Red and Alcian Blue stained slides. Increasing impairment on fracture healing observed with increasing GCV dosing duration. 45 Quantifying the Relationship Between Fixation Strategy & Distal Femur Fracture Site Motion Elmer Vazquez, Aerie Grantham, Joseph Davis, William R. Ledoux, and William D. Lack RR&D Center for Limb Loss & MoBility (CLiMB), VA Puget Sound, School of Medicine, Departments of Mechanical Engineering, & Orthopaedics & Sports Medicine, University of Washington, Seattle, WA INTRODUCTION: Nonunion occurs in up to 32% of distal femur fractures, compared to a rate of 5 to 10% for other long bone fractures. Qualitative assessments of postoperative distal femur fracture site motion have been associated with later fracture healing. However, strategies to optimize fracture healing remain limited by an inability to quantify motion in the clinical setting. To overcome this, we developed an in vivo method of quantifying distal femur fracture site motion via weightbearing CT. METHODS: Seven fresh frozen cadaveric femurs underwent distal femoral osteotomies and surgical fixation. Common surgical fixation strategies were applied including locked plates with short bridge spans (specimens 2 and 7), medium bridge spans (specimens 4 and 5), and long bridge spans (specimens 1 and 3). Specimen 6 was treated with retrograde nailing. Stainless steel beads were implanted in the cadaveric femur segments to allow for bead-based validation of bonebased tracking. Matching the conditions of our ongoing in vivo study, the cadaveric specimens underwent unloaded and loaded CT. Loading through the hip was replicated through a custom load platform that was attached to the head of the femur via a pseudoacetabulum. Relative fracture site motion was determined using image analysis software (Mimics) for four paired points (anteromedial-AM, anterolateral-AL, posteromedial-PM, and posterolateral-PL). The technique included manual registration of the injury CT data (devoid of implant artifact) to the cone beam CT data. RESULTS: Motion profiles differed by fixation strategy (Table 1). While retrograde nailing resulted in < 1mm of motion at all four locations, plate fixation resulted in much greater motion (all plate strategies resulted in ≥ 3mm of motion at one or more locations). Pooling all plate fixation strategies, motion was twice as great medially as it was laterally (3.3 vs 1.6mm, p = 0.018). This varied markedly by fixation strategy, with medium and long bridge spans being particularly associated with asymmetric motion (Figure 1). DISCUSSION: Beyond allowing validation of bonebased tracking for in vivo assessment of fracture site motion, the results provide insight into how fixation strategies relate to fracture site motion. Fracture healing has been more consistent following retrograde nailing than with plate fixation. The variability in magnitude and asymmetry (medial > lateral) of fracture site motion we found for plate fixation are possible causes of the inconsistent healing reported clinically with this strategy. Contrary to anecdotal reports that locked plate fixation is “too stiff”, we found universally greater motion with plate fixation than retrograde nailing, and longer plate bridge spans accentuated this magnitude and asymmetry of fracture site motion. Lateral plate fixation provides limited resistance to bending moments associated with distal femur fractures, resulting in significant shear motion as well as delayed and asymmetric fracture healing. Translational research has suggested short bridge, titanium plate fixation of distal femur fractures promotes radiographic callus formation. Our results demonstrate that such short bridge plate fixation yields a motion profile most similar to retrograde nailing. CLINICAL RELEVANCE: In vivo quantification of fracture site motion will inform observational & clinical trial research into whether biomechanical variables (fixation strategies and rehabilitation protocols) can be modified on a patient-specific basis to optimize fracture healing outcomes. We are actively enrolling human subjects for in vivo bone-based tracking of distal femur fracture site motion via weightbearing CT. REFERENCES: 1. Henderson CE, et al. JOT. 2011;25:S8–14, 2. Tzioupis, C. & Giannoudis, P. www.elsevier.com/locate/ injury (2007), 3. Elkins, J. et al. JBJS American Volume 98, 276–284 (2016), 4. Lujan TJ, et al. JOT. 2010;24:156–62. Table 1. Differences in motion are reported for four paired points across the osteotomy between unloaded (0N) and loaded (225.5N) tests. Figure 1. Longer plate bridge spans resulted in greater, but asymmetric fracture site motion (medial > lateral). Short bridge spans resulted in a motion profile most similar to a retrograde nail. 46


NICHD, NIH, Bethesda, MD Ϯ=co-corresponding authorship
Multinucleated osteoclasts, essential for skeletal remodeling in health and disease, are formed by the fusion of osteoclast precursors, where each fusion event raises their bone-resorbing activity. Here we show that the nuclear RNA chaperone, La protein has an additional function as an osteoclast fusion regulator. Monocyte-to-osteoclast differentiation starts with a drastic decrease in La levels. As fusion begins, La reappears as a low molecular weight species at the osteoclast surface, where it promotes fusion. La's role in promoting osteoclast fusion is independent of canonical La-RNA interactions and involves direct interactions between La and Annexin A5, which anchors La to transiently exposed phosphatidylserine at the surface of fusing osteoclasts. Disappearance of cell-surface La, and the return of full length La to the nuclei of mature, multinucleated osteoclasts, acts as an off switch of their fusion activity. Targeting surface La in a novel explant model of fibrous dysplasia inhibits excessive osteoclast formation characteristic of this disease, highlighting La's potential as a therapeutic target.  Single-cell RNA sequencing (scRNA-seq) has been used as a high-resolution tool to identify cell populations in the bone microenvironment. However, there are some disagreements on annotation of mesenchymal cell types among the published studies. We performed a comparative analysis across multiple published datasets with our in house generated scRNA-seq data of cells targeted by Osx1-Cre and Dmp1-Cre driver strains, which are commonly used to study osteoblasts and osteocytes. To accomplish this, we reprocessed the datasets and performed data harmonization and integration aimed at identifying a consensus of murine bone-associated cells. In total, over 100,000 high quality mesenchymal cells were included and projected on UMAP coordinates to uncover the structure and clusters of different cell types. We found that mesenchymal cells segregated into 11 clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and a cluster we refer to as osteo-X. Osteo-X cells were more abundant in preparations from periosteal bone and are defined by the Postn transcript marker. Based-on the identified mesenchymal cell types, we did not observe major differences in the clusters targeted by Osx1-Cre and Dmp1-Cre in endosteal bone. In contrast, Osx1-Cre efficiently targeted osteo-X cells more than Dmp1-Cre in periosteal preparations. Localization of osteo-X cells by Postn in situ hybridization showed that they were abundant at the periosteum but also present near the trabecular bone surface. We found fibro-2, whose transcript marker is Clec3b, localized to the periosteum but not to the endosteum or trabecular bone. Osteo-CAR cells, defined by Limch1 expression, were also predominantly associated with endosteal and trabecular bone. We developed a reference model of mesenchymal cell types from the integrated data using Azimuth workflow and used it to investigate the suppression of bone remodeling by Denosumab treatment at single cell resolution. Denosumab-treated mice completely lacked osteoblasts and exhibited altered gene expression in pre-osteoblasts in remodeling bone but there was no change in the abundance or location of osteo-X and osteo-CAR cells. These results suggest that differences between the Osx1-Cre and Dmp1-Cre transgenes may be due in part to differential targeting of a progenitor cell population defined in part by Postn expression.     3 Department of Orthopaedics, Division of Microbiology and Immunology, University of Utah, USA. Introduction: Up to 50% of type 3b compound fractures are complicated by biofilm infections 1 . Staphylococcus aureus, the most common cause of infection, resides within a protective biofilm. Lactoferrin (Lf), a natural animal milk glycoprotein, has antibiofilm properties 2 . Lf's ability to chelate iron and release bacteria from their protective biofilm, compliments antibiotics. The goal of this study was to test the ability of Lf as an adjuvant to antibiotics in the context of compound fracture infections. Specifically, we aimed to: 1) Demonstrate the in vitro anti-biofilm properties of bovine Lf. 2) Translate the in vitro antibiofilm activity of bovine Lf to a clinically relevant animal model of open fractures. Methods: 1) S. aureus Xen-36, a bioluminescent strain, biofilm was generated in a CDC Biofilm Reactor® (CDC-BR) over 4days culture on stainless steel coupons treated with varying concentrations of Lf and flucloxacillin (Flu). Treatment took place over 3 days. Day 0 and Day 3 bacterial colony forming unit (CFU) enumeration was completed from the stainless-steel coupons. Daily bacterial CFU enumeration was completed from the broth. 2) S. aureus Xen-36 biofilm generated in a CDC-BR was dispersed and stored at -80°C. Surgery was performed on adult male rats. Under sterile technique, a 1.5 cm skin incision was made to expose the craniomedial tibia. A 1.5 mm defect was created, and the tibia reamed with a 23-gauge needle. Ten µL (≈10 6 CFU) of S. aureus biofilm was inoculated into the defect. A 0.6 mm diameter Kirschner wire (k-wire) was placed within the medullary cavity of the tibia and the wound was closed. We tested the effect of Lf in lavage solutions. All rats received subcutaneous Flu 200 mg/kg immediately prior to surgery. After application of the inoculum and a 5-min holding time the defect was subject to 2x 50 mL lavage with 4 treatment groups (saline only, Lf only, Bactisure with Lf, Bactisure with saline). At day 3 and 7 post-op, rats were re-anaesthetised for bioluminescent and X-ray imaging. At day 7 post-op, rats were euthanized, and tibial explants collected for homogenization and bacterial colony enumeration. Implanted k-wires were collected for bacterial enumeration and scanning electron microscopy (SEM) to confirm the presence of bacteria. Results: 1) The minimum inhibitory concentration of Flu for S. aureus Xen-36 was established at 0.5 µg/mL. Lf at a concentration of 8 mg/mL with Flu concentrations (2 µg/mL, 20 µg/mL, and 200 µg/mL) after 3 days of treatment, demonstrated enhanced biofilm bactericidal effect compared with Lf and Flu alone. A 1 log10 reduction in CFU was achieved with 2 µg/mL Flu + Lf. Complete eradication (limit of detection 10 2 CFU) of bacteria was achieved at 20 µg/mL and 200 µg/mL Flu + Lf (resulting in >5 log10 reduction in CFU compared with the no treatment control). Flu only at 2 µg/mL, 20 µg/mL, and 200 µg/mL demonstrated 0, 3, and 4 log10 reductions, respectively. Lf alone resulted in no CFU reduction. Similar outcomes were achieved with Lf in combination with cefazolin. 2) Pilot results within rats (n=2) without experimental treatments demonstrated established S. aureus infection after 7 days with a mean of 2x10 6 CFU enumerated per homogenized tibia. Negative blood cultures confirmed no systemic infection with bioluminescence imaging confirming S. aureus localized at the generated tibial defect. No radiographic signs of osteomyelitis were detected. Results within the 4 treatment groups with a day 0, one-off lavage (saline only, Lf only, Bactisure with Lf, Bactisure with saline) of rats (n=10), demonstrated >95% reduction in the bacterial load 7 days post inoculation, with a reduction in S. aureus CFU from 1x10 6 per tibia down to 1x10 4 . There was no statistically significant difference between each treatment group (p = 0.55 with one way ANOVA). Intravital imaging of osteocytes with locally injectable fluorescent nanoparticles reveals sex differences and allows lacuno-canalicular network visualization. Melia D. Matthews 1 , Emily S. Cook 1 , Alexander Safarri 1 , Nada Naguib 1 , Uli B. Wiesner, PhD 1 , Karl J. Lewis, PhD 1 1 Cornell University, Ithaca, NY, USA, INTRODUCTION: Osteocytes are the resident mechanosensory cells in bone, maintaining skeletal health and homeostasis. Integrin mediated strain amplification has been shown to be a key functional mechanism for osteocyte activation to physiological fluid flow conditions 1,2 . These integrins are dynamic molecules, with endocytosis and recycling having been shown in vitro 3 but how those dynamics impact the function and mechanosensitivity of osteocytes has been challenging to interrogate in vivo. The imaging limitations imposed by the dense optical scattering matrix of bone can now be overcome using intravital imaging with multiphoton microscopy (MPM), which represents a powerful tool to study osteocyte mechanobiology 4 . A novel way to reach subcellular resolution with MPM osteocyte imaging involves surface functionalized fluorescent silica core-shell nanoparticles known as Cornell Prime Dots (C'Dots), which have been developed as an intravital imaging tool 5,6 . These extremely bright nanoparticles can be functionalized in many ways and can act as a targeted probe for integrins via surface RGD motifs (RGD-C'Dots). In previous work we validated the use of non-targeted and integrin-targeted C'Dots as novel in vivo osteocyte and integrin imaging tools 7 . Here we continue that work, exploring dynamin related integrin dynamics as well as using C'Dots to visualize dendritic processes and subcellular localization within the lacuno-canalicular network (LCN) in vivo.
METHODS: Silica core poly(ethylene glycol) shell (core-shell) nanoparticles covalently encapsulating Cy5 dye were created using a modified Stöber condensation in water 6 . RGD-C'Dots were created by surface functionalizing C'Dot surface with multiple amino acid motifs that bind to the extracellular RGD domain of integrin proteins. A control of non-functionalized C'Dots were also generated. Skeletally mature C57BL/6J mice age 16-20 weeks were used for these studies (n=5/group/sex). C'Dot Injections: Under inhaled isoflurane anesthesia, control or RGD-C'Dots were injected subcutaneously above the third metatarsal (MT-3) in the mouse hind paw at a concentration of 10μM. For the clearance study, C'Dots were incubated for 1 hour with mice allowed normal cage activity. Pre-incubation with Dyngo-4a for 30 minutes prior to C'Dot injection was used to inhibit dynamin-mediated endocytosis in treated groups. For LCN imaging, a 5 minute C'Dot incubation was used. Imaging: After incubation, the MT-3 was surgically isolated and stabilized using a metal 3-pin system in re-anesthetized mice 4 . The entire paw was submerged in a PBS bath and placed under a multiphoton microscope (MPM). MT-3 osteocytes were imaged using MPM (Bergamo II, Thorlabs), with a 20x water immersion objective (XLUMPLFLN, Olympus), excitation wavelength of 1090nm, and >647nm long pass filter acquisition. Image Quantification: Mean intensity and cell count of the C'Dot tagged osteocytes were quantified in ImageJ (NIH) using 3D segmentation and were normalized to background intensity. For statistical analyses GraphPad Prism software was used (ANOVA, p<0.05). All procedures were IACUC approved.

RESULTS
: Locally injected C'Dots can successfully localize to osteocytes in vivo, allowing visualization with MPM. (Figure 1). They can also allow observation of canaliculi and osteocyte processes within the LCN as well as nanoparticle subcellular localization ( Figure 2). A time-course study showed that clearance of C'Dots was sex-dependent ( Figure 3) (Linear Regression). Females had more C'Dot uptake and retained signal longer compared to males. When dynamin mediated endocytosis was pharmacologically inhibited the response was also sex-dependent ( Figure 3). Treated males had higher C'Dot uptake and retained signal, even above the original female clearance curve. Treated females had a reduction in uptake on the other hand, and the trend was most notable for both sexes in the RGD-C'Dot group ( Figure 3). DISCUSSION: C'Dots provide a novel opportunity to interrogate osteocyte integrin dynamics in vivo, with 3D microarchitecture and fluid environment in place, as well as providing enough resolution to visualize and interrogate the LCN in living bone. These experiments expand the use of C'Dots as a local, injectable, fluorescent osteocyte marker in vivo. This implicates novel applications for anatomical and functional studies of osteocyte and integrin dynamics. Our results also indicate that endocytosis dynamics or cell mediated clearing varies between RGD/control C'Dots and by sex.
SIGNIFICANCE: Fluorescent C'Dots are a novel method to study osteocytes and integrins in vivo, allowing for investigation of a range of structural, functional, and mechanobiological questions. This new tool offers an alternative to genetically modified mouse lines, reducing time and expense in the study of osteocyte/integrin mechanobiology, and could be translated to different species as well. Additionally, C'Dots can be used to visualize the LCN in vivo and interrogate biologically relevant topics in osteocytes such as endocytosis of integrins and focal adhesions.    2 Swim training induces differential osseous gene expression in anosteocytic and osteocytic teleost fish Josephine T.  (23)). In terms of the major regulator of mechano-adaptation in mammals, the Wnt signalling pathway, we found one DEG related to the canonical Wnt pathway in zebrafish at 1h, four at 8h, and seven at 24h. In contrast, ricefish did not demonstrate any Wnt signalling pathway-related significantly expressed DEGs at any time points. Further, we explored the expression profile of key regulator of the Wnt pathway, sclerostin (sost), and the main bone matrix protein collagen type I, col1a1 and col1a2. While their expression pattern did not reach statistical significance, zebrafish exhibited a decrease in sost in response to swim training, whereas ricefish did not exhibit a response. Further, zebrafish showed an overall upregulation of collagen type I, which increased in expression with swim training. Conversely, ricefish displayed relatively low collagen expression, which was not altered by swim training. Analysis of shared DEGs between zebrafish and ricefish revealed three overlapping DEGs at 1h, seven at 8h, and 92 DEGs at 24h. Narrowing these overlapping DEGs down to bone-related genes, identified again myocilin. Myocillin was expressed positively in both species, but more predominantly in ricefish. Conclusions: Our results demonstrate that zebrafish benefit from numerous bone-embedded osteocytes and their capacity to induce bone formation in response to mechanical stimuli. In contrast, for anosteocytic ricefish, swim training resulted in delayed effects on skeletal modelling. Previously, it was concluded that in medaka, nonosteocytic cells such as bone lining osteoblasts, chrondrocytes, and chordoblasts, sense and respond to mechanical load but in a different time-dependent manner (Ofer et al. PLoS Biol, 2019:17 (2)). Our results challenge the current paradigm of osteocytes exclusivity in bone-(re)modelling regulation, suggesting the existence of multivariate feedback networks in bone (re)modelling.
Non-invasive quantification of bone remodeling dynamics in adults with osteogenesis imperfecta using time-lapse HR-pQCT Seyedmahdi Hosseinitabatabaei, MS 1,2,3  Time-lapse imaging using high-resolution peripheral quantitative computed tomography (HR-pQCT) has emerged as a noninvasive imaging biomarker of bone (re)modeling. 1,2 In contrast to the density, microstructure, and strength measurements that indicate net overall changes in bone, timelapse HR-pQCT can additionally elucidate the cellular mechanisms behind the observed bone changes. Although this imaging biomarker has several advantages compared to non-site specific serum biomarkers and invasive histomorphometry of bone biopsies, there is no consensus on how to perform the procedure, as different studies have used different settings. Thus, we aimed to use same-day repeated scans from adults with osteogenesis imperfecta (OI) to 1) examine the influence of various imaging parameters on bone formation and resorption to identify a preferred methodology and longitudinal scans of adults with OI to 2) examine the effect of Setrusumab treatment) as part of a phase-2b dose finding multicenter trial (ClinicalTrials.gov Identifier: NCT03118570). In repeated scans from 29 adults (19-65yrs) without motion (n=13), any bone formation (mineralized volume fraction (MV/BV)) or resorption (eroded volume fraction (EV/BV)) indicates error. Thus, we first evaluated several parameters to minimize error in repeated scans. We first compared the input image (binary or grayscale) using two image registration methods (3D or matched-angle [MA]). Unlike 3D registration, MA rotates both images in opposite directions, thus propagating the error associated with image rotation to both images. The binary method considers bone formation when a voxel is absent in the first image, but present in the second image, while the opposite holds for resorption. 6 The grayscale method identifies formation when the density of a voxel increases past a certain threshold from the first to the second image. Thus, for the grayscale method, we also studied different values for the density difference between voxels to be considered formed or resorbed. 2 Additionally, for the grayscale approach, we also examined different strategies to reduce noise by removing clusters of formed/resorbed bone that are smaller than a certain size and by reducing noise using Gaussian smoothing. Regardless of image registration method, using binary input images resulted in large errors of ~13% and ~8% for XCT and XCT2, respectively. For the grayscale method, errors were almost identical but slightly smaller for the 3D compared to MA image registration. For both XCT and XCT2 scanner generations, a density threshold of 200 mgHA/cm 3 and a cluster size of 0 resulted in formation/resorption volumes approaching zero when combined with Gaussian noise reduction (Gaussian sigma of 0.8 for XCT and 1.2 for XCT2). We then validated the selected method by pairing either of the repeated scans with the follow up longitudinal scan which showed similar regions identified as formation and resorption. This confirmed that the formation and resorption sites were true biological changes rather than random noise.
Next, we used the method to evaluate the effect of different doses of Setrusumab on bone formation and resorption at the distal radius and tibia of adults with OI. The participants received Setrusumab on a monthly basis until 12 months, and were scanned every 6 months until 24 months (followed by 12 months off-treatment). Overall, we observed a positive dose-dependent effect of Setrusumab on bone formation and resorption at the radius and tibia. We also observed that the net difference between formation and resorption tended toward larger formation for the higher dose of Setrusumab. The net difference between formation and resorption was moderately and positively correlated with changes in bone density and strength outcomes, that were acquired from finite-element analysis.
Overall, this is the first study to perform comprehensive parametric study to identify the preferred settings for timelapse HR-pQCT analysis, and the first study to validate this method. This non-invasive imaging biomarker for bone formation and resorption can be used instead of invasive bone biopsies for dynamic histomorphometry, or non-site-specific serum biomarkers. Ex vivo studies suggest OA-associated cartilage softening may contribute to elevated shear and friction forces at the articular surface and predispose chondrocytes to acute cell death. [1][2][3][4] Thus, there has been great interest in the potential of stiffening mechanisms, such as collagen crosslinking (CXL), to slow OA progression. [5][6] Traditional stationary contact area (SCA) tribology studies support softening as a driver of elevated cartilage friction. In depressurized and slowly slid (~1mm/s) SCA contacts, equilibrium friction coefficients of OA-like cartilage (µk=0.2-0.3) are 2X higher than healthy tissue. 7 However, the clinical relevance of SCA-based findings is unclear given that cartilage operates at higher average sliding speeds (30-80mm/s) and markedly lower frictions (µk<0.005) in vivo. 8 Recent studies employing the convergent SCA (cSCA) reveal that modest sliding speeds (>30mm/s) facilitate interstitial fluid recovery and sustain near-physiological friction (µk≤0.02 in saline and µk≤0.005 in synovial fluid (SF)) via tribological rehydration. 7 Critically, cSCA tribological behaviors are largely insensitive to mechanical properties in healthy 8-9 and OA-like cartilage. 10 Here, we examined whether CXL-mediated stiffening alters cartilage lubrication under biofidelic sliding conditions in the cSCA. Methods: Osteochondral explants (AE19mm) were extracted from mature bovine femoral condyles. Mechanical properties, including tensile modulus (Ey+) and compressive modulus (Ey-), were determined from Hertzian indentation (50µm/s, 150mN normal force). 8 Tribological testing comprised ramp compressive loading to 7N followed by 30min static compression and 15min compression+sliding at 80mm/s. After characterization in the naïve state, effects of CXL were examined in (i) healthy or (ii) OA-like cartilage (approximating ICRS grade 2). 1-2 OA-like cartilage was generated via chondroitinase-ABC (chABC) digestion. 9 Explants were crosslinked via 0.6% glutaraldehyde (GTA) or 10mM genipin (GP). Results Fig. 1A). Tribological rehydration drove strain recovery of 6.09±0.83% in PBS-lubricated naïve cartilage. Strain recovery decreased to 4.54±0.56% with GTA treatment (p=0.016), but was not significantly altered by GP (p=0.30). Naïve cartilage sustained near physiological µk (median 0.009), whereas µk was significantly elevated after CXL (median 0.23 and 0.13 in GTA-and GP-treated cartilage, respectively; Fig. 1B). We examined whether synergy between tribological rehydration and SF 8 could mitigate the impaired lubrication seen after CXL. Interestingly, there were no differences in SF-lubricated strain recovery between naïve and CXL cartilage (p>0.1). Nonetheless, µk increased significantly following CXL. Whereas SF-lubricated naïve cartilage supported physiological µk (≤0.005), median µk values of 0.042 and 0.022 were observed after GTA and GP treatment, respectively (p<0.05, Fig. 1C). Digestion with chABC reduced Ey-from 0.66±0.19 to 0.48±0.13MPa but had no significant effects on strain recovery or friction (median µk=0.01 and 0.003 in PBS-and SF-lubricated contacts, respectively). Subsequent GP treatment increased Ey-to 0.91±0.20MPa (p<0.0005, Fig. 1D). Although GP had no effect on strain recovery, median equilibrium µk were elevated to 0.11 in PBS-and 0.021 in SF-lubricated contacts, respectively (p<0.005, Fig. 1E-F). Conclusions: Here, we introduce the first biofidelic assessment of cartilage lubrication following after CXL agents proposed for "reinforcing" native and engineered cartilage. [5][6] Our findings reveal incontrovertible detrimental impacts of CXL on cartilage lubrication. Despite persistent tribological rehydration (and synergistic lubrication in SF), friction coefficients after CXL exceeding those in paired naïve tests by 23-and 13-fold in PBS and SF, respectively. Exposure to such elevated friction coefficients is likely detrimental to chondrocytes, as multiple studies report marked cell death after just 30-60min of sliding at µk>0.05. [3][4][5] The markedly elevated friction coefficients of crosslinked explants indicates that CXL is ill-suited for cartilage preservation or joint resurfacing. More importantly, this work demonstrates that proposed OA treatments must undergo biofidelic lubrication testing to assess their suitability for use in the joint microenvironment.   Maroteaux-Lamy Syndrome, also referred to as Mucopolysaccharidosis VI (MPS VI), is a rare lysosomal disease arising from impaired function of the enzyme Arylsulfatase B (ARSB). This impairment causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in growth plates, cartilage, and extracellular matrix. While clinical presentation is variable in terms of age at first symptom manifestation and disease severity, MPS VI classically presents at early ages and strongly affects the skeleton. Current treatment guidelines recommend enzyme replacement therapy (ERT), which is known to provide incomplete or ineffective recovery from the skeletal phenotype. We can postulate this may be due to the inability of the exogenous enzyme to reach affected cells, or that disease may not be reversible at the time therapy is delivered. To date, no models of disease exist that separate treatment efficacy from reversibility.
To this end, we have generated a conditional by inversion (COIN) mouse model of MPS VI. In this design, gene expression is initially "off" due to the inversion of exon 5 in the Arsb gene. We have crossed these mice with a Rosa Cre-ER T2 line, such that after tamoxifen administration, the exon is flipped back to its native orientation, restoring gene expression. This approach allowed us to turn back "on" endogenous expression of the missing enzyme at several points during disease progression. By restoring Arsb expression at postnatal days 7, 21, and 56-70 (P7, P21, and P56-P70), we determined long bone length and other skeletal phenotypes can be fully rescued if gene restoration occurs at P7, while partial rescue occurs with later interventions. This work highlights the importance of early intervention in Maroteaux-Lamy patients, as maximal therapeutic impact would likely be seen with earlier treatment. Purpose: The purpose of this study is to assess the ability of various machine learning models to predict the occurrence of major medical complications following cervical decompression. Study Design: Crosssectional Database Study. Patient Sample: All patients (n=7743) undergoing cervical spine decompression (not including anterior cervical discectomy) in 2019 as identified in the National Inpatient Sample as sponsored by the Agency for Healthcare Research and Quality. Outcome Measures: Postoperative major medical complications: death, pulmonary embolism, deep vein thrombosis, respiratory failure, myocardial infarction, and cerebrovascular accident. Methods: Underlying patient demographic factors and comorbidities were utilized to train multiple machine learning algorithms to predict major post-operative complications. Training data was used to generate classifiers in a 10-fold cross-stratified manner. A randomly generated subset of 20% of the initially identified patients were withheld as a test set to generate final error values and confusion matrices. These groups were then analyzed with the Waikato Environment for Knowledge Analysis (WEKA) software using the classifiers logistic regression and random forest, as well as the meta-classifier vote. Results: The random forest model was most precise overall in discriminating between those patients who did and did not suffer major post-operative complications following cervical decompression, followed by the logistic regression model. The higher-order metaclassifier (vote) failed to predict any occurrences of major complications. Conclusion: A random forest machine-learning algorithm was the most successful at predicting major post-operative complications, followed by a logistic regression model, with both of these outperforming the higher-order metaclassifier. This demonstrates that increasing sophistication of a machine-learning model does not necessarily correlate with improved predictive power. As the influence and complexity of these models continue to expand, both within medicine and society writ large, it will be important to continue to hold their results under close scrutiny. Low back pain afflicts up to 80% of Americans and accounts for over $100 billion in healthcare and social costs annually. While intervertebral disc (IVD) degeneration and injury have been strongly associated with low back pain, the causation between pathoanatomical features of the IVD and clinical presentation of low back pain remains poorly understood. Recent rodent work show that axial low back pain symptoms can be recapitulated by the targeted injury of the lumbar intervertebral disc. The injury evokes a degenerative sequala in the IVD and produces neurite infiltration that are associated with chronic behavioral symptoms of axial low back pain. This is corroborated by human studies that observed increased innervation in degenerated IVD from humans. Thus, understanding the molecular events that drives IVD innervation may provide insights to therapeutic opportunities prior to the transition to chronic LBP symptoms. The proliferation of sensory nerves is tightly regulated by Nerve Growth Factor (NGF) and Vascular Endothelial Growth A (VEGFA). NGF serves critical homeostatic functions in neurons, while VEGFA plays a crucial role in spontaneous neurite extension and dendritic outgrowth in connective tissues, in addition to initiating angiogenesis. Degenerate and injured IVD cells express VEGFa as a part of the inflammatory cascade, but the mechanistic effects of VEGFA on neoinnervation in the IVD and subsequent low back pain behavior have not been investigated. Our central hypothesis is that the ablation of VEGFA attenuates neurite growth and vascularization into the injured IVD and alleviates ensuing low back pain symptoms. In this talk, we will share our recent findings on whether the deletion of VEGFA after injury prevents IVD innervation and vascularization, and low back pain symptoms 3-and 12-weeks following a targeted injury. The mechanistic understanding of VEGFA's role in modifying the pathoanatomy will pave the way as a disease modifying therapy for low back pain. The ease for delivery anti-VEGF therapies makes it an attractive candidate for the diseased IVD, and anti-VEGFA has already been approved for the treatment of macular degeneration and cancer in humans. Every day, billions of cells in the body undergo apoptosis and their efficient clearance by phagocytes (efferocytosis) has been shown across multiple systems to be crucial for tissue homeostasis and development. In bone, a proportion of osteoblasts undergo apoptosis yet efferocytosis in this organ remains underexplored. To investigate this process, we generated a murine model with conditional expression of inducible caspase-9 (iCasp9) triggered in the presence of Cre (1). Caspase-9 is an apoptosis-initiating enzyme and iCasp9 activity can be induced via a dimerizer (AP20187/AP). We generated mice with inducible osteocalcin (OCN)+ cell apoptosis by crossing iCasp9 mice with OCN-Cre. 3-week-old OCN-Cre+/-iCasp9+/+ male and female mice were injected intraperitoneally with AP/vehicle. Immunofluorescence and histomorphometry of the vertebrae confirmed a significant increase in cleaved caspase-3+ apoptotic cells (p<0.05) and reduction in OCN+ osteoblasts after AP (p<0.01). Examination of known efferocytic cells showed no changes in TRAP+ osteoclasts and Ly6G+ neutrophils, but increased F4/80+ osteal macrophages associated with bone surfaces (p<0.01). Zstack imaging confirmed that many of these macrophages contained intracellular OCN+ remnants, indicative of efferocytosis (p<0.01). Treatment with AP for 5 weeks resulted in a paradoxical increase in vertebral bone volume fraction (Fig1a) and bone mineral density (Fig1b) irrespective of sex. Dynamic histomorphometry confirmed an increase in bone formation rate with AP treatment (Fig1c). TRAP+ osteoclasts remained unchanged (Fig1d-e). We performed single-cell RNA sequencing of bone marrow-derived macrophages cultured with AP-treated apoptotic osteoblasts. Ossification genes including Igf2 and amphiregulin, which are mesenchymal stem cell osteogenic differentiation factors, were upregulated (log fold change=6-7) when macrophages efferocytose apoptotic osteoblasts (data not shown). Accordingly, previously used pre-osteoblast or osteoprogenitor markers were examined. There was an elevation of osterix+ (Fig1f) and amphiregulin+ (Fig1g-h) cells in the bone marrow near bone surfaces after AP treatment. Moreover, alpha smooth muscle actin (αSMA)+ cells directly associated with bone surfaces were also increased in AP-treated mouse vertebrae (Fig1i-j). Collectively, this work not only establishes the importance of osteoblast apoptosis but also macrophage efferocytosis in regulating bone formation likely via osteoprogenitor recruitment. Importantly, it exposes the potential of targeting these mechanisms to promote bone formation in clinical setting.   Characterization of the bone mechanoresponse in birds is limited. Numerous studies have reported the effects of exercise on bone in avian species including chickens, however the mechanical stimulus was not controlled in these experiments. Early controlled loading studies were performed on the ulna of turkeys and roosters; however, these experiments were invasive and focused only on skeletally mature animals. Since then, the bulk of controlled loading studies have been performed on mice and rats and have shown that the bone's mechanoresponse is influenced by age (1), genotype (2), and loading history (3). Whether or not this extends to avian species and chicken tibiotarsi specifically, remains unclear. We therefore aimed to investigate the effect of controlled in vivo tibiotarsal loading on bone mass and microstructure in young female chickens of different genotypes and loading history. We hypothesized that controlled loading would lead to improved microstructure, and that a strain-matched load stimulus would have similar effects across genotypes but would have a stronger effect in chickens with a more sedentary loading history.
Female chickens of two genetic strains (Lohmann Brown, B; Lohmann LSL Lite, W) were raised in either cages (Cage) that only allow for standing and sitting, or two styles of aviaries (Low, High), which have been shown to lead to low and high levels of physical activity, respectively (4). At 14 weeks of age, prior to onset of sexual maturity, the right tibiotarsus (n=7 chickens/genetic strain [2]/housing [3]) was subjected to controlled in vivo axial compressive loading (peak load of 170N, which engenders medial strain of -2,800 με, determined by strain gauging another cohort), while the left tibiotarsus served as a non-loaded control. After two weeks of once daily loading (4 Hz, 216 cycles/day, 5 days/week), chickens were euthanized. Tibiotarsi were μCT-imaged (9um voxel size) to assess the mid-diaphyseal cortical and medullary bone mineral density and microstructure. Results were analyzed by repeated measures ANOVA followed by Tukey's post-hoc tests and pairwise t-tests, with significance at p ≤ 0.05. Our results surprisingly show a negative effect of in vivo loading on mid-diaphyseal tibiotarsal microstructure and density. Our findings also show that there is a genetic strain and loading history-dependent effect of loading. Further microstructural analysis at different bone regions, fluorochrome-based histomorphometry to examine mineral apposition rates, as well as enzyme histochemistry to analyze osteoclast surface area are underway to assess cellular mechanisms behind this response. Future parametric studies are necessary to determine how load magnitude, frequency and duration may influence the mechanoresponse in lower limb bones of chickens. Development of a novel inorganic polyphosphate-releasing thermoresponsive hydrogel to improve fracture healing outcomes. Rayan Ben Letaifa 1,2 , Deepak Chauhan 3 , Chang-sheng Wang 3  INTRODUCTION: Bone fractures are among the most common musculoskeletal injuries. They severely disrupt the quality of life of the individual and increase healthcare costs. Approximately 5-10% of patients experience complications such as delayed unions and non-unions and consequently may require invasive revision surgeries, which are not always successful. Immune cells such as macrophages, neutrophils, and mast cells play a crucial role in fracture healing, and dysregulation of their function can lead to impaired healing. Among immunocompetent cells, mast cell granules have the broadest range of stored growth factors and play an essential role in bone healing, as shown by our team. Inorganic polyphosphates (polyP) have been shown to modulate immune function by closely regulating the functions of macrophages, monocytes, and mast cells. At the fracture site, polyPs released by degranulating platelets play a crucial role in initiating coagulation and, thereon, are thought to recruit immune cells. Accordingly, the objective of the study is to develop a thermosensitive hydrogel with sustained release of polyP to be implanted at the site of fracture to enhance the homing and degranulation of mast cells at the fracture site.
METHODS: Hydrogel formulations and sol-to-gel transition times were tested using several poloxamers, Gellan gum, and poly-N-vinyl-caprolactone (PNVCL). Varying concentrations of each component were tested in different combinations using the tube inversion method. Drug loading of hydrogel formulations was accomplished by mixing dehydrated polymer components with polyP-45-containing distilled water. A polyP release study was undertaken by inserting a gel in a dialysis membrane with a 6-8 kDa molecular weight cut-off. Samples of the supernatant were subsequently collected at designated several designated time points. Rheological assessments were performed to characterize the viscoelastic properties of the hydrogel. Assays for cell adhesion, cell proliferation, cell survival, and cellular metabolic state to assess the cytocompatibility of the fabricated hydrogel were performed using RBL-2H3 cells (murine mast cell analog), MC3T3 cells (murine pre-osteoblasts), and primary murine bone marrow-derived stem cells (BMSCs).
RESULTS: Tested hydrogel formulations incorporating poloxamers alone exhibited sol-to-gel transition times below 90 seconds, while those incorporating chitosan alone showed sol-to-gel transition times greater than 5 minutes. Poloxamer sol-to-gel transition times were also concentration and subtype dependent. PF-88 and PF-108 showed transition times of below 2 minutes, while those of PF-31 and PF-237 were greater than 5 minutes. The hydrogel formulation consisting of 20% PF-108, 1.5% PNVCL, and 3% Gellan gum (w/v) showed a sol-to-gel transition time of 60 seconds. This formulation was chosen for further analyses and development for its sol-to-gel transition time, as this would be optimal for in vivo implantation and rapid wound closure. This formulation was used to conduct the polyP release study, which produced a polyP concentration of 84.5 µM during the rapid-release phase and maintained a concentration of approximately 37 µM during the extended-release phase in the supernatant. Furthermore, approximately 60% of initially loaded polyP was released after a period of 5 days. Rheological analyses showed a significantly greater elastic modulus than a viscous modulus. Furthermore, the temperature sweep reveals that the sol-to-gel transition occurs at 35°C. The seeded on the hydrogel showed 92% cell adhesion and 95% cell viability (RBL-2H3, MC3T3, and primary BMSCs).
DISCUSSION: This study will generate a novel drug delivery system augmented with a bioactive compound, serving to modulate the body's own immune system to enhance bone healing, improve clinical outcomes, and reduce clinical strain. Finally, after the exhaustion of polyP from the hydrogel, the hydrogel remaining at the site can act as a bio-scaffold, allowing cells to migrate and enhance the development of the fracture callus, thus serving as a dual-purpose bioscaffold. Our next steps are to assess the immunoattractant properties of this polyP-releasing hydrogel in vitro to assess the migration of isolated primary murine mast cells and macrophages using a microfluidic device, with cell migration assay performed using AI-powered single-cell tracking software. Autophagy is a recycling pathway in which cellular components, including damaged or dysfunctional proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Reduction or insufficiency of autophagy is thought to contribute to a number of skeletal diseases such as skeletal aging. Accordingly, the elimination of autophagy from the entire osteoblast lineage or only from osteocytes reduces osteoblast numbers, decreases bone formation, and causes low bone mass 1,2 . However, whether increasing autophagy in osteoblast lineage cells would be beneficial for bone health under physiological or pathological conditions is unknown. To address this, we generated a novel model to increase endogenous Tfeb expression in osteoblast lineage cells. Transcription factor EB (Tfeb) is a master transcriptional regulator of genes involved in autophagy and lysosomal biogenesis. Consistent with this, Tfeb overexpression induces autophagy in cultured osteoblasts. We stimulated expression of the endogenous Tfeb gene in mice via CRISPR activation. Specifically, we expressed a sgRNA targeting the Tfeb transcription start site from the Rosa26 locus and activated a dCas9::SPH transgene using the Osx1-Cre transgene. This maneuver increased Tfeb expression 2.5-fold in RNA isolated from whole bones. At 8 weeks of age, the skeletal phenotype of mice overexpressing Tfeb was similar to littermate controls. However, at 4 months of age, Tfeb over-expressing mice displayed increased femoral and vertebral cortical thickness. The increase in femoral cortical thickness was due to increased periosteal circumference, suggesting an increase in periosteal bone formation. We also observed increased femoral, but not vertebral, cancellous bone volume. Femoral bone strength was increased as determined by threepoint bending analysis. In conclusion, Tfeb overexpression in osteoblast lineage cells increases cortical thickness and results in mechanically stronger bones and may represent an effective approach to combat age-associated bone loss.

In vivo induction of
Characterizing the limits of 3-photon microscopy as a tool for deep imaging of whole, cortical bone tissue Cornell University, Ithaca, NY Introduction: Osteocytes have emerged as the primary mechanosensors in bone, regulating its ability to adapt to mechanical stimulus. These cells form dense, interconnected tunnels within the bone tissue, and their function in health and disease is intrinsically tied to this native microenvironment; thus there is a need to study them in vivo to understand their mechanobiology. One major challenge in reaching this goal is the fact that mineralized bone is a dense and highly scattering material which is not easily amenable to optical techniques. Two-photon (2P) microscopy has been successfully used to observe osteocyte molecular signaling in-vivo in the mouse metatarsal (Lewis et al. 2017). Three-photon (3P) microscopy is a novel imaging platform that offers better depth penetration over 2P fluorescence imaging along with third and second harmonic generation. Wu and colleagues have successfully observed osteocytes in vivo in mouse calvaria, capturing lining cells within marrow and the canaliculi of the lacunar-canalicular space (LCS) (Wu et al. 2018). Further characterization is needed to investigate 3P use in long bones to assess maximal imaging penetration with longer wavelength excitation. In this study, we hypothesized that 3P microscopy in intact cortical bone ex vivo will yield better depth penetration and increased data variety compared to 2P in long bones. The development of 3P microscopy in bone reported in this abstract represents a meaningful advancement of intravital imaging approaches for the study of osteocytes in situ. . Bilateral third metatarsals, tibiae, femurs, and humeri were dissected and fixed for 24 hours in zinc formalin. Bones were then soaked overnight in 1mM CaCl2 to maximize GCaMP6f fluorescence brightness. Samples were divided into three groups based on excitation wavelength: 2P excitation at 920nm, 3P excitation at 1320nm, and 3P excitation at 1700nm (n=4/sex/group). Images were collected using an Opera-F amplifier pumped by a Monaco laser (Coherent) with a 25x water immersion objective (Olympus, NA=1.05) in ScanImage (MBF Bioscience). Z-stacks at 512x512 pixel resolution were taken at the mid-diaphysis through the anterior-coronal plane, except for the tibia, which was imaged through the medial-sagittal plane.

Results:
We found that 3P microscopy can penetrate to a depth of over 500 microns, revealing the marrow space in all bones studied. By contrast, 2P microscopy is limited to about 80 microns maximum. Third harmonic signal, which indicates changes in material interface, returned the brightest signal and provided morphological information about the LCS. Second harmonic signal resolved collagen structures and brightly revealed the bone matrix. Both harmonic signals are achieved without the need for exogenous label. GCaMP6f fluorescence reveals osteocyte bodies and the periosteum. Bone marrow only appears as a weak and diffuse signal without labeling.

Conclusion:
Three-photon fluorescence microscopy in conjunction with the third and second harmonic visualization can produce comprehensive images of both bone tissue and osteocytes from whole specimens with enough resolution to distinguish canaliculi ( Figure 1). Moreover, exogeneous labeling is not needed to observe the morphological structure of the LCS microarchitecture or bone matrix. Third harmonic imaging capabilities represent an important added dimension of intravital imaging capabilities compared to 2P systems. Future work includes in vivo loading paired with 3P imaging to interrogate differences in mechanical activation of osteocytes at the periosteal and endosteal surfaces. Three-photon imaging paired with exogenous labeling opens new pathways to concurrently explore the interdependent roles of osteocytes and their processes, related musculoskeletal cells, bone marrow, and bone tissue itself as a collective system. The NAD salvage pathway is required for both articular and growth plate chondrocytes of the perinatal appendicular skeleton Introduction: Nicotinamide adenine dinucleotide (NAD) is an essential co-factor for energy metabolism, redox homeostasis, and numerous other cellular processes. Systemic NAD deficiency has been implicated in skeletal deformities during development in both humans and mice. Cellular NAD levels are maintained by multiple synthetic pathways, but which ones are critical during skeletal development and homeostasis is currently unknown. The goal of this study was to determine the importance of the NAD salvage pathway during appendicular skeletal development.
Methods: Animal studies were conducted as preapproved by the Institutional Animal Care and Use Committee at UAMS. nicotinamide phosphoribosyltransferase (Nampt), a critical enzyme in the NAD salvage pathway, was deleted within all mesenchymal lineage cells of the limbs by crossing Nampt fl/fl with Prx1-Cre mice, generating Nampt ΔPrx1 progeny and Cre-negative littermate controls. To circumvent the need for the salvage pathway during gestation, dams were provided approximately 650 mg/kg of the NAD precursor nicotinamide riboside (NR) through their drinking water. The resulting pups, which did not receive further NR following birth, were examined indiscriminant of sex at P2, P7, and/or P21 by whole mount staining as well as histology with terminal deoxynucleotidyl transferase nick end labeling (TUNEL) or in situ hybridization. Hindlimb epiphyses and joints from additional P2 mice (2-3 per genotype) were digested into single cell suspensions, depleted of hematopoietic lineages, and submitted for single cell RNA-sequencing using a 10X Genomics workflow. After quality control, >8,000 cells per genotype underwent cluster and differential gene expression analysis.
Results: Nampt ΔPrx1 mice exhibited dramatically shortened limbs at birth due to the death of chondrocytes within the growth plate (Fig. 1A).
Administration of NR to dams prevented the majority of in utero defects while introducing the opportunity to study the importance of the salvage pathway during postnatal limb development (Fig. 1B). Postnatal NAD depletion led to progressive growth plate and articular chondrocyte death, preventing further endochondral ossification and joint development; pronounced articular chondrocyte death was noted as early as P2 in Nampt ΔPrx1 hindlimbs (Fig. 1C). Single cell analysis revealed sparsely-populated articular and growth plate chondrocyte clusters from prenatal-rescued Nampt ΔPrx1 hindlimbs, in favor of a distinct chondrocyte cluster characterized by reduced expression of cartilage anabolic genes and increased expression of stressrelated genes ( Fig. 2A). Performing differential gene expression between Nampt ΔPrx1 and control chondrocytes, several genes upregulated in the Nampt ΔPrx1 cells have been associated with osteoarthritis pathogenesis (Fig. 2B). This including Ptgs2 (COX-2), as confirmed to be upregulated in Nampt ΔPrx1 articular chondrocytes by in situ hydridization (Fig. 2C).
Discussion: These findings define a critical role for cell-autonomous NAD homeostasis during bone and joint development. Remarkably, chondrocytes forming the articular surfaces of hindlimb joints were especially sensitive to Nampt deletion. Many of the markers upregulated in stressed neonatal chondrocytes also characterize osteoarthritic articular chondrocytes, suggesting a possible role for NAD dysregulation in articular chondrocyte homeostasis.
Blood vessel associated osteoclasts: identification and functional characterization Sara G. Growth plate cartilage contributes to the generation of a large variety of shapes and sizes of skeletal elements in the mammalian system. The removal of cartilage and how this process regulates bone shape are not well understood. Here we identify a non-bone-resorbing osteoclast subtype termed vessel-associated osteoclast (VAO). Endothelial cells at the bone/cartilage interface support VAOs through a RANKL-RANK signalling mechanism. In contrast to classical boneassociated osteoclasts, VAOs are dispensable for cartilage resorption and regulate anastomoses of type H vessels.
Remarkably, proteinases including matrix metalloproteinase-9 (Mmp9) released from endothelial cells, not osteoclasts, are essential for resorbing cartilage to lead directional bone growth. Importantly, disrupting the orientation of angiogenic blood vessels by misdirecting them results in contorted bone shape. This study identifies proteolytic functions of endothelial cells in cartilage and provides a framework to explore tissue-lytic features of blood vessels in fracture healing, arthritis and cancer. , and may serve as a promising therapeutic target to reduce fracture burden. FDA-approved Raloxifene (RAL) is a promising matrix-modulating therapeutic that directly interacts with collagen-mineral interface to increase hydration and improve mechanical properties in a cell-independent manner. Synthetic salmon calcitonin (CAL) may also have matrix-modifying capacity like RAL due to its ability to reduce fracture risk clinically with little change in bone mineral density. Therefore, under CKD conditions, we sought to 1) determine whether CAL could modify hydration in a cell-independent manner like RAL (ex vivo), 2) elucidate if a matrix-modifying therapeutic could improve hydration/mechanical properties in vivo, and 3) extend the bone water interrogation to CKD iliac crest biopsies.
METHODS: Ex vivo: Femora from 31-week-old male C57BL/6 mice induced with chronic kidney disease (CKD) beginning at 16 wks via 0.2% adenine-laced casein-based (0.9% P, 0.6% C) chow, and their non-CKD control littermates (Con), were utilized. Femora were randomly assigned to the following ex vivo experimental groups: RAL (2 µM, n=5 CKD, n=5 Con), CAL (100 nM, n=5 CKD, n=5 Con), or Vehicle (VEH; n=4 CKD, n=5 Con). Bones were incubated in PBS+drug solution+1% penstrep at 37°C for 14 days using an established ex vivo soaking methodology followed by 3-pt bend testing (mechanical properties) and assessment of total and bound water via solid state nuclear magnetic resonance spectroscopy (ssNMR). Data were analyzed via 2-way ANOVA. In vivo: 16 wk male C57BL/6 mice were induced with CKD (n=48) or remained as non-CKD littermate controls (Con, n=48). At 26 wks, all mice underwent right tibial loading to a maximum tensile stress of 2050 μƐ 3x wk/5wks. Half of the mice (equal number CKD and Con) received s.q. RAL (0.5 mg/kg/5x wk) and the other half were untreated (UN). Mice were sacrificed at 31 wks, and bi-lateral tibiae underwent 4-point bending and ssNMR. Data were analyzed using 2-way RM ANOVA and Pearson correlation coefficients. Biopsies: A transiliac crest biopsies were obtained from a stage 3 CKD patient and from one non-CKD donor and assessed via ssNMR.
RESULTS: Detailed results from ex vivo CKD, in vivo CKD, and preliminarily experience with biopsies are found in Figure 1.
DISCUSSION: Total and bound water is compromised in CKD and its therapeutic modulation improved mechanical properties in ex vivo and in vivo experiments. Early biopsy experience shows that assessing water in clinical biopsies is feasible and appears to be compromised and work is underway to determine impact of CKD disease stage on bone water. for several 4-pt bend test properties including postyield displacement. ssNMR water analysis showed a main effect of disease and treatment for total water (F) and a main effect of disease, treatment, and a sig. interaction term for bound water (G). Post-hoc testing (displayed on figure) showed RAL treated CKD animals had sig. higher bound water vs. Un CKD animals. H) Pearson correlation coefficient between post-yield displacement and bound water was sig. demonstrating a positive relationship in CKD. Biopsy Data: I) 1 H ssNMR spectra from our Initial experience using ssNMR in patient iliac crest biopsies. Additional biopsy experiments are underway to elucidate water content.

Lymphatic Endothelial Cell Secretome and Gorham-Stout Disease Mouse Model leads to Enhanced Bone Resorption
Ernesto Solorzano 1  Complex Lymphatic Anomalies (CLA) are rare conditions characterized by malformed lymphatics in soft tissue and ectopic lymphatics in bone. Gorham-Stout Disease (GSD) is an aggressive type of CLA characterized by regional bone involvement resulting in cortical bone loss. Aberrant lymphatic vessels in CLA patients have been linked to KRAS somatic activating mutations. These mutations induced lymphatic bone invasion and destruction. Lymphatic endothelial cells (LECs) and their secretome are likely responsible to induce bone destructions as observed in GSD. To better understand the mechanism associated with CLA-induced bone destruction, we evaluated the effects of LEC-conditioned medium (L-CM) on osteoblast (OB, bone forming cells) and osteoclast (OC, bone resorbing cells) differentiation and function. OB and OC differentiation was assessed in the absence or presence of varying concentrations of L-CM. L-CM treatment inhibited OB proliferation, differentiation, and function while stimulating OC differentiation and function. These results were confirmed through bone formation and resorption assays in vivo. L-CM inhibited bone formation while accelerating bone resorption in mice. Next, we sought to determine the effects of the mutant KRAS in bone homeostasis in vivo. We generated a mouse model overexpressing inducible mutant KRAS in LECs and characterized their skeletal phenotype. Our preliminary studies showed a decrease in bone mass, bone mineral density, as well as bone-related markers in mutant animals compared to controls. We are currently assessing mutant KRAS mice OBs and OCs for their differentiation and function. In summary, our study revealed that mutant KRAS lymphatic secretome may lead to dysregulation in bone homeostasis observed in CLA patients. Gain of function mutations in the intracellular domain of the type I BMP receptor ACVR1 are associated with fibrodysplasia ossificans progressiva (FOP), a rare but catastrophically debilitating disease of heterotopic ossification (HO) of skeletal muscles, tendons, ligaments, and fascia. Although, the vast majority of FOP cases are caused by a missense mutation that alters Arginine 206 to a Histidine in the GS domain of ACVR1 (ACVR1[R206H]), FOP-causing mutations can occur throughout the GS and the kinase domain of ACVR1. Of particular note, are three patients carrying a mutation in the ACVR1 kinase domain that alters Arginine 258G to a Glycine (ACVR1[R258G]). These patients exhibit more severe developmental skeletal and non-skeletal defects and more robust post-natal HO than is observed for ACVR1[R206H] bearing FOP patients. In an effort to model this phenotypic variance, we engineered a Cre-regulated 'conditional-ON' allele of(Acvr1 INTRODUCTION: GFPT1 and GFPT2 are two isoforms of the enzyme glutamine-fructose-6-phosphate transaminase, which is the first and rate-limiting step in the hexosamine biosynthesis pathway (HBP) [1] . This pathway generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a crucial substrate for protein glycosylation and other cellular processes [1] . In chondrocytes, the main cell type in cartilage, the HBP has been shown to be involved in regulating cellular functions that are critical for cartilage homeostasis [2] . Emerging evidence has suggested that dysregulation of the HBP and GFPT in chondrocytes may contribute to the development and progression of osteoarthritis (OA) [2] . However, the relationship among the HBP, chondrocyte metabolism, and the pathogenesis of OA remains intricate and not fully understood, making the development of effective interventions for OA treatment challenging. In this study, we aim to investigate the role of GFPT1 and GFPT2 in chondrocyte metabolism and their involvement in OA progression. METHODS: All animal experiments in this study were performed in accordance with approval of the Committees on Animal Resources in Washington University in St Louis. Gfpt1f/f mice and Gfpt2f/f mice were generated by our laboratory. Agc1CreERT2; Gfpt1f/f mice and Agc1CreERT2; Gfpt2 f/f mice were viable and produced in Mendelian ratios. Tamoxifen was administrated daily at a dose of 1 mg/10g body weight for five consecutive days via intraperitoneal injection to 2-month-old Gfpt1f/f and Agc1CreERT2; Gfpt1f/f male mice, 2-month-old Gfpt2f/f mice and Agc1CreERT2; Gfpt2 f/f male mice, respectively. Following tamoxifen injection, meniscal ligament injury (MLI) surgery was performed unilaterally in the right knee joints of male mice at 3-month-old. Right knee joints were harvested for Micro-CT analysis and histological staining at 4 weeks and 12 weeks following MLI surgery. Moreover, articular chondrocytes were isolated from the femoral head of 2-week-old pups of Gfpt1f/f and Gfpt2f/f pups, respectively. The next day after plating, cells were transduced with adenoviruses expressing GFP or Cre at a multiplicity of 50 in the presence of polybrene (10 μg/ml). Following transfection and recovery, cells were treated with vehicle or TGFβ for the following experiments. Real time qPCR was conducted to analyze the relative gene expression. All data were expressed as means ± SD, and were analyzed using GraphPad. A p value < 0.05 was considered statistically significant. RESULTS and DISCUSSION: In Figure 1 and Figure Fig.3. Four weeks after MLI surgery, Gfpt1 and Gfpt2 loss-of-function (LOF) mice exhibited decreased resistance to knee joint injury, with more severe cartilage degeneration observed compared to the control mice, as indicated by higher OARSI scores. Overall, our findings indicate that both Gfpt1 and Gfpt2 are responsible for HBP and chondrocytes homeostasis, and disruption of the HBP in chondrocytes could potentially contribute to the pathogenesis and advancement of OA. In our future studies, we will explore the intracellular metabolite using 13C-labeled glucose/glutamine, and generate transgenic mice with double knock out of Gfpt1 and Gfpt2 to further investigate the mechanisms underlying the regulation of HBP pathway in chondrocyte homeostasis and its potential role in OA progression, and to identify specific molecular targets to OA therapy. Osteoarthritis (OA) of the knee is a degenerative condition of the skeletal extracellular matrix (ECM) marked by the loss of articular cartilage and necrosis of the underlying bone. Treatments for OA beyond full joint replacement are lacking primarily due to a lack of molecular knowledge concerning the biological drivers of the disease. OA commonly displays spatial discrimination preferring either the lateral (outer) or medial (central) joint, and importantly, histologic evidence suggests that early degeneration can appear and gradually progress across the entire joint and even in 'healthy' joints of aged individuals. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) offers the ability to spatially investigate the molecular landscape (proteins) of OA severity and progression to more fully understand the biological mechanisms of joint degeneration.
Human tibial plateaus from BMI matched male donors aged 60-75 with and without diagnosed medial OA (N=5 patients each) were collected and formalin fixed to prepare for paraffin embedding and sectioning. Mounted tissue sections were prepared for MS Imaging with a collagenase treatment to target ECM components and imaged utilizing the Bruker timsTOF fleX mass spectrometer. Untargeted analyses of collected MS1 mass spectra were processed using the Bruker SCiLS Lab software package.
We demonstrate the first collection of spatial ECM proteomic spectra from human bone and cartilage from tibial plateaus of healthy and OA patients which resulted in over 3,000 identified m/z features detected associated with the skeletal ECM. Visualization of detected ions revealed striking separation in the abundance of features assignable to human cartilage and bone via principal component analysis (PCA). Further, natural biologic boundaries within the joint not normally seen histologically were easily identifiable in generated ion maps of healthy tissue, improving upon details found with traditional microscope-based imaging. Unsupervised spatial segmentation of the top 500 abundant MS1 precursor ions from diseased joint tissue revealed new gradients in underlying bone degradation increasing from the less affected lateral joint to the more severe medial side of the joint where OA progression and cartilage loss was the greatest. Comparison of the identified sclerotic bone regions directly underneath areas of cartilage loss, segmented by SCiLS, to less degenerated tissue surrounding the lesion with AUC>0.75 found a minimum of 56 ions specific to the most severe OA regions. The identity of several peptides was confirmed as derived from the bone and cartilage collagen chains α-1(I) and α-1(II) while new unannotated peptides were discovered and identified by MS/MS as belonging to other ECM proteins including aggrecan, biglycan, several other collagen species and other proteins. This novel strong signature of 56 candidate ions for OA was consistently found in all five OA patients at high abundance in the medial (diseased) joint. These signature markers were also identified at much lower abundance in the transitional region between cartilage and bone in lateral OA joints and in both sides of aged-control joint samples, providing a 'gradient' of disease progression. The visualization and spatial up-regulation of collagen and ECM-derived peptides specific to OA and their unexpected appearance in aged-healthy joints reveals new molecular details about the development and progression of OA with age even in non-symptomatic individuals and allows for new characterization of disease severity. . AFSCs derive much of their therapeutic potential from the growth factors and cytokines they contain. These growth factors and cytokines can be isolated in the media of conditioned AFSCs. Preclinical and clinical studies with intra-articular injections of AFSC-conditioned media (AFSC-CM) have shown mitigation of PTOA and OA symptoms. In this study, we used a rat PTOA model evaluating the effects of AFSC-CM on functional and histological outcomes. We also measured cytokine levels to elucidate the inflammatory pathway AFSC-CM specifically targets.
METHODS: All animals used for the study were approved by IACUC at Wake Forest University School of Medicine. PTOA was introduced in both female and male rat cohorts (n=12 per group) with destabilization of the medial meniscus (DMM) and anterior cruciate ligament (ACL) transection on the left knee. Right knee was used as contralateral control. Intraarticular injection of AFSC-CM or saline was administrated at 2 weeks post injury. Animals were followed up for 1, 2, or 3 months for outcomes (n=4 per time point in AFSC-CM and saline group, respectively). Gait analysis was performed using Digigait Imaging system (Mouse Specifics Inc.) to calculate various kinematic and gait dynamics related to the rat's motion. Knees were decalcified, embedded then sectioned for histological staining. Slides were stained with hematoxylin and eosin (H&E; Abcam) and safranin-O (SigmaAldrich) for Articular Cartilage Structure (ACS) scoring to evaluate arthritis severity. Multiplex cytokine analysis was performed on the serum at different time points following treatment. To compare differences in saline control versus AFSC-CM cohorts, unpaired and paired t-tests were performed on ACS scores. T-tests were also performed on cytokine levels at 2 weeks post injury prior to the treatments. One-way analysis of variance test was performed on Digigait parameters between groups. Probability level of 0.05 was considered significant for both tests. DISCUSSION: There was improved cartilage structure at 2 months on histological safranin-O staining with superior preservation of cartilage in the AFSC-CM treated group compared to saline control, which had obvious compromise of tibial cartilage surface and vertical fissures (Figure 1a and 1b). Decrease in cytokine inflammation was significant at 3 months for TNF-α and at 1 month for IL-1α providing a promising response to AFSC-CM treatment given the widely reported effects of TNF-α and IL-1α driving OA pathology. Gait analysis is a validated method of analyzing locomotive parameters such as mechanical allodynia and evaluating the functional effects of biomechanical pathophysiology as in the case of DMM and ACL transection with AFSC-CM treatment. Differences in paw length, paw area and increased paw variability all possibly suggest temporal and spatial differences in gait due to increased pain in the absence of AFSC-CM treatment in rats.   Introduction: Humans have limited regenerative potential of the musculoskeletal system following limb loss. To that end, the murine digit is used to investigate the pathways regulating complex tissue regeneration after amputation [1][2][3][4].
Following distal amputation of the terminal phalanx (P3) bone, stem/progenitor cells (the 'blastema') regrow the resected tissues, but the mechanisms controlling skeletal elongation and patterning remain unclear. Although regeneration may not completely recapitulate embryonic development [3], activating elements of a limb-like developmental program, such as Hox genes, may be critical for bone outgrowth [4,5]. We hypothesized that the expression of Hox genes, and especially Hoxa13, modulates skeletal outgrowth and patterning during digit regeneration in adult mice.
Methods: To test this hypothesis, we generated novel Osx-Cre ERT2 ;HoxA flox/flox mice to conditionally delete HoxA cluster genes from Osx+ cells (HoxA cKO) and compared them to HoxA flox/flox littermates without Cre (Control) [6]. For these studies, tamoxifen was given at 7-8 weeks of age to induce Cre-Lox recombination. We also utilized mice heterozygous for a global Hoxa13 null allele (Hoxa13 +/-) and compared them to wild type (WT) littermates [7]. Regenerative digits were established by resecting 25% of the P3 length at 10-12 weeks of age. Digits were imaged with microcomputed tomography (microCT) pre-and post-surgery at 0 days post-amputation (DPA), and at various DPA thereafter to quantify P3 length, volume, and pattern restoration (% volume overlap with pre-amputated bone). 5-ethynyl-2'-deoxyuridine (EdU) was added to drinking water 5 days before sacrifice to label proliferating cells and Alizarin Red was administered 2 days before sacrifice to visualize mineralization. Animals were euthanized at 14, 21, or 56 DPA, and digits were fixed, decalcified, and sectioned for histological analysis and confocal imaging. Significance was assessed by 2-way ANOVA with Tukey's post-hoc test (p<0.05).
Results: Morphologic and histologic measurements showed that resecting the distal portion of P3 led to different quality of bone regrowth for each mouse genotype (Fig. 1). After distal amputation, bone elongation was reduced in HoxA ckO mice compared to Control (Fig. 1B), as well as in Hoxa13 +/mice compared to WT (Fig. 1D). Furthermore, digit patterning was disrupted in mice with Hox defects. 3D image registration revealed that Control samples consistently restored the preamputated digit morphology, whereas HoxA cKO samples had comparatively lower volume overlap with pre-amputated states at 56 DPA (Fig. 1C). Similarly, P3 bones of Hoxa13 +/digits exhibited a disorganized, porous structure and lacked the organized collagen bundles that anchor the newly formed bone to the dermis in WT digits (Fig. 1E).  Fluorapatite Scaffolds as the Engineered Bone Grafts for Repairing Critical-Sized Pig Calvarial Defects Jeyapalina S 1,2 ; Nielson C 1,2 ; Shea J 1,2 ; Beck JB 1, 2 ; Agarwal JP 1,2 ; Yagas LC 2 ; Gociman B 2 1 Veteran Affairs Medical Center, Salt Lake City, UT, 2 University of Utah, Salt Lake City, UT INTRODUCTION: While autograft bone remains the gold standard, allografts are commonly used in orthopedic and dental surgeries to regenerate bone tissue. Although preferred, the downside of autograft bone includes the morbidity and cost of additional surgical sites for tissue harvest and the limited volume of available bone. To address the increasing demand for bone substitutes, demineralized bone matrix, xenograft bone, as well as engineered scaffolds are used but have had limited effectiveness in repairing critical-size bone defects-which are the defects that would not heal spontaneously despite surgical stabilization and require further surgical intervention with bone grafts. Although engineered bone grafts can be sourced in large volumes, they do not possess the intrinsic biological attributes of autografts or allografts. Our previous research has suggested that fluoridated apatites can improve bone formation within small defects. Moreover, in this rat model, we have also shown that fluorapatite (FA) scaffolds induce reduced immune responses when compared to hydroxyapatite (HA) while depositing more volume fraction of new-bone tissue compared to autografts. Based on these findings, we hypothesized that mechanically stable, porous FA scaffolds would have the ability to repair critical-size craniofacial defects as effectively as autografts. This hypothesis was tested using a pig-cranium model. METHODS: Using the previously established aqueous precipitation synthesis technique [1], FA was synthesized and characterized in-house. A modified [2] "lost foam" technique was then used to fabricate porous FA scaffolds, and they were dried prior to sintering at 1150°C (using a ramping procedure previously optimized for the foam burnout). Post-sintering, micro-CT images were obtained, and pore-size distribution and porosities of each scaffold were calculated (Materialize Mimics). Scaffolds were then cleaned with alcohol and sterilized. Using an IACUC-approved protocol, ten Yucatan miniature pigs were either implanted with autografts (n=5) or FA scaffolds (n=5). These pigs survived for 12 weeks. At necropsy, the bone tissue around the defects was removed, imaged using micro-CT, and then fixed in 10% formalin. After fixation, the samples were dehydrated in ascending grades of alcohol and xylene prior to embedding in polymethyl methacrylate. Embedded samples were cut into thin sections and polished to obtain optically clear sections for staining with Sanderson's rapid bone stains, counterstained with Van Gieson, and then imaged. RESULTS and DISCUSSION: Figure 1 shows representative micro-CT images of the scaffold, confirming the presence of interconnecting pores with an average size of ~800 microns. Figure 2 shows that, at 12 weeks post-implantation, while new bone tissue uniformly filled the FA scaffolds, the autografts group presented a center region devoid of bone. The semiquantitative micro-CT analysis revealed 67.8% ± 7.3 and 75.8% ± 9.5 of the bone volumes within the defects of autograft and FA groups, respectively. Statistical analysis revealed no significant difference (P>0.05) in bone formation between the two groups. Histological analysis revealed that new bone, without interposing fibrous tissue, was in apposition with the FA scaffolds ( Figure 3). Importantly, the quantitative bone volume data and histological images supported the tested hypothesis. Both data at twelve weeks showed that bone tissue completely infiltrated FA scaffolds. Thus, it could become a suitable autograft replacement material. It is also interesting to note that there was no foreign body response within newly deposited bone tissue in the FA scaffolds group. These images further highlighted the presence of healthy bone tissue within the FA scaffolds, with osteocytes and networks of canaliculi. On the other hand, in the middle of the cage, a region of dense fibrous tissue with necrotic bones and osteoclasts was visible within the autograft group.     Cartilage formation and maintenance are tightly regulated by chondrocyte differentiation. Chondrocytes can respond to a variety of external signaling via the transmembrane G protein-coupled receptors (GPCRs), however, how the cartilagespecific GPCR signaling pathway regulates chondrocyte differentiation is still incompletely understood. We have recently identified a cartilage-enriched GPCR named ADGRG6 (Adhesion G Protein-Coupled Receptor G6), which plays a crucial role in cartilaginous tissues for articular cartilage and intervertebral disc homeostasis. Using conditional knockout mice (Col2Cre; Adgrg6 f/f , called Adgrg6 cKO for short) and spatial transcriptomics approaches, we show that ADGRG6 is required postnatally to prevent hypertrophic differentiation and osteogenic transition in the growth plate. Ablation of Adgrg6 in osteochondral progenitor cells does not obviously affect embryonic limb development (Fig. 1A-D) but leads to severe growth plate defects at P20, including loss of resting zone cells, presence of acellular clefts in the proliferative growth plate, and persistent of hypertrophic chondrocytes in the hypertrophic growth plate (Fig. 1E-F''). To map the molecular mechanism of Adgrg6 in postnatal chondrocyte differentiation, we established a spatial transcriptomics workflow on formalin-fixed, paraffin-embedded (FFPE) knee joint tissues at P20 ( Fig. 2A-B). This approach identified 12 unsupervised tissue populations that are well correlated with the histological morphologies ( Fig. 2D-E). Local distinguishment analysis on the spatial spots mapping to the growth plate regions (Fig. 3A) identifies 256 differentially expressed genes between control and Adgrg6 cKO mutant mice (Fig. 3C). We observed reduced expression of anabolic genes but abnormally activated catabolic enzymes and increased osteogenic gene expression in the mutant growth plate (Fig. 3B). These results suggest that loss of Adgrg6 leads to an accelerated ossification of the growth plate chondrocytes, characterized by increased expression of hypertrophic marker (Col10a1) and osteogenic genes (Spp1, Col1a1, Runx2, Sp7, Postn), coupled with upregulated matrix enzymes (Mmp9, Mmp14, Prtn3, Elane). Analyses of gene ontology (GO) enrichment for biological processes showed that ossification and extracellular matrix organization were predominantly affected (Fig. 3D-E). The expression pattern of some genes has been validated with immunohistochemistry (IHC) analyses (Fig. 3F). We further demonstrated that this abnormal hypertrophic differentiation phenotype is correlated with a region-specific reduction of SOX9 expression and diffused upregulation of IHH signaling. Altogether, we identified a GPCR that is required for postnatal chondrocyte differentiation with a newly established spatial transcriptomics approach in mouse knee joints. These findings expand our knowledge of the cellular and molecular mechanisms that ensure appropriate postnatal differentiation of the growth plate and reveal a GPCR-mediated chondrocyte hypertrophic differentiation and transition process.

Mercer University School of Medicine
Introduction: The patient populations eligible for a lumbar spinal fusion (LSF) or a primary total hip arthroplasty (THA) tend to overlap and symptoms of one of the conditions may mimic or exacerbate the other, making the creation of a treatment plan challenging. Therefore, the sequential order of these procedures must be carefully considered to maximize patient outcomes.
Methods: The National Library of Medicine online database was searched for peer-reviewed articles published within the past 5 years (2018-2023) relating to the timing of lumbar spinal fusion and primary total hip arthroplasty.

Results:
Those that had an LSF after THA had greater wound complications, implant loosening, and osteolysis than those that had a THA after LSA [1]. Those that had a THA after LSF had a greater risk of adjacent segmental disease, periprosthetic fracture, periprosthetic joint infection, implant dislocation, cellulitis, heterotopic ossification, cup-liner dissociation, THA revision, and THA arthrotomy [2,3,4]. Those that experienced dislocation within the THA after LSF group had an increased PI-LL mismatch than those that did not experience dislocation [3]. There was no difference in the sequential order of LSF and THA on the Charlson Comorbidity Index, vascular injury incidence, bearing surface wear, and breaking of the implant [1]. Furthermore, the surgical approach of the total hip arthroplasty (direct anterior or direct lateral) had no significant effect on THA dislocation in both LSF after THA and THA and LSF groups [5]. Overall, dislocation is the most common etiology for hip arthroplasty revision for both LSF after THA and THA after LSF groups [4].

Discussion:
There are a greater number of complications associated with THA after LSF than LSF after THA, therefore LSF after THA should be the preferred surgical sequence. Nevertheless, taking into account the risk of dislocation for THA after LSF and the overall prevalence of dislocation, studies in the future should be directed toward how implant positioning can be optimized to a patient's unique anatomy, including a spinal fusion.  Osteoarthritis (OA) is a degenerative joint disease that causes progressive loss of articular cartilage. Current treatment options for OA are pain management, intra-articular injection of hyaluronic acid (HA) or invasive total joint replacement surgery. Despite the efforts to develop disease-modifying osteoarthritis drugs (DMOADs) in the last decade, there is yet an FDA-approved DMOAD available for patients. Cellular senescence is characterized by a loss of cell proliferation and an acquired senescence-associated secretory phenotype (SASP), a pro-inflammatory secretome that can pathologically alter the function of neighboring cells and tissues. Senescent chondrocytes significantly contribute to OA by inducing local inflammation and extracellular matrix degradation. Here, we demonstrate that activation of the canonical Wnt/β-catenin signaling pathway contributes to cellular senescence in healthy and osteoarthritic human chondrocytes. To target and prevent chondrocyte senescence in OA, we developed StemJEL, a new combination hydrogel comprised of the Wnt inhibitor sclerostin and hyaluronic acid. The StemJEL treatment to osteoarthritic human chondrocytes down-regulated the expression of genes/proteins associate with senescence, inflammation and ECM degradation in human chondrocytes. We next evaluated the therapeutic efficacy of StemJEL in three independent knee joint OA animal models, including a surgically-induced rat knee joint OA model, the Prg4 knockout OA mouse model, and in an age-induced knee joint OA model. We evaluated the knee joint function of all three models using rotarod performance test, which allow us to quantitatively measure the knee joint function based on the running time of animals on the rotarod. In histology analysis, we determined the severity of OA in each model based on the OARSI scoring system. And we quantified the percentage of p21/p53-positive chondrocytes in knee joint frontal section as a measurement of chondrocytes senescence. The StemJEL treatment preserved the knee joint function, ameliorated knee joint OA and decreased the number of senescent chondrocytes in all three independent knee joint OA animal models in vivo. Based on these outcomes, we believe that the StemJEL is a very promising treatment for knee joint OA and could possibly be the first DMOAD.
INTRODUCTION -We present the first case of spontaneous Neisseria Elongata septic arthritis (SA) of a large joint in a patient without known risk factors. A 50-year-old male presented to an outside emergency department (ED) due to four-day history of increased right knee pain, swelling, and difficulty ambulating. The patient described tremendous pain, swelling, and difficulty in ambulation. The patient denies any recent trauma or past injury to the affected knee. The patient denies any past medical history, relevant surgical history, medications, recent dental work, or substance use. The patient denied fever, chills, or recent infection at this time. Arthrocentesis and blood work were completed in the ED revealing C-Reactive Protein (CRP) of 25 mg/l and white blood cell count of 48,000 cells per µl. The patient was indicated for arthroscopic knee washout and recovered well with an uneventful post operative course. METHODS -Case Report. RESULTS -Case Report. DISCUSSION N. Elongata is a gram negative, rod-shaped, intracellular bacterium that is commonly isolated from the human nasopharynx. Due to its intracellular nature, gram stain is often negative. It is commonly described as a non-pathogenic organism, but cases of endocarditis, osteomyelitis, and sternoclavicular septic arthritis have been described. Fortunately, this bacterium is susceptible to various antibiotic regimens, although b-lactamase producing N. Elongata species have recently been isolated. This case demonstrates the importance of a high clinical suspicion for SA. The lack of risk factors, lack of positive gram stain, and lack of antecedent event all convolute the clinical picture. Fortunately, cell count was elevated sufficiently to warrant operative washout, but had the patient presented earlier in his four-day course, this may not have been the case. In uncertain clinical presentations of knee pain and effusion, clinical suspicion for SA must remain high. Patients undergoing posterior spinal fusion (PSF) for neuromuscular scoliosis (NMS) are at high risk of massive blood loss due to their small blood volume, subclinical coagulation abnormality, medications, and possible vascular smooth muscle dysfunction. While iv tranexamic acid (TXA) has proven to be effective, concerns are present regarding its systemic thrombogenecity. This study aims to determine the role of TXA when used as topical soaked sponges (tTXA) on peri-operative blood loss and changes in hemoglobin following PSF for NMS.

Methods:
A single center retrospective review of NMS patients who underwent PSF was conducted. The initial set of patients where no tTXA (control) was used were compared to consecutive NMS patients in whom tTXA was used. In the tTXA group, sponges soaked in 1g TXA in 100ml NS were packed in the wound instead of dry sponges. Estimated blood loss (EBL) was calculated intraoperatively using a standard way. Pre-operative, intra-operative and immediate post-operative variables were collected and compared between the 2 groups.

Results:
Thirty-four patients were included (mean age-13.5yrs., mean BMI-21, 17 females). Seventeen patients were included in each group. Pre-op demographic and radiographic variables were similar between the 2 groups (figure). EBL, EBL per level, EBVL, operative time and number of levels fused were similar in both groups. tTXA group received less intra-operative pRBC transfusion as compared to the control group (150 ± 214 vs. 363 ± 186ml, p=0.004). Post-operative hemoglobin and hematocrit at day 1 and day 2 were higher in tTXA group compared to the control group (p< 0.05); however, no difference was noted in post-op blood transfusion and drain out put for 3 days in both the groups. tTXA group had lesser hospital (5.1 vs 8.9 days) and ICU length of stay (2 vs. 4.2 days) and fewer immediate postoperative complications (23.5 vs. 52.9%) compared to the control group but not statistically significant (p> 0.05).

Conclusion:
Administration of tTXA-soaked sponges is an effective and safe method to reduce intraoperative blood transfusion requirements in the correction of spinal deformity in patients with neuromuscular scoliosis. Heterotopic ossification (HO) can occur in an acquired form due to severe trauma, or in the rare autosomal-dominant genetic disease Fibrodysplasia Ossificans Progressiva (FOP). Both forms of HO proceed via endochondral ossification, resulting in ectopic bone formation in soft tissues, including muscles and tendons; hence the molecular mechanisms that drive these processes are postulated to be similar. FOP-causing mutations in ACVR1 impart neofunction to the receptor by rendering it responsive to Activin A; inhibition of Activin A with a neutralizing antibody abrogates HO in FOP mice and human FOP patients. However, we have shown previously that blocking Activin A did not alter either the incidence or severity of trauma HO 1 , suggesting that Activin A induced signaling does not drive HO in trauma patients who have WT ACVR1. On the other hand, previous data has also shown that inhibiting BMP signaling using an ALK3-Fc ligand trap or small molecule inhibitors can reduce ectopic bone formation in mouse models of trauma and burn-induced HO 2 . This led us to hypothesize that inhibiting BMP signaling mediated by ACVR1 should prevent the formation of trauma-induced HO (tHO). We used a burn/tenotomy mouse model to determine the efficacy of ACVR1 inhibition on trauma-induced HO using an ACVR1 neutralizing antibody. Treating prophylactically with ACVR1 antibody in this model significantly reduces tHO burden and its progression. Unlike in FOP patients, tHO patients sometimes undergo resection surgery to remove the heterotopic bone, hence we also tested whether blocking ACVR1 post-resection prevents its recurrence. In our HO surgical resection study, the model was induced and mice were allowed to develop heterotopic bone lesions for 6 weeks, after which the mice underwent lesion resection and ACVR1 antibody treatment was initiated. We observed significant inhibition of tHO recurrence and its progression post-resection surgery in ACVR1 antibody treated mice. These data suggest that ACVR1 signaling plays a major role in the development of tHO and blocking it offers an effective therapeutic option. Aging and obesity are the leading causes of osteoarthritis (OA). Although the mechanisms are not fully understood, impaired cellular metabolism is one of the central features of OA chondrocytes. Emerging evidence shows that aging and obesity can impair metabolic enzyme activity and function through accumulation of post-translational modifications (PTMs). SIRT5 is a NAD+ dependent demalonylase that catalyzes the removal of malonyl residues from metabolic proteins to increase their activity. We previously discovered an age associated decline of Sirt5 in cartilage, and Sirt5 deficiency caused a global increase of protein posttranslational malonylation (MaK). The goal of this study is to investigate the role of Sirt5 in chondrocyte cellular metabolism and OA development during aging and obesity. We hypothesized that Sirt5 deficiency upregulates malonylation of metabolic enzymes leading to chondrocyte dysfunction, and consequently cartilage degeneration. METHODS: Human cartilage tissue from young (29, 36, 45 years old) and old (61, 66, 74 years old) donors were collected and immunohistochemically stained for SIRT5 (Lifespan Biosciences) and MaK (CST, #14942s). The staining intensity for both SIRT5 and MaK was semi-quantified, and the ratio of positively stained chondrocytes was calculated. Primary chondrocytes were isolated from 7 days old WT and Sirt5 -/mice and cultured until confluency. P1 primary chondrocytes (60,000 cells per well) were subject to ''Glycolytic Rate Assay'' and ''Mitochondrial Stress Assay'' using a Seahorse XFe24 Analyzer (Agilent). An untargeted proteomics analysis was used to quantify the protein abundance in both WT and Sirt5 -/chondrocytes. Sirt5 -/and WT mice were fed either a low-fat diet (LFD) (10% kcal fat; D12450Ji) or high-fat diet (HFD) (60% kcal fat; D12492i) beginning at 6 weeks of age until 26 weeks of age. Body fat and glucose tolerance testing were also performed. Knee joints from WT and Sirt5 -/mice were then processed for OA histopathology using standard methods and blinded semi-quantitative OARSI and Mankin scoring. RESULTS: We firstly confirmed the aging associated decline of SIRT5 in human cartilage tissue. It was found that the ratio of SIRT5 strongly stained chondrocytes decreased nearly 70% in old donors comparing with young donors, while MaK displayed an upregulation in the old donors (Figure 1). Both basal and compensatory glycolytic rate were 20% lower in Sirt5 -/chondrocytes compared to WT ( Figure 2A&B). At basal level, oxygen consumption rate (OCR) as an indicator for mitochondrial function is significantly lower in Sirt5 -/chondrocytes (p<0.01), the majority of this difference is due to a lower level of ATP-linked respiration ( Figure 2C&D). Comparing with WT, Sirt5 -/mice gained less weight in response to HFD while they developed increased glucose intolerance (Fig 3A&B). Knee joint histopathology showed that comparing with WT with HFD, Sirt5 -/mice with HFD developed more severe OA characterized by larger cartilage lesions, more well-developed osteophytes, and significantly higher OARSI (p<0.05) and Mankin (p<0.01) scores. The global proteomics analysis revealed a total of 2,843 quantifiable proteins, among which 204 were significantly altered. Several types of collagen that are enriched in cartilage tissue (including the predominant type Col2a1) are significantly decreased, while mediators in the inflammatory pathway (e.g., PTGS1, CSF-1, HMGB1) are increased in Sirt5 -/chondrocytes ( Figure 4). DISCUSSIONS: In conclusion, our study has identified that Sirt5 and protein post-translational malonylation are playing an important role in chondrocyte metabolic dysfunction and OA development during aging and obesity. This metabolic perturbation is associated with lower extracellular collagen synthesis and activation of inflammatory pathways. On-going efforts in lab include: 1. Conditional knockout of Sirt5 specifically in cartilage tissue and examine OA development in response to HFD; 2. Identify downstream metabolic targets of MaK.
Materials and Methods: The HyA hydrogels were synthesized using previously reported methods. [2] [3] Briefly, Nacryloxysuccinimide was reacted with an HyA derivative carrying hydrazide groups (HyA-ADH) to generate acrylate groups on the HyA (AcHyA). hFAPs were harvested from deltoid muscle biopsies from patients undergoing arthroscopic rotator cuff repair. Cells were encapsulated by mixing the cells into the macromer precursor solution containing cell binding peptide. Gelation was initiated by addition of a bis-cysteine terminated peptide-cleavable crosslinker of varying degradation rates (i.e., different Michaelis-Menten kinetics). BAT phenotype was assessed by immunostaining for UCP-1 and SMA after 1, 3 and 7 days. For in vivo experiments, ten 9-month-old NSG mice underwent unilateral right suprascapular nerve denervation and combined supraspinatus and infraspinatus tendon transection, described previously. [6] The mice were randomized to treatment with 10μL saline (PBS) or 10μL HyA-hydrogel and sacrificed at 6 weeks, at which point bilateral supraspinatus muscles were harvested and analyzed using immunohistochemistry. Results, Conclusions, and Discussions: Conjugation of different integrin engaging peptides impacted spreading and protein expression of FAPs after 7 days in culture as indicated by immunostaining (Fig 1A). Quantitative analysis of immunostaining demonstrated increased expression of UCP1 in the HyA-bsp-RGD(15), -C16, and -P3 peptide hydrogels compared to HyA-T1 (Fig. 1B). Secretion of promyogenic cytokine IL-10 from the media of hFAPs encapsulated within HyA-bsp-RGD(15) hydrogels was significantly increased after 7 days (Fig 1C). In vivo, HyA-bsp-RGD(15) hydrogel implantation in a rotator cuff injury model resulted in a significant decrease in relative muscle weight loss compared to the PBS control ( Fig 1D). Trichrome staining showed fibrosis was also significantly decreased in the HyA-bsp-RGD(15) hydrogel group compared to PBS (Fig 1E). Furthermore, fatty infiltration in the HyA-bsp-RGD(15) group was reduced to the level of the control group. To measure vascularization, laminin and CD31 staining revealed a significant increase of both markers in the HyA-bsp-RGD(15) group compared to PBS treatment. Together, our results suggest that hydrogeldriven FAP-BAT differentiation may contribute to the positive regenerative outcomes after treatment of muscular injury with our hydrogel, highlighting the potential for this material to serve as an effective therapeutic for rotator cuff diseases.
The impact of aging on osteocyte lacunar canalicular turnover depends on intracortical strain environment Ghazal Vahidi 1 & Chelsea Heveran 1 : 1 Montana State University, Bozeman, MT The expansive osteocyte lacunar-canalicular system (LCS) has an active role in mineral homeostasis 1 . Osteocytes can form and remove bone along their LCS in a process called LCS turnover 2,3 . Because the LCS surface is immense 4 , genetic mouse models that impede LCS turnover decrease bone fracture toughness 5 , and these genetic models produce an aging-like phenotype to LCS geometry, a current hypothesis is that LCS turnover has an important role in maintaining bone quality but that the frequency or extent of this process diminishes with aging. Recent work demonstrates that LCS turnover impacts bone quality immediately around individual osteocytes 6 . However, the impact of LCS turnover on whole bone tissue is not known because the scale and frequency of this process have not been determined. Furthermore, osteocytes are mechanosensitive cells, and their abundance depends on tissue strain 7 . However, it is not known if LCS turnover differs with the tissue strain environment. We hypothesize that the prevalence and persistence of LCS bone mineralization decrease with aging and that LCS bone mineralization depends on tissue strain and osteocyte location. We utilized skeletally-mature young adult (5 mo) and early old age (22 mo) female C57BL/6Nia mice. Each mouse received two injections of fluorochrome labels, at two specific dates. These dates included 16d, 8d, 4d, or 2d before the euthanasia. The injections were administered in a manner where each mouse received one injection of alizarin and one injection of calcein, but the specific timing and sequence of the injections varied for each group. Proximal and distal femurs were embedded, sectioned in transverse (for cortical midshaft) or sagittal (for metaphysis) directions, and polished. Confocal laser scanning microscopy, along with accompanying surface images, was used to visualize bone-mineralizing (labeled) and non-bone-mineralizing (not labeled) osteocyte lacunae. The percentage of bonemineralizing osteocytes was measured for cortical bone within A/P/M/L regions of interest (ROI) at the midshaft ( Figure 1A-B) and for cancellous bone ( Figure 1C) at the metaphysis, using custom Matlab analysis. We assessed the impact of intracortical strain on bone-mineralizing osteocytes (just for 2d labels) by dividing each cortical ROI into three distance sections: first 30%, middle 30-70%, and last 70-100% of cortical thickness ( Figure 1G). Mixed model ANOVA was used to test if the percentage bone-mineralizing osteocytes depends on ROI, label date, or aging. All animal procedures were approved by the university IACUC. We found that aging reduced the percentage of bone-mineralizing osteocytes in cortical bone (-50%, p<0.001, Figure  1E) as well as cancellous bone (-50%, p<0.001, Figure 1F). The dynamics of LCS turnover appeared to depend less on age as 2d labels were very abundant (cortical bone: >80% for 5 mo, >50% for 22 mo) and label presence decreased with time in both ages (i.e., 16d vs 2d: -58%, p<0.001 for cortical and -63%, p<0.001 for cancellous bone). The decrease in prevalence of labels administered at later dates implies that these labels were resorbed. We also found that aging decreased (~-55%, p < 0.001) the highly-abundant (>70%) presence of double-labeled lacunae in both cortical and cancellous bone ( Figure 1D). The effects of both cortical strain environment (age-ROI interaction p<0.001) and intracortical strain (age-ROI-intracortical distance interaction p<0.001) on dynamics of LCS turnover depended on age (interaction p<0.001). In 22 mo mice, labels diminished (16d label prevalence vs 2d label prevalence) much slower in the medial and lateral cortexes (neutral axis, smaller strain) compared to the posterior and anterior cortexes (higher, compressive strain). Moreover, osteocytes located at the lowest intracortical section (closest to endocortical, first 30% of cortical bone) had most consistently the lowest participation in bone mineralization (except for lateral ROI). In anterior and posterior ROIs, osteocyte located at the high intracortical strain section (largest distance from endocortical, 70-100% cortical thickness) had most consistently the highest participation in bone mineralization (for 2d labels). However, in 5 mo bones, there were no significant differences in LCS turnover among ROIs or at different intracortical distances ( Figure 1H). This study presents novel evidence that osteocyte participation in mineralizing their surroundings is highly abundant in both cortical and cancellous bones of young mice but decreases with aging. Aging decreases the prevalence of LCS bone turnover but not the LCS turnover dynamics. Labels are less persistent with time in both young and old mice, indicating potential LCS bone resorption. The large decline in cortical bone LCS turnover in aging suggests significant implications for bone quality. These data add to other evidence for the same strain, sex, and ages of mice that LCS turnover increases the compliance of bone local to osteocytes 6 . Together, these datasets demonstrate that LCS turnover likely influences the quality of a substantial quantity of bone tissue and that a decrease in LCS turnover with aging has the potential to impact whole bone fracture resistance. We also demonstrate that the impacts of aging on LCS turnover depends on both cortical strain environment and intracortical strain. LCS turnover rate may be slower in low strain regions of old bones, which could indicate the need for higher loads to activate osteocyte LCS turnover. All in all, LCS turnover has the potential to have an essential role in the decline of bone tissue fracture resistance in aging and could represent an important, overlooked therapeutic target. Step 3. From a point 1.5 cm away a rectangular cortical bone 5 mm wide and 1.5 cm long was formed, Step 4, Step 5. The defect was treated using the prepared scaffolds. Goat Step 1. The goat was laid in a right lateral recumbency position, Step 2. The femoral shaft was exposed using a retractor and a longitudinal incision was made on the left posterior thigh, Step 3. Cortical bone defects, Step 4, Step 5. Scaffolds were implanted into the bone defect area. Figure 2. BFP-5 increases osteogenic differentiation markers in cells. RT-PCR analysis and relative mRNA expression level for RUNX2, ALP, Osteopontin, Collagen type I, DLX-5, and Osterix. The above data represent the mean ± SD from triplicate measurements (# p < 0.05 ODM vs negative control; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 peptide vs ODM). A novel periosteal cell type, identified by single cell RNA sequencing, senses mechanical loading and in response to it differentiates into osteoblasts Xuehua Li, PhD, Intawat Nookaew, PhD, Jinhu Xiong, PhD

University of Arkansas for Medical Sciences, Little Rock, AR, United States
Mechanical loading stimulates periosteal bone formation and osteocytes are considered to be the major cells sensing and transducing mechanical signals in bone. However, a complete understanding of the identity of mechanosensitive cells in periosteum is lacking. To determine which cell types are responding to mechanical stimulation in periosteum, we isolated periosteal cells from loaded tibia at different time points and performed 10X-based single cell RNA sequencing (scRNA-seq).
The left tibiae of 7-month-old female C57BL/6J mice were loaded with 9N at 1 bout per day for 1, 2, and 5 days. Periosteal cells were then isolated 12 hours after loading for 12-hour time point and 24 hours after the last set of loading for the 2and 5-day time points. scRNA-seq identified 8 clusters: Fibro-1, Fibro-2, Osteo-X, Osteoblasts, Chondrocytes, Articular Chondrocytes, Tenocytes, and a new cluster we refer to as Fibro-L. Comparing clusters between the control and 12-hour groups, we found that the number of Fibro-L is greatly increased within 12 hours after loading. The dramatic change of the cell number in this cluster within 12 hours cannot be explained by cell proliferation but the change in gene expression profile induced by mechanical loading. Differential gene expression analysis of this cluster showed increased expression of Ptgs2, Lif, and Il-6 which are classic early response genes to mechanical loading. To confirm the increased expression of these genes in periosteum, we localized their mRNA using RNAscope based in-situ hybridization. Tibiae collected 4 hours after loading were used for RNAscope. Consistent with scRNA-seq analysis, we found increased Ptgs2, Lif, and Il-6 expression in osteocytes as well as cells at the periosteal surface, suggesting that periosteal cells are also capable of sensing mechanical stimulation. Differential gene expression analysis of this cluster at later time points showed that proliferation related genes such as Ki67 and Top2a were significantly elevated. In addition, RNAscope showed a dramatic increase of Top2a expression in the tibial periosteum 5 days after loading, demonstrating increased cell proliferation in periosteal cells. More importantly, cells in the Fibro-L cluster became Sp7 and Spp1 positive at day 5 consistent with their differentiation towards osteoblasts. Taken together, our results provide evidence that a subset of periosteal cells may directly sense mechanical loads and differentiate into osteoblasts. Introduction: Up to 5% of bone fractures exhibit delayed healing or non-union (1). Long bone critical size defects (CSD) model several aspects of nonunion, but most current therapies show suboptimal efficacy in such models (2). Mes-1022, a novel hydroxyapatite-targeted prostaglandin E2 receptor 4 agonist prodrug, has been shown to promote bone healing when applied locally with bone graft material (3), but it is unclear whether systemic administration enhances healing. The aim of this study was to evaluate the efficacy of systemically-administered MES-1022 on the healing of a femoral segmental CSD in the absence of bone grafting and on the mass and microstructure of the intact contralateral femur in a rat model.
Methods: Ten-week-old female Sprague-Dawley rats (7/group) were randomized to receive 12 weekly subcutaneous injections of MES-1022 at 5 mg/kg (high dose), MES-1022 at 1.7 mg/kg (low dose), or Vehicle (PBS + 25 nM EDTA). Rats underwent a 5 mm osteotomy of the left femoral midshaft, stabilized by a unilateral external fixator. Bone markers (P1NP, TRAcP-5b) and open field activity were measured at baseline and throughout the study. Rats were sacrificed on day 84 and the osteotomized and unoperated femora were imaged via micro-CT at 8 µm voxel size. Volumes of interest (VOI) for the unoperated femur were 5% of bone length at mid-diaphysis and 10% at the distal metaphysis. The osteotomized femur VOI was 7 mm in length. A threshold of 819 mg HA/cm 3 was determined by Otsu in the unoperated femur. For osteotomy region analyses, a range of 35% -70% of this density was used to distinguish newly mineralized tissue (i.e., new bone and calcified cartilage), referred to as non-quiescent bone (NQ), and a range of 70-100% of the density identified existing mineralized bone (quiescent bone; Q). Defect site healing parameters for Q, NQ, and total (T) included tissue volume (TV, mm 3 ), mineralized callus volume (BV, mm 3 ), mineralized callus volume fraction (BV/TV, mm 3 /mm 3 ), callus tissue mineral density (TMD, mg HA/cm 3 ), and callus tissue mineral content (TMC, mg). Standard cortical and trabecular microCT parameters were calculated for the unoperated femur (n = 5). All MicroCT analyses were performed using Xamflow software. Three-point bending (day 84) was used to assess bone strength of the unoperated femora. Data were analyzed using an ANOVA followed by post-hoc Tukey testing, with significance at p ≤ 0.05.   1 Among the many risk factors for non-unions, age is most significant. 2 Current non-union management typically involves surgery, often with autograft and fixation, 3 are costly with varied success rates. 4 As such, there is a need to develop non-surgical options. To this end, we have pioneered the development of a nanoparticle (NP) drug delivery system functionalized with a targeting peptide to promote non-union healing. NPs composed of poly(styrene-alt-maleic anhydride-b-poly(styrene) (PSMA-b-PS). NPs are then functionalized with a peptide (TBP) that specifically binds to tartrate resistant acid phosphatase (TRAP), a protein deposited by osteoclasts during fracture healing and non-union. 5 The NPs have been designed to deliver a GSK-3b inhibitor (AR28) to improve fracture healing by activating the Wnt/b-catenin pathway. 6 Drug loaded nanoparticles (TBP-NPAR28) enhanced fracture healing. These results motivate future studies of the effect of TBP-NPAR28 in impaired fracture healing models using aged mice. Methods-The PSMA-b-PS diblock polymers were synthesized and self-assembled as previously described. 5 Briefly, excess styrene was added along with maleic anhydride ( The reaction was purged with nitrogen for 45 minutes before being placed in a 60 °C oil bath for 72 hours. The reaction mixture was then dissolved in acetone before precipitation in petroleum ether after which they were dried under vacuum and taken for further characterization. The TBP was conjugated to the surface of the TBP-NPs through maleic anhydride ring opening, as previously described. 7 These NPs were evaluated in a middiaphyseal femur fracture Balb/c mice models. Results-Characterization of the polymer revealed the molecular weight (MW) of 1 st block (PSMA) as 21 kDa and MW of 2 nd block (PS) as 24 kDa with a PDI of 1.0. The synthesized TBP-NPAR28 have a hydrodynamic diameter of 67 ± 7 nm and a zeta potential of -24 ± 1 mV. TBP-NPAR28 were injected three days post-fracture into mice. Enhanced bone healing was observed 4 weeks post-injection through microcomputed tomography (µCT) in the TBP-NPAR28. Bone volume was 1.8-and 1.5-fold higher in the TBP-NPAR28 group compared to untreated control and free drug. Immunofluorescence of the fracture site after 10 days revealed an increased number of M2 macrophages in the TBP-NPAR28 group compared to the control group, which was confirmed by flow cytometry. Further investigation by bulk RNAseq revealed that the TBP-NPAR28 enhanced fracture healing by down-regulating pro-inflammatory pathways, including TNF, NFkB, and IL17 and enriched genes that were involved in angiogeneisis and arteriogenesis, which are essential for fracture healing. Discussion-In sum, data suggest that TBP-NPAR28 modulates and rebalances M2/M1 ratio resulting from upregulation in Wnt/B-catenin signaling, which is associated with enhanced fracture healing in both bone formation volume and mechanical properties after TBP-NPAR28 treatment. With these results, we believe this TBP-NPAR28 can be used to restart the healing process in aged mice with impaired fracture healing conditions that lead to nonunions, including age. Premature, or preterm, birth occurs in about 10% of all live births and leads to various short-and long-term health consequences. Since about 80% of skeletal mineral accrual occurs during the final trimester of gestation, preterm birth disrupts a critical period of skeletal development and leaves preterm neonates at increased risk for osteopenia and metabolic bone disease of prematurity (MBDP). Preterm birth has also been associated with reduced peak bone mass and increased risk for osteoporosis later in life. While many studies have measured bone mineral content or density in preterm neonates, mechanistic insight into how preterm birth affects the mechanical competence of bone requires a large animal model that is compatible with human skeletal development. The pig model of preterm birth has already been used to examine multiple developing tissues and may provide a resource to study how prematurity affects postnatal skeletal development. Thus, the purpose of this study was to evaluate the pig as a model for postnatal skeletal development after premature birth.

Results
Preterm pigs (Landrace x Yorkshire x Duroc; all raised in Denmark) were born on day 106 of ~117 of gestation by cesarean section and were reared artificially. Term control pigs were born vaginally on day 117 ± 2 of gestation and reared naturally by their mother sow on a farm. Groups of term and preterm pigs, respectively, were sacrificed on postnatal days 1 (n=6 and 7), 5 (n=7 and 17), and 19 (n=7 and 13). Pigs were weighed, blood and urine were collected (only on days 5 and 19), and hindlimbs were dissected. Plasma was used to determine levels of alkaline phosphatase, calcium, phosphate, and creatinine. Calcium, phosphate, and creatinine were also measured in urine to calculate the rates of calcium excretion and tubular reabsorption of phosphate. Serum was used to measure parathyroid hormone (PTH), vitamin D (25-OHD), osteocalcin, C-terminal crosslinks of type I collagen (CTX-I), and procollagen type I N-terminal propeptide (PINP). Microcomputed tomography was used to assess femoral mid-diaphysis cortical bone geometry and bone mineral density (BMD), distal metaphysis integral BMD and trabecular bone BMD. Femurs were tested in three-point bending to determine wholebone stiffness and maximum load to failure. Data were collapsed across sex since initial statistical tests indicated that sex was not significant. Statistical analyses included multivariate two-way analyses of variance (ANOVAs), allometry to determine the relationship between bone cross-sectional properties and length, and multivariate one-way ANOVAs in pigs of similar post-conceptual age.
Preterm pigs were smaller and gained less body mass over time than term pigs (p<0.001). Femur length was reduced in preterm compared to term pigs (p<0.001). Calcium and phosphate were reduced in preterm pigs (p=0.013 and p<0.001, respectively). Alkaline phosphatase levels decreased between day 5 and 19 in term pigs but remained constant in preterm pigs (p=0.005), leading to elevated levels at day 19 in preterm compared to term pigs. Calcium excretion was elevated in preterm pigs (p<0.001). Tubular reabsorption of phosphate was depressed in preterm compared to term pigs on day 5. PTH remained low in preterm pigs while there was a large increase in term pigs between days 5 and 19 (p=0.029). 25-OHD increased from day 5 to 19 in preterm pigs while levels remained stable in term pigs over time (p=0.001). Preterm pigs had higher osteocalcin levels (p=0.004), lower PINP (p=0.008), and higher CTX-I levels than term pigs. Cortical bone area, medullary area, and total area were each reduced in preterm compared to term pigs at each time point (all p<0.001) as was cortical thickness (p=0.022). By day 19 in preterm pigs compared to term pigs, metaphyseal integral BMD was reduced (p=0.021). Whole-bone stiffness(p=0.009) and maximum load to failure (p<0.001) were reduced in preterm pigs compared to term pigs at all 3 time points. Preterm pig femurs were slender for their length. When pigs of a similar post-conceptual age were compared many of the skeletal phenotypes persisted.
A major barrier to understanding the skeletal consequences of preterm birth is the lack of a translational animal model. We report here that the pig model recapitulated many commonly observed features of preterm birth, including altered mineral metabolism, reduced cortical bone area, and increased bone resorption. Clinical features of MBDP were also recapitulated, including reduced phosphate and increased alkaline phosphatase. We also show here, for the first time, that additional properties of bone structure and turnover, as well as whole-bone mechanical properties were affected by preterm birth in pigs. While additional long-term studies are needed, the pig model of preterm birth has utility for studying skeletal consequences and interventions after preterm birth. Atrophic nonunion occurs when an osteochondral callus fails to form after fracture. Proliferation of periosteal cells is a key feature of fracture healing. We have previously shown that proliferation of periosteal cells during the first 2 weeks of fracture healing is essential for normal healing 1 . This was determined using mice expressing a thymidine kinase (TK+) 'suicide gene' driven by the 3.6Col1a1 promoter, which is active in osteoblast lineage periosteal cells. In this mouse model, proliferating TK+ cells die when dosed with ganciclovir (GCV), without affecting non-dividing cells. Here, we sought to investigate the role of proliferating 3.6Col1a1 expressing cells in early stages of fracture healing and determine their critical proliferation window. This was done using the 3.6Col1a1-TK mouse model to study how fracture healing is affected when GCV is administered for different durations following a full femur fracture in 12-week-old TK+ mice and their wild type (WT) littermates. This study was IACUC approved. First, mice were dosed with GCV daily for 3, 7, and 14 days starting on the day of fracture and healing was evaluated at 21 days post fracture (DPF). Radiographs and histology at 21DPF showed that GCV dosing in TK+ mice for 3 days led to a slightly smaller, yet fully bridged mineralized callus. In contrast, GCV dosing for 7 and 14 days led to a significant impairment in callus formation and failure to bridge by 21DPF, compared to the control group ( Fig. 1, right). Mice in the TK+ group dosed with GCV for 7 days developed calluses composed mostly of woven bone, yet still had 20% less woven bone, 10% more cartilage, and 10% more fibrous tissue than the control group (Fig. 2C). The greatest effect on healing was observed in the TK+ group dosed with GCV for 14 days, where calluses had 70% less woven bone, 15% more cartilage, and 45% more fibrous tissue than controls (Fig. 2C). We then focused on earlier time points to further understand the role of proliferating osteoblast lineage cells in the first 10 days after fracture. Histology at 5DPF (Fig.  1, left) showed that GCV dosing for 3 and 5 days resulted in TK+ mice with slightly smaller fracture calluses that had ~15% less woven bone compared to control mice ( Fig. 2A). µCT analysis at 10DPF showed a trend where TK+ mice developed smaller calluses with less mineralization as GCV dosing duration increased. Histology at 10DPF (Fig. 1, middle) showed TK+ mice dosed with GCV formed calluses composed largely of cartilage (30%) and fibrous (40%) tissues, with only 10%-15% being woven bone (Fig. 2B). In contrast, control mice formed larger calluses composed mostly of woven bone (>40%) with less cartilage (25%) and fibrous (15%) tissues. We conclude that proliferation of osteoblast lineage cells between 3 and 10 days post fracture is required for normal fracture healing in mice.     3 However, strategies to optimize fracture healing remain limited by an inability to quantify motion in the clinical setting. To overcome this, we developed an in vivo method of quantifying distal femur fracture site motion via weightbearing CT. METHODS: Seven fresh frozen cadaveric femurs underwent distal femoral osteotomies and surgical fixation. Common surgical fixation strategies were applied including locked plates with short bridge spans (specimens 2 and 7), medium bridge spans (specimens 4 and 5), and long bridge spans (specimens 1 and 3). Specimen 6 was treated with retrograde nailing. Stainless steel beads were implanted in the cadaveric femur segments to allow for bead-based validation of bonebased tracking. Matching the conditions of our ongoing in vivo study, the cadaveric specimens underwent unloaded and loaded CT. Loading through the hip was replicated through a custom load platform that was attached to the head of the femur via a pseudoacetabulum. Relative fracture site motion was determined using image analysis software (Mimics) for four paired points (anteromedial-AM, anterolateral-AL, posteromedial-PM, and posterolateral-PL). The technique included manual registration of the injury CT data (devoid of implant artifact) to the cone beam CT data. RESULTS: Motion profiles differed by fixation strategy (Table 1). While retrograde nailing resulted in < 1mm of motion at all four locations, plate fixation resulted in much greater motion (all plate strategies resulted in ≥ 3mm of motion at one or more locations). Pooling all plate fixation strategies, motion was twice as great medially as it was laterally (3.3 vs 1.6mm, p = 0.018). This varied markedly by fixation strategy, with medium and long bridge spans being particularly associated with asymmetric motion (Figure 1). DISCUSSION: Beyond allowing validation of bonebased tracking for in vivo assessment of fracture site motion, the results provide insight into how fixation strategies relate to fracture site motion. Fracture healing has been more consistent following retrograde nailing than with plate fixation. The variability in magnitude and asymmetry (medial > lateral) of fracture site motion we found for plate fixation are possible causes of the inconsistent healing reported clinically with this strategy. Contrary to anecdotal reports that locked plate fixation is "too stiff", we found universally greater motion with plate fixation than retrograde nailing, and longer plate bridge spans accentuated this magnitude and asymmetry of fracture site motion. Lateral plate fixation provides limited resistance to bending moments associated with distal femur fractures, resulting in significant shear motion as well as delayed and asymmetric fracture healing. 3,4 Translational research has suggested short bridge, titanium plate fixation of distal femur fractures promotes radiographic callus formation. 3 Our results demonstrate that such short bridge plate fixation yields a motion profile most similar to retrograde nailing. CLINICAL RELEVANCE: In vivo quantification of fracture site motion will inform observational & clinical trial research into whether biomechanical variables (fixation strategies and rehabilitation protocols) can be modified on a patient-specific basis to optimize fracture healing outcomes. We are actively enrolling human subjects for in vivo bone-based tracking of distal femur fracture site motion via weightbearing CT  DISCUSSION: To advance targeted drug delivery for the joint, tools are needed to quantify the pharmacokinetics and biodistribution of drug carriers in the body in both preclinical and clinical studies. MPI technology is being used pre-clinically in other fields such as cell-based therapies and oncology and has the potential to be clinically translated, as it is scaled for imaging brain perfusion in humans . Here we demonstrate a formulat ion method for successful incorporation of MPI tracers into PLGA NPs engineered for cartilage localization. The MPI signal was detectable and quantifiable within the cartilage explants, suggesting this emerging imaging technology could be a tool to track NPs localization within the joint. Likewise, MPI was able detect and quantify signal in the joint on a longer timeline versus fluorescent tracking. This data suggests that the mode of tracer -SPIONs or fluorophore association with particles, and method of imaging modality can affect interpretation of nanoparticle retention in the joint. This project is the first to demonstrate the MPI utilization and feasibility to evaluate NP uptake and localization into articular cartilage. This represents the first steps towards leveraging MPI to understand how NPs delivery system and potential therapeutics are biodistributed, localized and retained in the joint as OA progresses. SIGNIFICANCE/CLINICAL RELEVANCE: The application of the MPI allows us to improve our tracking and assessment of NPs drug delivery systems. With this information, we can better understand how therapeutic efficacy in terms of retention, localization and clearance are affected during osteoarthritis progression. Manganese Dioxide Nanozymes as a Chondroprotective Therapy for Osteoarthritis Jessica Aldrich 1 *, Arjun Panicker 1 , Brenda Sanchez 1 , Jillian Jacek 1 , Robert Ovalle Jr. 1 , Kyle D. Allen 1,2 , Blanka Sharma 1 1. J. Crayton Pruitt Department of Biomedical Engineering, University of Florida, Gainesville, FL 2. Department of Orthopedics and Sports Medicine, University of Florida, Gainesville, FL Introduction: Nanozymes, antioxidant-enzyme mimicking nano-scale biomaterials, are an emerging therapeutic strategy for medical applications including neurological conditions, cancer, and more recently orthopedic diseases. Antioxidantmimicking therapies are a promising approach for modifying disease states caused by oxidative stress, such as osteoarthritis (OA). Current treatment strategies for OA are limited by poor bioavailability and retention in the joint. Our group has reported on manganese dioxide nanoparticles (MnO2 NPs) as an ROS scavenging therapeutic for OA that may overcome limitations of conventional therapeutics. The MnO2 NPs were engineered to localize within cartilage, specifically due to their small size (<15 nm) and cationic charge (+30 mV). They have been shown to integrate with the cartilage extracellular matrix, colocalize with chondrocytes, and provide chondroprotection by mitigating the loss of ECM components (glycosaminoglycan) in cartilage explants and decreasing the production of nitric oxide. These chondroprotective effects may be due to the antioxidant mimicking functions of MnO2, specifically by catalyzing the breakdown of H2O2 into water and oxygen. The objective of this work was to i) characterize the enzymatic functions of MnO2 NPs, ii) evaluate the use of MnO2 NPs as a therapy in human in vitro models, and iii) advance the application of MnO2 in an in vivo OA model. Materials and Methods: MnO2 NPs were synthesized following previously reported methods. Briefly, potassium permanganate and poly allylamine hydrochloride were mixed in a redox reaction then stabilized with PEG (MW 3400) and fluorescently tagged. Enzymatic functions were evaluated in an acellular model using commercially available kits designed to measure catalase and superoxide dismutase function. Human OA cartilage and chondrocytes were isolated from patients undergoing total knee arthroplasty to evaluate the biological effects of MnO2 on human cells. Cell viability was measured using an MTS cell metabolism assay with MnO2 concentrations ranging from 5-20 ug/ml. MnO2 uptake into chondrocytes was visualized with confocal microscopy and quantified via flow cytometry analysis. Passive and active methods of uptake were measured by modulating the culture conditions, exposing cells to different temperature environments (4C vs. 37C) to quantify changes in MnO2 uptake profiles. MnO2 fate in relation to uptake mechanism was evaluated by analyzing nanoparticle properties following exposure to pH environments relative to intracellular compartments. At a larger scale, chondroprotective effects in cartilage explants were measured via GAG loss and NO release from cartilage explants exposed to MnO2 (20 ug/ml) and IL-1B (10 ng/ml) simultaneously. Lastly, the therapeutic effects of MnO2 NPs were evaluated in a non-invasive injury model of OA. Following injury, weekly intra-articular injections of MnO2 NPs (15 mg/ml) were given for four weeks. After four weeks, the joints were evaluated histologically using a modified-Mankin grading scale. Results and Discussion: In an acellular model, MnO2 mimics physiologically relevant levels of catalase (2.23 U/ml) that are found in a healthy adult (Fig 1a). Indicating that MnO2 nanozymes may rescue low levels of catalase found in OA patients. The addition of MnO2 (5-20 ug/ml) had no effect on cell viability and there were no significant differences between donors (n = 9) or sex. MnO2 were measurable in chondrocytes, with at least 60% of cells expressing MnO2, at both 1 and 3 hours regardless of culture conditions. Uptake was dose dependent, with increased uptake at 20ug/ml compared to 5 ug/ml. Uptake was quantifiable on confocal microscopy within 3 hours of MnO2 (20 ug/ml) exposure to chondrocytes. These results indicate that MnO2 enter chondrocytes through both active and passive transport mechanisms and may be subject to various intracellular fates. Cartilage explants treated with MnO2 (20 ug/ml) had decreased NO production (Fig 1c), consistent with results from bovine cartilage explants. This indicates that the therapeutic effects of MnO2 are translatable across species. Weekly injections of MnO2 (15 mg/ml) following a non-invasive knee injury in a rodent model of OA showed improved cartilage protection compared to a saline control (Fig 1d). Although there was not a statistically significant difference in modified Mankin scores between groups at this early timepoint, the trends suggest that chondroprotective effects may have been initiated and may be more measurable at later stages of the disease. This work showcases the antioxidant functions of MnO2 and their potential for use as a chondroprotective agent to slow the development of OA.   3 Orthopedics, University of Michigan.

IMAGES AND TABLES
Introduction: Musculoskeletal injuries are a major cause of severe disability. Each year, 5-15% of the 7 million fractures result in delayed union or nonunion. Hypertrophic nonunions are the clinically predominant form caused by high interfragmentary movement and tissue strain. Although acute inflammation is crucial for establishing the healing cascade, increased interfragmentary movement can give rise to aberrant immune cell phenotypes that can cause fibrosis and compromised functional healing. However, the specific cellular pathways and mechanisms leading to disruptions in the healing cascades are poorly understood. Therefore, elucidating the strain-mediated effects on the inflammatory response in a fracture milieu can help develop immunomodulatory therapies to prevent nonunion. We have developed a murine delayed healing model that mimics the etiology of hypertrophic nonunion. Here, we elucidate the dysregulated immune response leading to fibrosis in delayed-healing fractures and identify predictive biomarkers for early prognosis with the goal of improving clinical care for recalcitrant fractures. Materials and Methods: First, we created a diaphyseal femoral fracture in mice and then differentially stabilized the femurs using intramedullary nails made from a nickel-titanium (Ni-Ti ) with low (2.7 N/mm) and high stiffness (16.5 N/mm) that allowed higher (>15%) and lower interfragmentary strain (<5%), respectively. Blood and tissue samples were harvested longitudinally at various time points, and immune factors were analyzed using flow cytometry, multiplex ELISA, histology, immunohistochemistry, and single-cell RNA-seq analyses to characterize inflammatory and healing responses. Femurs were harvested and analyzed using microCT and a 3-point bending test to assess functional healing. Results and Discussion: The fractures with high intramedullary strain resulted in a significantly larger fracture callus (Fig.1A), with significantly higher bone volume (BV), tissue volume (TV), and mineral content (Fig.1B,  p<0.001) than the low-strain group. A 3-point bending test of the femurs at week 3 showed significantly lower elastic modulus (p=0.010), ultimate stress (p=0.011), and stiffness (p=0.054) in the high-strain group, indicating poor healing (Fig.1C). Histological analysis of the tissues revealed significantly more fibrosis of callus of high-strain fractures (Fig.1D, p=0.010). Then we analyzed various immune factors in the callus and circulating blood which showed significant differences between groups. Linear regression analysis revealed significant correlations between 32 circulating immune factors with functional outcomes assessed. For instance, MIP-1α on day 7 showed a strong positive correlation with elastic modulus (R=0.970, p=0.0013) and stiffness (R=0.917, p=0.009). Likewise, the CD8+ and CD25+ T cell populations on day 14 correlated with ultimate stress negatively (R=-0.624, p=0.017) and positively (R=0.58, p=0.03), respectively. On the other hand, monocytes in the callus on day 21 significantly correlated with ultimate stress. Our multivariate analysis also showed a clear separation between the low and high-strain groups and a strong linear correlation between the immune factors and functional healing (R=0.72, p=0.003). Further, our partial least squares regression revealed that the amount of CD206+ macrophages in the callus on Day 21 and CD25+ Tregs in the peripheral blood on day 14 are the best predictors of functional healing. Single-cell transcriptomic analysis of the callus also identified cell populations at varying ratios between strain groups (Fig.1E). For example, the percentage macrophage population at day 3 in the high-strain (18.3%) was 3.9% lower than in the low-strain group (22.2%). While a 5.7% increase in neutrophils is seen in the high-strain group (47.2%) compared to the low-strain group (41.5%) (Fig.1E). Together, our fracture model showed that higher strains led to fibrosis of the callus and impaired functional healing, while lower-strain support ossification and healthy bone healing. The analysis of the peripheral blood showed significant associations between early immune factors and functional bone healing. Hence, these factors can serve as early biomarkers for functional healing. Together, our work provides mechanistic insights into strain-driven immune dysregulation in recalcitrant fractures that can help develop immunomodulatory materials and strategies to improve clinical care. Figure 1: Age-related changes in body mass and non-fasting glucose before and after switching male mice from standard chow to 1 of 2 synthetic diets (medial ± IQR). (A) While the mice consumed a standard rodent chow, there were no pronounced differences in body mass between the 2 strains. When the mice started to consume a synthetic diet at 16 weeks of age, high fat increased body mass, but this depended on the strain. (B) From 11 weeks to 16 weeks of age, a few mice had circulating levels above 250 mg/dl, but this was not consistent as the matured. Corresponding to the consumption of the high fat diet, all NONcNZ010 mice had glucose levels well above 250 mg/dl, while glucose levels increased for some NON/ShiLtJ mice. Objectives Fibrous dysplasia (FD) is a rare, disabling disorder in which activating GNAS variants alter differentiation of multipotent skeletal stem cells, leading to expansile lesions characterized by fibro-osseous and fibro-cartilaginous tissue with prominent osteoclastogenesis. Though skeletal disease burden varies, patients often develop pain, fractures, and deformities that can lead to functional impairment. Surgery is the mainstay of treatment, and there are currently no approved medications for FD. The aim of this study is to identify the cell types within FD lesions that harbor GNAS variants to inform understanding of FD pathogenesis and the development of targeted therapeutics.
Methods Bone specimens (n=10) were obtained as surgical waste under IRB-approved protocols. All samples were processed for histology; of these, 5 were further divided and frozen for future analyses. GNAS p.R201C and p.R201H allele frequencies were determined using digital droplet PCR (ddPCR) following DNA extraction from frozen samples further dissected into fibrous tissue and cartilage. Histological preparations were stained using the BaseScope Duplex assay to visualize wild-type and p.R201C or p.R201H GNAS mRNA and proportions of cells expressing GNAS variants were quantified by three readers. Using the BaseScope assay, probes for GNAS p.R201C/H were paired with markers for chondrocytes (collagen, type 10A1; COL10A1) and osteoclasts (cathepsin K; CTSK) to assess whether they express GNAS variants. To determine potential effects of GNAS activation on osteoclastogenesis, primary cultures from wild-type mouse femurs were induced with RANKL and MCSF to initiate osteoclast differentiation and subsequently treated with vehicle, 0.1 mM, or 0.5 mM dibutyryl-cAMP (db-cAMP).
Results Of this cohort, 6/10 patients were female, 7/10 carried the p.R201H variant, and 3/10 carried the p.R201C variant. Eight samples were derived from long bones (femur, tibia) and 2 from flat bones (rib, scapula Conclusions We have demonstrated that ddPCR and BaseScope are reliable methods for genotyping and detecting GNAS variants in FD. Spatial resolution of GNAS using RNA-based techniques demonstrates that cartilage in FD lesions carries GNAS variants, while osteoclasts do not. This suggests that affected skeletal stem cells retain some level of multipotency, and that the increased osteoclastogenesis observed in FD is a result of aberrant cell signaling rather than a direct consequence of GNAS activation. Defining the variant cell types in FD addresses key knowledge gaps in the pathogenesis of this disease and will inform the development of targeted therapies. Articular cartilage in the tibiofemoral joint contains unique tissue microstructures that serve specific functions. Such functions include the reduction of friction and load distribution. A proficient understanding of these microstructures can lead to significant clinical advances in diagnosing orthopedic diseases such as osteoarthritis and improving cartilage repairs. The surface of tibiofemoral condyles can be roughly separated into load-bearing and meniscus-covered areas. Due to the difference in mechanical loading between the two regions, we hypothesize that their microstructures differ. To test this hypothesis, we used cartilage punches harvested from the tibial condyle of porcine knee joints as an example tissue and a custom nonlinear optical microscope for performing a label-free imaging study. The custom nonlinear optical microscope could acquire two-photon excitation auto-fluorescence (TPAF) and second harmonic generation (SHG) images simultaneously. Through the TPAF channel, elastin fibers are visible along with chondrocytes. The SHG channel was utilized for observing the vast collagen network and its evident orientation throughout the tibial condyle. Images were analyzed by ImageJ (OrientationJ) to reveal alignment angles of the collagen network and elastin fibers. Figure 1 shows typical images and punches acquired from various locations. A dense, uniform collagen network with few elastin fibers characterizes the load-bearing region. On the other hand, the meniscus-covered areas have a distinctive collagen orientation with a higher concentration of co-localized elastin fibers. The articular cartilage microstructure has a similar "C" shape to the meniscus, with most of the microstructure aiming toward the loadbearing region (see Figure 1H). The biological differences are likely derived from their different biomechanical environments in the tibiofemoral joint.

Introduction
The use of tourniquets for extremity hemorrhage in tactical combat casualty care has significantly reduced mortality. However, prolonged tourniquet application can lead to increased risk of complications, such as threatened limb loss, due to ischemia-reperfusion (I/R) injury. Delayed return of blood flow to ischemic muscle leads to mitochondrial dysfunction, increased production of reactive oxygen species (ROS), and cell apoptosis. I/R injury also contributes to the impairment of satellite cells to regenerate damaged muscle and leads to long-term deficits in muscle strength. The purpose of this study is to determine whether inhibition of the pro-oxidant PKCβII-p66shc pathway can mitigate oxidative stress in satellite cells to enhance muscle fiber repair following simulated I/R injury. We hypothesize that Ruboxistaurin (RBX), a selective PKCβII inhibitor, will decrease mitochondrial ROS production using an in vitro model of chemically induced oxidative stress.

Methods
Phorbol 12-myristate 13-acetate (PMA) was used to stimulate ROS production in C2C12 myoblasts (i.e., activated satellite cells) with and without RBX pre-treatment. Cells were incubated with 2,2'7dichlorofluorescin (DCFH) for PMA-induced ROS detection with a fluorescent microplate reader. Cell viability was assessed using a colorimetric assay and microplate reader to determine toxicity of treatments. Live-cell imaging was performed with Hoechst nuclear stain, DCFH, and Mitosox for visualization of nuclei and ROS regions of interest using fluorescent microscopy. Fluorescence intensity was quantified into corrected total cell fluorescence (CTCF) units using ImageJ software. The CTCF units were calculated as follows: integrated density -(area of selected cell x mean fluorescence background). All data were analyzed using the student-test and ANOVA Tukey method.

Results
PMA stimulation resulted in a four-fold increase in ROS levels compared to untreated controls (p<0.05). Cell viability was similar in all test groups. RBX significantly decreased intracellular ROS levels in DCFHstained cells by 45%, from 9578±737 to 5282±1306 CTCF units (p<0.05). Mitosox-stained cells treated with RBX demonstrated a 61% reduction in mitochondrial ROS levels (59±4 vs. 151±31 CTCF units) compared to PMA controls (p<0.05). CTCF units for nuclei regions of interest were similar for all PMA and RBX-treated samples.

Discussion
The data suggests that RBX attenuates PMA-stimulated ROS production in myoblasts. Reductions in ROS were not likely due to cell death since neither PMA nor RBX were toxic to cells. PKCβII inhibition may represent a novel therapeutic approach for the prevention of tourniquet I/R injury in settings of prolonged tourniquet application. In future studies, we will compare Western blots of cytosolic and membrane fractions to confirm the mechanism of RBX to inhibit PKCβII redistribution and subsequent p66shc activity in C2C12 myoblasts. We will also test the effects of RBX on muscle fiber regeneration after 24h hypoxic injury.