A Multifunctional Scaffold for Bone Infection Treatment by Delivery of microRNA Therapeutics Combined With Antimicrobial Nanoparticles

Treating bone infections and ensuring bone repair is one of the greatest global challenges of modern orthopedics, made complex by antimicrobial resistance (AMR) risks due to long‐term antibiotic treatment and debilitating large bone defects following infected tissue removal. An ideal multi‐faceted solution would will eradicate bacterial infection without long‐term antibiotic use, simultaneously stimulating osteogenesis and angiogenesis. Here, a multifunctional collagen‐based scaffold that addresses these needs by leveraging the potential of antibiotic‐free antimicrobial nanoparticles (copper‐doped bioactive glass, CuBG) to combat infection without contributing to AMR in conjunction with microRNA‐based gene therapy (utilizing an inhibitor of microRNA‐138) to stimulate both osteogenesis and angiogenesis, is developed. CuBG scaffolds reduce the attachment of gram‐positive bacteria by over 80%, showcasing antimicrobial functionality. The antagomiR‐138 nanoparticles induce osteogenesis of human mesenchymal stem cells in vitro and heal a large load‐bearing defect in a rat femur when delivered on the scaffold. Combining both promising technologies results in a multifunctional antagomiR‐138‐activated CuBG scaffold inducing hMSC‐mediated osteogenesis and stimulating vasculogenesis in an in vivo chick chorioallantoic membrane model. Overall, this multifunctional scaffold catalyzes killing mechanisms in bacteria while inducing bone repair through osteogenic and angiogenic coupling, making this platform a promising multi‐functional strategy for treating and repairing complex bone infections.


Introduction
3][4][5][6] The infection, primarily caused by Staphylococcus aureus (S. aureus) or other less common bacteria such as Escherichia coli (E.coli) or Pseudomonas aeruginosa (P.aeruginosa), [4,7,8] results in challenging consequences impairing two vital processes for bone regeneration, vasculogenesis and osteogenesis. [9,10]Traditional treatment of osteomyelitis, which lasts between 4 and 6 weeks [11] and involves systemic administration of broad-spectrum antibiotics such as beta-lactams, fluoroquinolones, or sulfonamides, [7,12] often fails due to the presence of thick biofilm, which hampers antibiotic penetration, leading to prolonged, difficult-to-treat pathogenic infections. [5,13,14]Substantial surgical debridement is typically needed to remove infected tissue, leaving behind a large, debilitating bone defect which requires further management to support, repair, and restore bone functionality.Hence, the optimal treatment for bone infection should incorporate a two-pronged approach, targeting bacterial infection while simultaneously promoting the regeneration of the often very large bone defect that remains post-debridement.
To add further challenges, the treatment of osteomyelitis faces additional complications due to the escalating pandemic of antimicrobial resistance, an issue that has been overshadowed in recent years by the emergence of COVID-19. [15]The increasing prevalence of highly dangerous Methicillin-resistant strains [3,16,17] causing up to 50% of infections, together with diminished antibiotic effectiveness due to over-prescription, highlights an urgent need for developing alternative, non-antibiotic-dependent therapies. [5][31] The potential of these alternative therapies is promising; yet, their effective delivery to the infection site faces substantial challenges due to the presence of natural physiological barriers, such as the host immune response and the presence of biofilm within the infection site, underlining the need for local delivery strategies.
To address these challenges, our research has turned to a potential solution: localized delivery using biomaterial scaffoldbased systems, which offers not only numerous benefits such as the use of lower antimicrobial concentrations, faster action, and enhanced bacterial sensitivity for increased therapeutic efficacy but also provides much-needed structural support to encourage cellular infiltration for bone regeneration.We have previously developed a series of bioactive 3D collagen-hydroxyapatite (CHA) scaffolds and demonstrated their potential as an off-the-shelf platform for bone regeneration in pre-clinical studies, [32][33][34][35][36][37][38][39] equine clinical cases, [40] and clinical studies involving over 30 human patients.These scaffolds, with their inherent versatility, open the possibilities for the integration of ions, [41] growth factors, [42] and other bioactive molecules [43] to induce specific tissue responses and regulate cell recruitment, proliferation, and differentiation.Therefore, in this study, we posit that these scaffolds represent a potentially ideal system for the delivery of antibiotic-free antimicrobial copper-doped bioactive glass (CuBG) nanoparticles, thereby addressing the challenge of eradicating bone infection. [4]urther, the utilization of copper-doped bioactive glass nanoparticles in these scaffolds not only imparts antimicrobial properties but also offers advantages in promoting osteogenesis and angiogenesis.Specifically, it enhances mineralization by stimulating the osteogenesis of mesenchymal stem cells [31,44] and promoting collagen maturation through lysyl oxidase crosslinking; [45] while, also inducing the generation of vascular endothelial growth factor (VEGF), a potent angiogenic factor. [46,47]hile the CuBG particles may have some potential for the treatment of large, traumatic bone defects, a further stimulus may be required.The most commonly employed clinical growth factor delivery approach for bone defects involves the delivery of recombinant bone morphogenetic protein-2 (BMP-2). [48]However, this is beset with limitations, including uncontrolled release, short half-life, risk of ectopic bone formation, and a singlepathway focus in the multifaceted bone healing process. [49]Given these drawbacks, microRNAs (miRs), small, non-coding RNA therapies, may provide a compelling alternative due to their ability to modulate the expression of multiple target proteins within host cells. [50]MicroRNAs modulate protein expression at the post-transcriptional level and have the capability to silence up to 100 mRNA targets at the same time, which enables control over entire gene cohorts. [43,51]These synthetic therapeutic miR-NAs can either mimic or inhibit gene function, leading to the overexpression or downregulation of specific targets and proteins.Importantly, this approach does not require supraphysiological dosages; thus, reducing the occurrence of aberrant effects.Multiple miRs have been identified to play a role in bone regeneration, including miR-133a, [52,53] miR-16, [54] or miR-210. [55]mong these, microRNA-138a (miR-138a) has emerged as an intriguing candidate, particularly for bone-related applications. [56]t negatively influences angiogenic and osteogenic processes, acting as a negative regulator of the hypoxia-inducible factor [57] and -catenin. [58]Therefore, by delivering an inhibitor of miR-138 (antagomiR-138), we can potentially stimulate both angiogenesis and osteogenesis, essential processes in bone regeneration, at the same time.
One challenge and a major reason for the lack of clinical translation of microRNA therapeutics is the inherent instability of miRNAs due to potential enzymatic degradation and short half-lives, coupled with the insufficiency of a single dosage.We propose a solution in the form of a gene-activated scaffold to deliver the therapeutic and provide sustained, controlled release; [33,43,59,60] while, simultaneously acting as a template for tissue repair.By complexing the miRNA therapeutic with effective non-viral nanoparticle (NP) vectors for intracellular delivery, such as hydroxyapatite NPs with proven potential for bone repair, [52,[60][61][62] such scaffolds protect the nucleic acid from degradation and provide a robust strategy to avoid rapid in vivo clearance and off-target effects.We posit that the combination of antagomiR-138, which targets both angiogenic and osteogenic pathways, with a scaffold-based delivery system could serve as a potent platform for inducing bone regeneration in bacteriainfected defects where natural healing processes are significantly compromised. [43]he ultimate goal of this project was thus to design a multifunctional platform that exhibits angiogenic, osteogenic, and antimicrobial features via combined delivery of antagomiR-138 and antimicrobial antibiotic-free copper-doped bioactive glass particles.To achieve this, the study had the following specific aims: 1) Initially, we focused on validating the osteogenic and angiogenic potential of antagomiR-138, utilizing a previously optimized collagen-hydroxyapatite scaffold (CHA).The antagomiR-138 was complexed with hydroxyapatite NPs as the non-viral vector and incorporated into the CHA scaffold.To evaluate both the transfection efficiency and osteogenic efficacy, we performed in vitro studies using human mesenchymal stem cells (hMSCs).Following demonstration of in vitro potential, an in vivo loadbearing femoral defect model in rats was used to assess both the regenerative capacity and presence of blood vessels induced by an antagomiR-138-activated scaffold.2) We engineered an antimicrobial scaffold by incorporating antibiotic-free copperdoped bioactive glass nanoparticles into a collagen scaffold.We first sought to optimize the concentration of copper that strikes a balance between effective bacterial killing and minimal cytotoxicity to hMSCs.Following the determination of this optimal concentration, the biological performance of the CuBG scaffold was assessed in vitro using hMSCs, and two representative bacterial strains, gram-positive S. aureus and gram-negative E. coli.
3) Following the demonstration of the osteogenic and angiogenic potential of antagomiR-138 nanoparticles in the scaffold (Aim 1) and the antimicrobial potential of CuBG in the scaffold (Aim 2), Aim 3 combined both successful technologies to design a multifunctional scaffold which eradicates bacteria and stimulates bone growth simultaneously.The ability of the novel antagomiR-138activated CuBG scaffold to exhibit antimicrobial, osteogenic, and angiogenic properties was demonstrated using bacterial strains, hMSCs, and an in vivo chick chorioallantoic membrane model.

AntagomiR-138-Activated Collagen-Nanohydroxyapatite (CHA) Scaffolds Enhance Mesenchymal Stem Cell-Mediated Osteogenesis In Vitro and Foster Highly Vascularized Bone Regeneration in a Load-Bearing Defect Model in Rats In Vivo
As part of the multi-stage development of the final product, our first step focused on the creation and validation of osteogenic and angiogenic properties of an antagomiR-138-activated collagennanohydroxyapatite (CHA) scaffold.The genetic cargo was first complexed with hydroxyapatite nanoparticles (i.e., the non-viral vector) and incorporated into a CHA scaffold for controlled, local delivery.The antagomiR-138-activated CHA scaffold effectively transfected human mesenchymal stem cells without affect-ing their metabolic activity (Figure 1A).The success of transfection was evident from the significant 2.7-fold downregulation of miR-138 expression when compared to the un-transfected group, which consisted of a miR-free CHA scaffold (Figure 1B).Importantly, this reduced expression of miR-138 had a notable impact on osteogenic signaling, leading to a 1.3-fold increase in calcium production compared to the miR-free CHA scaffold and the scaffold containing the vector only (nHA-CHA) (Figure 1C).As calcium production serves as a surrogate marker for osteogenic differentiation, our findings demonstrate that the delivery of antagomiR-138 on the scaffold effectively enhanced osteogenic signaling pathways within human MSCs.
Having demonstrated the robust capability of antagomiR-138activated CHA scaffold to effectively transfect hMSCs and augment osteogenic differentiation and mineralization in vitro, we then proceeded to evaluate its regenerative potential in a loadbearing femoral defect model in rats by assessing the bone repair over 8 weeks (Figure 2A-C).The histological evaluation showed that both the antagomiR-138-activated CHA scaffolds and miRfree CHA scaffolds were infiltrated by host cells throughout their depth (H&E staining, Figure S1, Supporting Information), demonstrating the excellent integration of the CHA scaffolds within the host tissue with no effects on osteoclastogenesis (no TRAP + cells, Figure S2, Supporting Information).
The μCT analysis showed progressive bone repair in the miRfree CHA scaffold (Figure 2D; Figure S3, Supporting information).However, the antagomiR-138-activated CHA induced the formation of new bone to a greater extent than the miRfree CHA scaffold.Of note, antagomiR-138-activated CHA scaffolds showed significantly higher bone production as early as 2 weeks post-implantation.Specifically, the antagomiR-138activated CHA scaffold reached the values of 10 mm 3 ± 4 and 30 mm 3 ± 24 at weeks 2 and 8, respectively; this represented a 1.7-fold and 1.6-fold increase over the miR-free CHA scaffold (6 mm 3 ± 2 at 2 weeks and 18 mm 3 ± 7 at week 8) (Figure 2D).Further, the antagomiR138-activated CHA scaffold showed a greater amount of glycosaminoglycan deposition (i.e., GAG + cells), suggesting that newly formed bone is partially produced by endochondral ossification (Figure S4, Supporting Information).Importantly, the antagomiR-138-activated CHA scaffold also stimulated angiogenesis within the defect.Specifically, it fostered the formation of blood vessels resulting in 12 ± 4 of blood vessels per mm 2 , guaranteeing a highly vascularized defect site (Figure 2E).This represented a 1.5-fold increase compared to the miR-free CHA scaffold.
Both scaffolds, the antagomiR-138-activated CHA scaffold and miR-free scaffold, provided an environment which supported angiogenesis and osteogenesis, which was confirmed through the presence of positively stained cells for angiogenic (CD31, Figure S5, Supporting Information) and osteogenic (Runx2, Figure S6, Supporting Information; Osterix, Figure S7, Supporting Information) markers.Overall, the antagomiR-138-activated CHA scaffold showed superior osteogenic capability leading to enhanced osteogenesis in vitro and in vivo.Importantly, the antagomiR-138-activated CHA scaffolds repaired bone defects while providing an optimal environment which encouraged angiogenesis within the defect, confirming our hypothesis by demonstrating the dual role of antagomiR-138 in simultaneously promoting the most relevant processes in bone regeneration.The results show the significant promise held by the antagomiR-138 NPs for the repair of extensive traumatic defects through osteogenic-angiogenic coupling when combined with a scaffold tailored to support bone repair.

The Incorporation of Copper-Doped Bioactive Glass Particles into a Collagen Scaffold Imparts Antimicrobial Properties Against Gram-Positive and Gram-Negative Bacterial Strains
Having validated the osteogenic potency of antagomiR-138 nanoparticles using our optimized CHA scaffold system within Aim 1, we proceeded to the second aim of the work to develop an antibiotic-free antimicrobial scaffold through the incorporation of copper-doped bioactive glass particles.
To harness the antimicrobial potential of copper while ensuring minimal cytotoxicity to human mesenchymal stem cells (hMSCs), we first evaluated the effects of various concentrations of copper chloride, aiming to identify an optimal balance that would facilitate effective minimal bacterial inhibition and minimal bactericidal concentration (MIC and MBC, respectively) for its subsequent integration into the bioactive glass.By assessing ion concentrations delivered from copper chloride, we found that 24-h exposure to copper chloride decreased the viability of gram-positive S. aureus and gram-negative E. coli.The reduction was observed at 2.5 and 5 mm for S. aureus (Figure 3A) and E. coli (Figure 3B), respectively.HMSCs, when exposed to lower copper chloride concentrations below 2.5 mm, maintained their metabolic activity (Figure 3C).This cell viability was further validated through fluorescence imaging, where cells robustly attached to the well plate (Figure 3D), demonstrating that lower copper chloride concentrations could be effectively used without compromising the health and activity of the cells.
Having demonstrated the antimicrobial potential of copper ions, we incorporated them into a bioactive glass. [28]We found that the CuBG nanoparticles induced bacterial death by inducing mechanisms related to oxidative stress, such as reactive oxy-gen species (ROS) generation and lipid peroxidation.Specifically, we found that the delivery of 1000 ppm of CuBG NPs significantly increased the ROS levels in S. aureus (Figure 3E) and E. coli (Figure 3G).No increase in lipid peroxidation was observed in S. aureus; while, E. coli exposed to 1000 ppm of CuBG showed increased levels of lipid peroxidase compared to the control indicating the disruption of the structure and function of the cell membrane of E. coli.Importantly, these results confirmed that the incorporation of copper ions into the amorphous structure of bioactive glass did not hamper its antimicrobial properties.
We then proceeded to develop the antibiotic-free scaffold for treating bacterial infection by incorporating optimized CuBG particles, containing the maximum achievable concentration of 2% (mol) copper into a collagen scaffold to guarantee a continuous and steady release of antimicrobial ions.We found that the CuBG particles were effectively incorporated into the scaffold structure resulting in CuBG scaffolds with a calcium content of 119 μg ± 15 per scaffold (Figure 4A).The release kinetics showed that CuBG scaffolds exhibited early burst release of calcium ions during the first 24 h (Figure 4C) followed by a slower diffusionmediated release (Figure 4C).Further, the copper content was 23 μg ± 12 per scaffold (Figure 4D).The initial burst release followed by a slower diffusion-mediated release was also observed for copper ions on the CuBG scaffold (Figure 4E,F).The results demonstrated the effective incorporation of copper-doped bioactive glass particles into a collagen scaffold, offering an antimicrobial biomaterial which catalyzes killing mechanisms in bacteria and therefore provides a compelling antibiotic-free alternative.

Multifunctional AntagomiR-138-Activated CuBG Scaffolds Eradicate Bacteria While Enhancing Osteogenesis of hMSCs In Vitro and Significantly Enhancing Blood Vessel Formation In Vivo
Having demonstrated the successful incorporation of antagomiR-138 (Aim 1) and CuBG particles (Aim 2) into a collagen-based scaffold, as well as their separate osteogenic and antimicrobial properties, we proceeded to the ultimate objective of our study: the development of a multifunctional scaffold which, for a first time, combined gene therapy and antibiotic-free antimicrobials in a single platform.This scaffold aimed to achieve dual goals in treating osteomyelitis -eradicating the bacterial infection and providing superior osteogenic and angiogenic properties to facilitate the regeneration of bone tissue.To ensure that the observed results are associated with the successful delivery of antagomiR-138 nanoparticles from CuBG scaffolds, the study used two control groups: miR-free CuBG scaffolds (CuBG) and CuBG scaffolds containing non-viral vector (nHA-CuBG).The in vitro evaluation with gram-positive (Figure 5A) and gram-negative (Figure 5B) strains showed that the antagomiR-138-activated CuBG scaffold effectively eradicated bacterial spread.Specifically, the antagomiR-138-activated CuBG scaffolds exhibited 78% ± 25% less of S. aureus (Figure 5C) and 61% ± 26% less of E. coli (Figure 5D) attached compared to the control group (a collagen scaffold), demonstrating strong antimicrobial potential through contact-kill mechanism.
To assess the osteogenic properties of the antagomiR-138activated CuBG scaffolds, we cultured them with human mesenchymal stem cells.All scaffolds supported cell viability within 28 days of the study (Figure 6A).AntagomiR-138-activated CuBG scaffolds effectively transfected hMSCs with genetic cargo resulting in a 28-fold downregulation of miR-138 compared to untreated cells cultured on CuBG scaffolds (Figure 6B).Im-portantly, this translated into enhanced production of calcium by the hMSCs compared to both control groups (Figure 6C).AntagomiR-138-activated CuBG scaffolds also enhanced the expression of multiple osteogenic (BMPR1A, BMPR1B, MMP2, ITGA1, SP7) and angiogenic (FGF2, FGFR1, EDN1, HIF1A, IL6, PDGFA) markers (Figure 6D).Overall, the results highlight that the combination of antagomiR-138 and CuBG nanoparticles is an effective strategy to enhance the osteogenic features of the scaffold.
Having demonstrated the superior antimicrobial and osteogenic properties of antagomiR-138-activated CuBG scaffolds in vitro, we next aimed to determine their angiogenic potential in a more biologically complex in vivo environment using the ex ovo, shell-less chicken embryo model [63,64] (Figure 7A).The efficacy of antagomiR-138-activated CuBG scaffolds was compared to the CHA scaffolds used within Aim 1 for assessing the osteogenic and angiogenic potential of antagomiR-138.In addition, the CuBG scaffold was incorporated as an extra control to distinguish the angiogenic effects of copper ions themselves from the combinatory effect of genetic cargo and copper ions in the antagomiR-138-activated CuBG scaffold.The antagomiR-13-activated CuBG scaffolds induced superior vessel production in the chick embryo model, demonstrating their angiogenic potential in vivo (Figure 7A).The antagomiR-138activated CuBG scaffold showed increased numbers of branches (45% ± 10%, Figure 7B), junctions (33% ± 17%, Figure 7C) and average branch length (28% ± 8%, Figure 7D) compared to the control scaffold (CHA scaffold).The antagomiR-138-activated CuBG scaffold showed a similar number of triple (Figure 7E) and quadruple (Figure 7F) points compared to the control groups (CHA and CuBG scaffolds).Overall, the antagomiR138-activated CuBG scaffolds significantly improved vasculogenesis in vivo in a chick chorioallantoic membrane model (CAM), demonstrating the tremendous angiogenic potential of our platform.In conclusion, the study showed that the scaffold combining antagomiR-138 and CuBG particles significantly reduced bacterial attachment while potentiating osteogenesis and angiogenesis, making this system very promising for treating and regenerating infected bone tissue.

Discussion
Bone infections present a demanding and complex clinical challenge, and there is a major need for new strategies that effectively eliminate bacterial infections while simultaneously fostering a conducive environment for bone regeneration.In this study, we have successfully developed a multifunctional scaffold which enhances bone regeneration in vivo via antagomiR-138-complexed nanoparticles and employs copper-doped bioactive glass particles as an antibiotic-free antimicrobial solution which reduces attachment of gram-positive S.aureus.The escalating concern of antimicrobial resistance requires innovative strategies to address these continually evolving threats.By integrating the strengths of diverse therapeutic modalities, our scaffold-based platform provided an integrated solution capable of confronting the multifaced nature of bone infections.
First, we assessed the osteogenic potential of antagomiR-138 using a scaffold with proven bone repair potential.The antagomiR-138 was complexed with hydroxyapatite nanoparticles as a non-viral vector and incorporated into a collagenhydroxyapatite scaffold (CHA) with the aim of producing an antagomiR-138-activated CHA scaffold, which could deliver genetic cargo locally to resident host cells providing transient expression of therapeutic genes.The antagomiR-138-activated CHA scaffold effectively transfected human MSCs in vitro, enhancing mineralization.Notably, the in vivo assessment showed that the antagomiR-138-activated scaffolds promoted bone healing in load-bearing femoral defects in rats.To address the bacterial spread, we then developed an antimicrobial scaffold by delivering copper ions from bioactive glass particles incorporated within the scaffold structure.The evaluation with S. aureus and E. coli confirmed the antimicrobial features showing that CuBG scaffolds catalyze killing mechanisms in bacteria inducing oxidative stress.Following these successful individual developments, we then merged these two therapeutically potent platforms into a unified and multifunctional scaffold system by incorporating antagomiR-138 NPs into CuBG scaffolds.Importantly, this fusion did not hamper the antimicrobial properties of the CuBG scaffold.Further, the antagomiR-138-activated CuBG scaffolds showed superior osteogenic properties stimulating hMSCmediated osteogenesis.Moreover, the in vivo evaluation with the chick embryo model demonstrated the ability of these scaffolds to induce angiogenesis.Collectively, our work shows that combining microRNA-based gene therapeutics with non-antibiotic biomaterial-based approaches is an effective strategy to simultaneously eradicate infection and repair traumatic large bone defects.
Among various miRNAs involved in bone healing, miR-138 has been identified as a negative regulator of osteogenesis [66] and angiogenesis by targeting -catenin [58] or hypoxia-inducible factors. [57]Thus, within Aim 1 of this study, we proposed to use a miR-138 inhibitor (antagomiR-138) to positively influence osteogenesis and bone regeneration.The antagomiR-138 was effectively delivered to the human MSCs in vitro using the non-viral nanohydroxyapatite vector in combination with the CHA scaffold.This system provides several significant advantages.First, the CHA scaffold serves as a stable structural support and osteogenic regenerative template, facilitating cell infiltration and spatial arrangement to promote effective bone repair.In addition, the CHA scaffold then functions as a reservoir for the antagomiR-138 NPs, controlling their release to the surrounding environment.This feature allows for more precise modulation of gene expression while mitigating the risk of uncontrolled bolus release.Finally, utilizing proven nonviral vectors such as nanohydroxyapatite [52,53] in the scaffold promotes transient expression of the gene of interest.This is highly desired while applying gene therapy for regenerative medicine purposes as it allows sustained gene expression for a predictable time but eventually stops host cells from prolonged protein production that could lead to ectopic tissue formation.
Our in vitro findings indicated that the antagomiR-138activated CHA scaffold effectively transfected human MSCs and markedly enhanced osteogenic differentiation and mineralization, evidenced by a significant increase in calcium production.This aligns with a number of studies underscoring the multiple pathways through which antagomiR-138 can induce osteogenic differentiation.Suppressing miR-138 expression in human [67] and primary calvaria osteoporotic osteoblasts [68] resulted in enhanced expression of osteogenic genes (Runx2, Osterix, ALP, and OC), greater ALP production, and increased mineralization.Similarly, Chen et al. explored the osteogenic potential of antagomiR-138 by reporting its stimulatory effect on the osteogenic differentiation of MC3T3-E1, a mouse osteoblastic cell line. [58]The study found that antagomiR-138 modulated the activity of -catenin, a crucial regulator of bone mass and osteoblast function.These changes in osteogenic gene expression triggered by antagomiR-138 mirror the enhanced osteogenic differentiation observed in our research.
Having demonstrated the osteogenic potential of antagomiR-138 in vitro, we determined the regenerative capacity of the antagomiR-138-activated scaffolds in vivo using a complex loadbearing femoral defect model in rats.The μCT analysis revealed that antagomiR-138-activated CHA scaffolds produced a higher Figure 7. AntagomiR-138-activated CuBG scaffolds showed superior angiogenic properties when implanted into the in vivo chick embryo model.A) To evaluate the effect of the scaffold on vasculogenesis, the adjusted scaffold regions of interest (yellow squares) were selected for further analysis.The white arrow indicates chick embryo, and the green arrow indicates scaffold.B) The quantification of blood vessels was performed using Fiji software. [65]he antagomiR-138-activated CuBG scaffolds enhanced C,D) the number of vessel branches, E) junctions, and F) augmented the average branch length.The antagomiR-138-activated CuBG scaffolds showed a similar number of G) triple and H) quadruple points compared to the control.** and *** correspond to p < 0.01 and p < 0.001, respectively (n ≥ 7).The p values were calculated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.
volume of bone as early as 2 weeks post-implantation.While our analysis primarily leaned on robust parameters such as bone volume and surface, providing a detailed analysis of the trabecular structure of the bone could offer additional insights into the effects of our scaffold system on bone regeneration.Moreover, histological analysis demonstrated an increased presence of glycosaminoglycans (GAG) + cells which suggests that newly formed bone is produced by endochondral ossification, which could be associated with the potential effects of antagomiR-138 on chondro-progenitor cells (Figure S4, Supporting Information).This is supported by previous research which found that miR-138 was more highly expressed in hypertrophic chondrocytes compared with chondro-progenitor cells demonstrating its impact on cartilage cells. [69]Further, the delivery of antagomiR-138 into chondrocytes induced osteogenic differentiation leading to enhanced calcium deposition, indicating its effect on chondromediated osteogenesis. [70]mportantly, the histological evaluation revealed a greater presence of blood vessels in the antagomiR-138-activated CHA scaffold group compared to the miR-free CHA group, indicating the involvement of miR-138 in vascularization.However, it is worth mentioning that there is considerable disparity in the literature regarding whether miR-138 exhibits positive or negative effects on angiogenesis.Zhang et al. showed that antagomiR-138 negatively affects angiogenesis and inhibits p-STAT3 protein which could negatively influence migration and tubule formation of endothelial cells. [71]On the contrary, Lui et al. found that miR-138 repressed vessel density in vivo by interacting with hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor A (VEGFA). [72] Similarly, Zhou et al. showed that miR-138 inhibits endothelial progenitor cell proliferation and signaling by targeting mitogenactivated protein kinase, AKT signaling, and HIF-1. [73]Thus, the delivery of antagomiR-138, which is an inhibitor of miR-138, can potentially reverse these effects potentiating angiogenesis within the bone or other tissues.Overall, our results indicate the multifunctionality of the genetic cargo, showing that antagomiR-138 regenerates bone through osteogenic-angiogenic coupling.
Having developed an antagomiR-138-activated CHA scaffold with superior osteogenic potential, we then sought to develop an antibiotic-free antimicrobial scaffold to tackle bacterial infections without contributing to the rising issue of antimicrobial resistance.[20][21] This multi-faceted approach made it challenging for bacteria to develop resistance.Our studies showed that the incorporation of CuBG enhanced levels of ROS and lipid peroxidation in both gram-positive S. aureus and gram-negative E. coli, toxic reactions known to inhibit bacterial activity.This finding underscores the potent antimicrobial action of copper, reinforcing its suitability as a key component in our scaffold design.Our CuBG scaffolds exhibited an initial burst release of copper ions within the first 24 h, followed by a more gradual, sustained release showing that our platform can create an antimicrobial environment.This translated into significantly reduced attachment of S. aureus -a primary strain responsible for bone bacterial infections.This initial inhibition of bacterial attachment is of paramount importance as the early stages of infection carry an increased risk of biofilm formation, typically between 2-48 h post-bacterial introduction to the wound site. [74]he biofilm can significantly reduce the effectiveness of the treatment leading to a chronic condition.The existing literature corroborates our findings, revealing the advantageous properties of copper-containing bioactive glasses to kill bacteria.For instance, Baro et al. reported the efficacy of bioglasses containing 2% copper, demonstrating a reduction in both gram-positive (S. aureus, S. epidermis) and gram-negative (E.coli) bacterial growth by ≈30-40% within a day compared to control of mesoporous bioactive glass groups.This reduction also resulted in the prevention of biofilm formation. [47]verall, our findings effectively underscore the significant promise of CuBG scaffolds in combating bacterial growth, showing them to be a great alternative to antibiotics.
After demonstrating the osteogenic role of an antagomiR-138activated scaffold and the antimicrobial features of a scaffold containing copper-doped bioactive glass (CuBG scaffold), we proceeded to the final and most important aim of this study which focused on developing a multifunctional scaffold combining both platforms.For the first time, gene therapy and antibiotic-free antimicrobials were included in a single platform that targeted two major challenges of osteomyelitis -eradicating bacterial infection and encouraging bone tissue regeneration through osteogenic and angiogenic coupling.Our in vitro results demonstrated the remarkable antimicrobial and osteogenic capability of the antagomiR-138-activated CuBG scaffold.The scaffold effectively eradicated bacterial spread, showing 78% ± 25% and 61% ± 26% reductions in gram-positive S. aureus and gramnegative E. coli attachment compared to the control group (collagen scaffold).Interestingly, the incorporation of nHA or nHA contained the antagomiR-138 granted antimicrobial features of CuBG scaffolds against E.coli.We propose this is a result of two factors.As the nHA particles are deposited onto the surface of the scaffold, they likely introduce increased roughness to which E. coli might be susceptible due to their higher motility. [75]Further, the nHA particles are negatively charged; [60] and therefore, might act as repellents when interacting with E.coli, which net surface charge is more negative than S.aureus.This significant decrease in bacterial adhesion can be attributed to the roughness of the scaffolds and to the release of copper ions from the bioactive glass particles, causing bacterial cell wall destruction, protein denaturation, [76] and DNA changes; [77,78] thus, substantially reducing bacterial growth rates. [79]While our findings demonstrated the effectiveness of our scaffold system against S. aureus and E. coli, other clinically relevant bacterial strains should be explored to confirm the broad effectiveness of our scaffolds.Further, the effectiveness of antagomiR-138 CuBG scaffolds should also be assessed in in vivo bone infection model to confirm its effectiveness.
Importantly, the incorporation of copper ions in the form of copper-doped bioglass into the scaffold design allowed us to exert control over the potential cytotoxic effects of copper ions on hM-SCs while retaining antimicrobial properties.Upon culture with hMSCs, the antagomiR-138-activated CuBG scaffolds exhibited efficient transfection and enhanced calcium production, which can be associated with the combined effect of antagomiR-138 and copper ions from the CuBG.AntagomiR-138 CuBG scaffolds also enhanced multiple osteogenic and angiogenic genes in human MSCs due to the combined effect of gene therapy and copper ions.Both BMPR1A and BMPR1B play crucial roles in osteochondral ossification and bone formation by phosphorylating specific receptor-regulated SMAD proteins. [80,81]SP7 (Osterix) is a master regulator in osteoblast differentiation. [82]The enhanced levels of these genes observed in our study are further supported by literature where BMP receptor signaling [83] was modulated by miR-138; while, enhanced levels of SP7 were induced by copper ion. [84]miR-138 was reported to decrease levels of SP7 and therefore delivering its inhibitor (antagomiR-138) can be responsible for enhancing the levels of this transcription factor. [85]miR-138 has been previously shown to modulate the expression of MMP2. [86,87]This could be particularly relevant in the context of bone regulation.For instance, Tang et al. demonstrated that the delivery of exosomal MMP2 from osteoblasts promotes migration, proliferation, and tube formation of endothelial cells in vitro by activating the VEGF/Erk ½ signaling pathway [88] which underlines the possible involvement of miR-138 in angiogenic-osteogenic coupling.Moreover, observed the enhanced levels of HIF1A, which regulates bone homeostasis and angiogenesis, [89] in hMSCs cultured on antagomiR-138 CuBG scaffolds.Previous work corroborates our results showing that both copper ions [90] and miR-138 target HIF1A activity. [72]verall, the literature supports our findings from our study showing that antagomiR-138 enhances osteogenesis through ERK ½ signaling pathways [67] and -catenin activity; [58] while, copper ions provide a favorable osteoimmunomodulatory environment via the Oncostatin M (OSM) pathway. [44]ubsequent to establishing superior antimicrobial and osteogenic properties of the antagomiR-138-activated CuBG scaffold, we then assessed the angiogenic potential of the scaffold in vivo, showing that it significantly improved vasculogenesis in a chick chorioallantoic membrane model (CAM) by increasing the number of branches, junctions, and average branch length.The underlying angiogenic effect of our scaffolds could be attributed to the dual influence of copper ions and antagomiR-138 nanoparticles.Consistent with our results, previous studies have demonstrated that copper-doped biomaterials can promote vascularization. [46,91]For instance, copperintroduced mesoporous bioactive glasses have been reported to augment endothelial cell migration as well as induce vessel formation in zebrafish embryos. [46]Similarly, copper-doped nanoscale bioactive glass was shown to enhance neo-blood vessel formation in chick embryo and rat subcutaneous models. [91]he angiogenic potential of antagomiR-138 was also supported by previous research; which found that antagomiR-138 was capable of modulating various signaling pathways and proteins, such as p-STAT3 protein, [71] VEGFA, [72] mitogen-activated protein kinase, AKT signaling, and HIF-1. [73]Overall, our study showed that the antagomiR-138-activated CuBG scaffold is a multifunctional biomaterial which catalyzes killing mechanisms in bacteria with no cytotoxicity to mammalian cells offering a compelling antibiotic-free antimicrobial alternative while inducing bone regeneration through osteogenic and angiogenic coupling, which makes this system extremely advantageous for treating and regenerating infected bone tissue.

Conclusion
This study developed a first-of-its-kind scaffold combining gene therapy (antagomiR-138) and antibiotic-free antimicrobial (CuBG) nanoparticles, providing an innovative and promising approach for treating and regenerating infected bone tissue.While traditional methods often focus on treating the infection first, followed by tissue regeneration, this work provides a holistic approach which addresses both challenges concurrently, potentially reducing treatment times and improving future clinical outcomes.This multifunctional scaffold leverages the inherent bactericidal properties of copper ions arising as a viable alternative to traditional antibiotics; while, stimulating bone healing through osteogenic-angiogenic coupling associated with incorporating antagomiR-138 and inducing multiple markers involved in bone healing and remodeling.Further, the study demonstrates, for the first time, the feasibility of combining non-antibiotic biomaterial based-approaches with gene therapeutics, which opens the door to new possibilities in a myriad of indications beyond bone repair.

Experimental Section
Development of AntagomiR-138-Activated Collagen-Nanohydroxyapatite (CHA) Scaffolds: Collagen-nanohydroxyapatite (CHA) scaffolds were developed at a 1:1 weight ratio following the previously established protocol. [53,54]Briefly, the bovine tendon collagen type I (Collagen Solutions, UK) was dissolved in 0.5 m of acetic acid (Sigma-Aldrich) at 5 mg mL −1 , and the in-house produced nHA particles were homogenously added to the slurry.This was followed by a lyophilization cycle in an Advantage Pro Benchtop Freeze Dryer (SP Industries) at a −40 °C final freezing temperature.The CHA scaffolds were sterilized using a dehydrothermal (DHT) treatment and cross-linked chemically using EDAC (1-ethyl-3-[3-dimethyl aminopropyl] arbodiimide) (6 mm) and NHS (Nhydroxysuccinimide) (2.4 mm) (both from Sigma-Aldrich) prior to the in vitro and in vivo experiments. [92]HA scaffolds were activated with antagomiR-138 using a non-viral osteogenic vector system (i.e., nanohydroxyapatite, nHA) previously optimized for bone applications.Nanohydroxyapatite particles were prepared following an established protocol. [52,54,60]Briefly, a mixture of sodium phosphate (12 mm) and Darvan 821A (0.017% v/v; RT Vanderbilt) was stirred with an equal volume of calcium chloride (20 mm), filtered using 0.2 μm sterile filters, and added to miR-138a miRIDIAN antagomiR (Dharmacon), yielding a final concentration of 20 nm. 30 uL of antagomiR-138 and non-viral nHA solution were added to the scaffolds (15 uL per side).
Effect of AntagomiR-138-Activated CHA Scaffolds on Human Mesenchymal Stem Cell-Mediated Osteogenesis In Vitro: For the in vitro evaluation, human mesenchymal stem cells in passages 3 and 4 were used.The bone marrow aspirates were purchased (Lonza, UK) to obtain hMSCs following stern phenotype analysis prior to functional experiments.To demonstrate the osteogenic properties of antagomiR-138 delivered on a CHA scaffold system, 3 × 10 5 of hMSCs were seeded onto the scaffolds, incubated in low-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% of foetal bovine serum and 1% of penicillin/streptomycin (all from Sigma-Aldrich, Ireland), and evaluated in terms of transfection efficiency, metabolic activity, DNA content and mineralisation.For assays assessing the osteogenic potential of hMSCs, regular cell culture media were additionally supplemented with osteogenic supplements consisting of 50 μg mL −1 ascorbic acid-2-phosphate, 10 nm -glycerophosphate, and 100 nm dexamethasone to yield osteogenic media (all from Sigma).
Analysis of the Capacity of AntagomiR-138-Activated CHA Scaffolds to Transfect hMSCs In Vitro: The expression of miR-138 was evaluated on day 3 post-transfection using Real-Time Polymerase Chain Reaction (qRT-PCR).Total RNA was extracted using a combination of QIAzol and a miRNeasy kit (Qiagen) and following the manufacturer's instructions.The levels of miR-138 were measured using the Taqman miR-PCR assay kit (Bio-Sciences), and relative expression was calculated against 18S ribosomal RNA using the 2(−ΔΔCt) method.
Evaluation of the Metabolic Activity of hMCSs Cultured on AntagomiR-138-Activated CHA Scaffolds: The metabolic activity of hMSCs cultured on the scaffolds was assessed at days 14 and 28 post-transfection using the alamarBlue assay (Invitrogen).The scaffolds were rinsed thrice with phosphate-buffered saline (PBS, Sigma-Aldrich) and incubated for 1 h with 10% AlamarBlue reagent.The absorbance was measured at 570 and 600 nm as per the manufacturer's instructions.
Evaluation of Mineralization of hMCSs: To determine if hMSCs cultured on antagomiR-138-activated CHA scaffolds deposit increased calcium, a surrogate for mineralization, the Ca 2+ content was quantified through a calcium o-cresol phthalein complexone method [93,94] using a colorimetric Calcium Liquicolor kit (Stanbio Laboratories), following the manufacturer's instructions and measuring the absorbance at 570 nm at day 28.Calcium deposits were dissolved by placing the scaffolds in 0.5 m HCl for at least 24 h.A background associated with calcium content in non-cultured CHA scaffolds was subtracted from all values.
Assessment of the Regenerative Capacity of AntagomiR-138-Activated CHA Scaffolds in a Load-Bearing Femoral Defect in Rats In Vivo: To evaluate the capacity of antagomiR-138-activated CHA scaffolds to regenerate weightbearing bone defects, the materials were implanted into 5 mm loadbearing femoral defects in 15 weeks old female Sprague Dawley rats (Janvier Labs Inc.).This study and all the protocols involved were approved by the RCSI Animal Welfare Body (AWB), Animal Research Ethics Committee (AREC), and the Health Products Regulatory Agency (HPRA, approval number AE19127/P069).
Surgical Procedure: All animal experiments were performed in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes.The pre-and post-operative analgesic regime consisted of 0.05 mg kg −1 of buprenorphine and 1.5 mg kg −1 of meloxicam.Buprenorphine was administered at least 30 min prior to the commencement of anesthesia.The rats (n = 8 per experimental group: antagomiR-138-activated CHA scaffolds and miR-free-CHA scaffolds) were anesthetized using a mixture of 5% gaseous isofluorane (2-chloro-2-(difluoro methoxy)−1,1,1-trifluoro-ethane) in oxygen and maintained using 2-3% isofluorane in oxygen applied by gas perfusion.The animals were placed on the heating pad before and during the surgery, and the temperature of the rat was monitored through a temperature controller attached to a rectal probe.
Surgical access to the femur was achieved via an anterolateral longitudinal skin incision and separation of the hind limb muscles, the vastus lateralis, and the biceps femoris.A weight-bearing polyether ether ketone (PEEK) internal plate was fixed to the anterolateral femur using metal screws inserted into four holes created in the femur with a surgical drill.A 5 mm defect was created using an oscillating saw under constant irrigation with saline solution.The antagomiR-138-activated CHA scaffolds and miR-free-CHA scaffolds were press fitted into the defect.After ensuring the proper placement and infiltration of the scaffold with fluids, the soft tissue around the defect was accurately readapted using 5-0 absorbable sutures (Johnson & Johnson, Ireland). 1 mL of pre-warmed sterile saline solution was administered subcutaneously to account for fluid loss during the procedure.All animals received postoperative analgesia, which consisted of subcutaneous injections of buprenorphine (0.04 mg kg −1 ) for 72 h and meloxicam (1.5 mg kg −1 ) for 120 h.The animals were scored using a scoring system approved by the RCSI Animal Welfare Body, Animal Research Ethics Committee and the HPRA.
To assess the capacity of antagomiR-138-activated CHA scaffolds to regenerate bone, in vivo micro-computed tomography (μCT) scans were performed on all the rats at 2 and 8 weeks post-surgery using Quantum GX2 micro CT Imaging System (USA) (Section 5.3.2).The 2-week time point was strategically chosen to observe the early stages of bone healing and initial scaffold integration.Conversely, the 8-week time point aimed to evaluate the effectiveness of the scaffold in facilitating progressive bone regeneration and remodeling.The animals were euthanized at 8 weeks using CO 2 , and the left femur, with the intact PEEK plate attached, was harvested for further histological and histomorphometric analysis (Section 5.3.3).

In Vivo Micro-Computed Tomography (μCT) Analysis:
To measure the ability of anrtagomiR-138-activated CHA scaffolds to regenerate loadbearing femoral bone defect, the mineralization of the defect site and the volume of newly formed bone were assessed using in vivo microcomputed tomography (μCT) following the HPRA-approved procedure were incubated with the same concentrations of CuCl 2 under standard culture conditions (37 °C, 5% CO 2 , and 95% relative humidity) for 24 h.The metabolic activity of hMSCs was measured using the alamarBlue assay (Invitrogen, USA).The hMSCs were rinsed trice with phosphate-buffered saline (PBS, Sigma-Aldrich) and incubated for 1 h with 10% alamarBlue reagent.The absorbance was measured at 570 and 600 nm as per the manufacturer's instructions.
Development of Bioactive Glass Particles Containing Cu 2+ Ions and Evaluation of Their Antimicrobial Properties: The copper-doped bioactive glass particles were synthesized following an established sol-gel process and physicochemical characterization was described elsewhere. [28]To unravel the potential mechanisms responsible for the antimicrobial properties of these copper-doped bioactive glass particles, the levels of reactive oxygen species and lipid membrane peroxidation were assessed in gram-positive S. aureus Newman and gram-negative E. coli after incubation with CuBG. 1 mL of S. aureus or E. coli was added at 5 × 10 5 colony forming units (CFU) mL −1 to the Eppendorf in brain heart infusion (BHI) broth with increasing concentrations of CuBG (1-1000 ppm) at 37 °C for 1 h.After the incubation, the eppendorffs were centrifuged at 4 °C at 100 rpm, and the supernatants were collected for further quantification.
[97][98] The supernatants were incubated with 10 μm of DCFH-DA solution at 37 °C for 1 h in the dark.The fluorescence was measured at  ex = 503 nm and  em = 523 nm.
To quantify lipid peroxidation, a thiobarbituric (TBA) acid test was employed [96,99] using Lipid Peroxidation (MDA) Assay Kit (Sigma-Aldrich) and following the manufacturer's instructions.The fluorescence was measured at  ex = 532 nm and  em = 553 nm.
Fabrication and Physiochemical Assessment of CuBG Scaffolds: The copper-doped bioactive glass particles were homogenously added to the collagen slurry, as described in Section 5.1.The CuBG scaffolds were lyophilized in an Advantage Pro Benchtop Freeze Dryer (SP Industries) at −40 °C, sterilized dehydrothermally and chemically cross-linked with EDAC (1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide) (6 mm) and NHS (N-hydroxysuccinimide) (2.4 mm) (both from Sigma-Aldrich) solution prior to the in vitro and in vivo assessment.
The scaffold was assessed in terms of calcium and copper content to confirm the effective incorporation of CuBG particles.The CuBG scaffolds were placed in 0.5 m HCl for at least 24 h.The calcium content in the supernatant was quantified as described in Section 5.2.3 using a colorimetric Calcium Liquicolor kit (Stanbio Laboratories) and measuring the absorbance at 570 nm.Copper content was determined by Copper Assay Kit (Sigma-Aldrich) and following the manufacturer's instructions.The absorbance was measured at 359 nm.
Evaluation of Release Profiles of Osteogenic and Angiogenic Ions from CuBG Scaffolds: Having demonstrated the effective incorporation of CuBG particles into collagen scaffolds, the release profiles of calcium and copper ions were assessed from the scaffold matrix.The CuBG scaffolds were placed in 24-well plates, and 1 mL of molecular water (Sigma-Aldrich) was added.The release study was performed at 37 °C in static conditions.The 200 μL of water was recovered at specific time points, and fresh 200 μL was added per well.The amount of calcium and copper in supernatants was quantified using, respectively, the Calcium Liquicolor kit (Stanbio Laboratories) and Copper Assay Kit (Sigma-Aldrich) following the manufacturer's instructions.
Assessment of Antimicrobial Properties of CuBG Scaffolds: The antimicrobial features of CuBG scaffolds were assessed using the agar diffusion assay method.Moreover, the viability of bacterial populations cultured on the scaffold using the LIVE/DEAD BacLight bacterial viability kit (Invitrogen, USA) was evaluated.
The viability of S. aureus or E. coli cultured on CuBG scaffolds was determined by incubating the material with 5 × 10 5 CFU per mL in brain heart infusion at 37 °C for 24 h.After that time, the scaffolds were fixed with 10% formalin solution for 30 min at 4 °C.The LIVE/DEAD BacLight was added, incubating the scaffolds for 3 min at room temperature.Each step was accompanied by three rinses with phosphate-buffered saline (PBS).A Zeiss LSM710 confocal equipped with a W N-Achroplan 10×/0.3 and Plan-Apochromat 40×/1.4objective were used for image acquisition.The live dye was excited using a 488 nm (detection range 493-577 nm), and dead dye excited using 561 nm (detection range 568-712 nm) lasers.Multiple single Z slice images were captured for each sample and analyzed using a FIJI macro to determine the percentage of live and dead bacterial cell area on the scaffolds.
Development of Multifunctional AntagomiR-138-Activated CuBG Scaffolds: Having demonstrated the osteogenic potential of antagomiR-138 nanoparticles in our CHA scaffold system (Aim 1) and developed a promising antibiotic-free antimicrobial technology through the incorporation of CuBG particles (Aim 2), the authors proceeded to fulfil Aim 3 of the study and develop, for the first time, multifunctional scaffolds which delivered antibiotic-free antimicrobials and gene therapy.
The antagomiR-138 nanoparticles were prepared as described in Section 5.1 and incorporated into a previously optimized antimicrobial CuBG scaffold.
Assessment of Antimicrobial Properties of AntagomiR-138-Activated CuBG Scaffolds Against Gram-Positive and Gram-Negative Bacteria: To confirm that the incorporation of genetic cargo does not interfere with antimicrobial features of the CuBG scaffold, gram-positive S. aureus and gramnegative E. coli were cultured as per Section 5.4.5, stained with the LIVE/DEAD BacLight and visualized with the confocal laser scanning microscope as per Section 5.4.5.
Evaluation of Osteogenic Capacities of AntagomiR-138-Activated CuBG Scaffolds in Vitro Using hMSCs: To show that antagomiR-138-activated CuBG scaffolds effectively transfected hMSCs in vitro and to assess their osteogenic potential, 3 × 10 5 of hMSCs were seeded onto the scaffolds and assessed in terms of expression of miR-138, metabolic activity, and mineralization following the methodologies described in Section 5.2.The expression of osteogenic and angiogenic markers in hMSCs was assessed at day 14 using qRT-PCR.RNA was extracted from hMSCs using RNeasy kits (Qiagen, UK) and transcribed using Quantitect Reverse Transcription Kits (Qiagen, UK).The reactions were prepared using 2.5 ng of cDNA and commercially available primers (Qiagen, UK), and following the manufacturer's protocols (Qiagen, UK).qRT-PCR was performed using a QuantStudio 3 Real-Time PCR System (Roche, UK).The ΔΔCT method was used to determine the fold change in the expression of genes of interest using GAPDH as a housekeeping gene and collagen scaffold as a control group.
Assessment of Angiogenic Properties of AntagomiR-138-Activated CuBG Scaffolds Using In Vivo Chick Embryo Model: Having assessed the antimicrobial and osteogenic properties of antagomiR-138-activated CuBG scaffolds, an established in vivo model was utilized -the ex ovo, shell-less chicken embryo model, to determine the angiogenic potential of the materials.All experimentation carried out on chick embryos was in accordance with the EU Directive 2010/63/EU for animal experiments.Fertilized chicken eggs (Ovagen Group Ltd, Co. Mayo, Ireland) were incubated horizontally at 37 °C until 3 days of development.On day 3, the eggs were cracked into 100 mm Ø Petri dishes (Corning Inc., New York, USA) and placed into a larger 150 mm Ø petri dish (Corning Inc., New York, USA) with 25 mL of sterile PBS to maintain humidity allowing chick embryos to grow.On day 7 of development, the samples: CHA scaffold, CuBG scaffold, and antagomiR-138-activated CuBG scaffold were placed on the membrane and incubated for additional 5 days.To assess the local effect of the scaffolds on the angiogenesis of the chick embryo model, the images were taken (n > 4 per embryo) and assessed using a FIJI macro which utilized a MorphoLibJ [100] Black Top Hat filter of a size 5 and 25 to accentuate the vessels of various diameters.These two filtered images were combined to give the final image that was skeletonized and analyzed.The analyzed vessels yielded the number of branches, junctions, triple, and quadruple points and average branch length.
Statistical Analysis: All statistical analyses were conducted using GraphPad Prism software.The data presented in the graphs are expressed as mean ± standard error of the mean (SEM).The in vitro experiments were performed at least in triplicate (n ≥ 3).Following the 3R principles, the in vivo experiments were performed once after performing power analysis and determining the number of animals (n ≥ 7) which would generate meaningful data.Normality was assessed using the Shapiro-Wilk test, and any outliers were identified and excluded using Grubbs analysis.Statistical differences between groups were assessed using either the Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons.The significance level for all tests was set at p < 0.05.

Figure 2 .
Figure 2. AntagomiR-138-activated CHA scaffold displayed early new bone formation at just 2 weeks post-implantation and promoted vascularization in a load-bearing rat femoral defect model.A,B) Schematics of rat femoral defect model.C) Goldner Masson Trichrome staining showed remarkable deposition of new tissue within the scaffold accompanied by enhanced vascularisation.Scale bar: 2000 μm (top panel) and 200 μm (bottom panel).B: bone, S: scaffold, BM: bone marrow, CT: cartilage.Arrows indicate blood vessels.D) The μCT analysis confirmed enhanced bone regeneration in the antagomiR-138-activated CHA.E) The vessel number was significantly higher on the antagomiR-138-activated CHA scaffold than on the miR-free scaffold.* indicates p < 0.05 (n ≥ 7).The p values were calculated using the Student's t-test.

Figure 3 .
Figure 3. Copper ions induced death of gram-positive S. aureus and gram-negative E. coli, showing their potency as an antimicrobial alternative to antibiotics.The effect of Cu 2+ free ions on A) gram-positive S. aureus, B) gram-negative E. coli, and C,D) human mesenchymal stem cells after 24 h incubation with CuCl 2 solution.The effect of Cu 2+ ions incorporated into bioactive glass nanoparticles (CuBG NP) on oxidative stress in E,F) S. aureus and G,H) E. coli.*, ***, and **** correspond to p < 0.05, p < 0.001, and p < 0.0001, respectively (n ≥ 4).The outliers were excluded using Grubbs analysis.The p values were calculated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.

Figure 4 .
Figure 4.Copper-doped bioactive glass particles were effectively incorporated into collagen scaffolds.This was confirmed by quantifying A) calcium ions on the scaffolds.The CuBG scaffold showed B) an initial release of Ca 2+ followed by C) a plateau phase for up to 28 days.The successful incorporation of copper-doped bioactive glass was also confirmed by quantifying D) copper ions.A similar release trend was observed for E,F) Cu 2+ ions compared to calcium.** and **** correspond to p < 0.01 and p < 0.0001, respectively (n = 3).The p values were calculated using the Student's t-test.

Figure 5 .
Figure 5. Incorporation of copper-doped bioactive glass particles imparted antimicrobial properties to the scaffolds.A) Cu-BG scaffolds reduced the attachment of gram-positive S. aureus; this was maintained when antagomiR-138 was added to the CuBG scaffolds.B) The incorporation of CuBG did not reduce the attachment of gram-negative E. coli, but the addition of antagomiR-138 inhibited the attachment of bacteria.Consequently, reduced bacteria coverage was observed on antagomiR-138-activated CuBG scaffolds cultivated with C) S.aureus and D) E. coli.* and **** correspond to p < 0.05 and p < 0.0001, respectively (n ≥ 4).The p values were calculated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.