Autologous chondrocyte grafting promotes bone formation in the posterolateral spine

Background context Pseudarthrosis following spinal fusion remains problematic despite modern surgical and grafting techniques. In surgical spinal fusion, new bone forms via intramembranous and endochondral ossification, with endochondral ossification occurring in the hypoxic zones of the fusion bed. During bone development and fracture healing, the key cellular mediator of endochondral ossification is the hypertrophic chondrocyte given its ability to function in hypoxia and induce neovascularization and ossification. We therefore hypothesize that hypertrophic chondrocytes may be an effective bone graft alternative. Purpose Spinal fusion procedures have increased substantially; yet 5% to 35% of all spinal fusions may result in pseudoarthrosis. Pseudoarthrosis may occur because of implant failure, infection, or biological failure, among other reasons. Advances in surgical techniques and bone grafting have improved fusion; however pseudarthrosis rates remain unacceptably high. Thus, the goal of this study is to investigate hypertrophic chondrocytes as a potential biological graft alternative. Methods Using a validated murine fracture model, hypertrophic chondrocytes were harvested from fracture calluses and transplanted into the posterolateral spines of identical mice. New bone formation was assessed by X‐ray, microcomputed tomography (μCT), and in vivo fluorescent imaging. Results were compared against a standard iliac crest bone graft and a sham surgery control group. Funding for this work was provided by the Department of Orthopaedics and Rehabilitation, the OREF (Grant #16‐150), and The Caitlin Lovejoy Fund. Results Radiography, μCT, and in vivo fluorescent imaging demonstrated that hypertrophic chondrocytes promoted bone formation at rates equivalent to iliac crest autograft. Additionally, μCT analysis demonstrated similar fusion rates in a subset of mice from the iliac crest and hypertrophic chondrocyte groups. Conclusions This proof‐of‐concept study indicates that hypertrophic chondrocytes can promote bone formation comparable to iliac crest bone graft. These findings provide the foundation for future studies to investigate the potential therapeutic use of hypertrophic chondrocytes in spinal fusion.


| INTRODUCTION
Spinal fusion procedures have increased significantly over the past 10 years. 1,2 While a successful spinal fusion rates have improved with modern implant and grafting techniques, complications can arise, with approximately 5% to 35% of all spinal fusions developing pseudoarthrosis. [2][3][4][5][6][7] Pseudoarthrosis, or nonunion, may lead to increased patient morbidity such as continued pain, segmental instability, and need for revision surgery. Additionally, revision surgeries following the development of a pseudoarthrosis are costly to the health care system, averaging $41 631 per revision. 8  Currently, autogenous bone graft (autograft) harvested from the iliac crest, commonly known as an iliac crest bone graft (ICBG), remains the gold standard for augmenting spinal fusion. 9 Although ICBG demonstrates high fusion rates, its use is hampered by donor site morbidity and limited supply-particularly for multilevel fusions. [9][10][11] Thus, there is a need for a bone graft alternative that can be clinically available in large quantities, provide ample bone formation, and subsequently result in low pseudoarthrosis rates and limited adverse side effects.
In spinal fusion, new bone formation has been previously described to occur via intramembranous and endochondral ossification-processes similar to long bone fracture healing. 10,[12][13][14][15][16] The key cellular mediator of endochondral ossification is the hypertrophic chondrocyte. 17 Along with their unique ability to survive in hypoxia, hypertrophic chondrocytes induce neovascularization and ossification through the release of vascular endothelial growth factor (VEGF), vesicles of hydroxyapatite, and bone-morphogenic proteins (BMPs). 5,[17][18][19] For these reasons, we hypothesize that hypertrophic chondrocytes offer a potential graft alternative for promoting spinal fusion. To test this hypothesis, hypertrophic chondrocytes were harvested from a soft fracture callus at day 10 postfracture and surgically implanted into the posterolateral spinal gutters of a genetically identical mouse recipient. Following implantation, the ability of these fracture callus chondrocyte grafts (FCCGs) to drive posterolateral bone formation was assessed and compared with either standard iliac crest bone graft (ICBG) or a sham implantation surgery control group ( Figure 1). Following adequate anesthesia and analgesia, a 10 to 12 mm long medial incision was made to expose the mid-shaft of the femur. The femur was then fractured in a controlled manner by scoring the bone with a beaver blade before inducing a clean break. The transverse fracture was stabilized with the intramedullary placement of a 30gauge needle, to induce a larger soft-tissue fracture callus, as compared with needles of a larger size (23G) with more stiffness (unpublished results); thereby allowing for a more efficient harvest.

| MATERIALS AND METHODS
The incision was then closed using 5-0 nylon sutures. Mice received analgesics every 12 h for 3 days following the fracture procedure to minimize pain and discomfort. Ten days following the fracture, when the soft-tissue callus was largest and amply expressing VEGF ( Figure 2), the mice were sacrificed by CO 2 inhalation. At this time, the FCCG was harvested, along with an ICBG, for the subsequent transplantation into the posterolateral spine of syngeneic mice. Harvested grafts were standardized by volume and a 2 × 2 × 2-mm section was obtained for implantation.

| Murine posterolateral spinal bone formation model
The purpose of this surgery model was to determine the capacity of FCCG or ICBG to promote bone formation following implantation to the posterolateral lumbar spine. Immediately following FCCG and separate, yet genetically identical, male C57BL/6J mice from Jackson Laboratory at 8 weeks of age ( Figure 3). Following adequate anesthesia and analgesic, the dorsal fur was removed ( Figure 3A) and a midline incision was made through the skin and dorsolumbar fascia to expose the perispinal musculature ( Figure 3B,C). Subperiosteal dissection was carried out using a beaver blade to expose the transverse processes of the L3 to L5 vertebrae prior to decortication ( Figure 3D). The laminae and spinous processes were then decorticated with a beaver blade. The previously harvested FCCG or ICBG was then transplanted into the posterolateral gutters ( Figure 3E-H).
Following this, the internal fascia was closed with absorbable monofilament suture while the skin was closed with 5-0 nylon suture in a simple interrupted fashion ( Figure 3I). Mice were then transferred to their respective cages and monitored until they regained normal ambulation. Analgesic was administered every 12 h for 3 days following surgery to minimize pain and discomfort.

| Assessment of posterolateral bone formation 2.3.1 | Radiographical assessment and quantification
To assess the development of bone formation between the transverse processes, digital radiographs were obtained longitudinally (4 s, 35 kV) beginning day one postsurgery and then weekly until 6 weeks postsurgery (Faxitron, Tucson, Arizona). Mice were placed in the prone position, aligning the spine vertically within the imaging plane. Images were used to quantify the amount of bone formation by 3 blinded independent observers. For each image, the area surrounding the transverse processes of vertebrae L3 through L5 was selected ( Figure S1A, Supporting information). The 6 selected areas were then scored for the amount of calcification present, with a score of "0" representing ≤25% of the total area becoming calcified, a score of "1" representing 26% to 50% of the total area becoming calcified, a score of "2" representing 51% to 75% of the total area becoming calcified, and a score of "3" representing >75% of the total area becoming calcified. The sum of the 6 boxes was then recorded per observer per mouse ( Figure S1B,C). Inter-and intraobserver error was assessed through the use of kappa statistics. On average, observers were found to be in fair to moderate agreement (κ = 0.255-0.489) per the Landis and Koch criteria. Additionally, when rescored with more than 7 days between analyses, intraobserver variability was found to be moderate with observers being in slight to moderate agreement (κ = 0.087-0.481). Further assessment of individual observer scores over time (ie, the slope of the line) were found to have no statistical difference between observers for any experi-

| Microcomputed tomography (μCT) analysis
To further assess bone formation qualitatively, mice were sacrificed

| Histological analysis
To assess the fracture calluses isolated from the donor mouse a subset of femurs were isolated, decalcified, processed, and embedded in paraffin prior to sectioning at 10 days following fracture injury, when the soft tissue callus was largest. Histological sections through the fracture callus were stained for the presence of VEGF.

| Statistical analysis
Inter-and intraobserver variability, for quantification of calcification surrounding the transverse processes, was assessed using kappa statistics and interpreted with the Landis and Koch criteria. 21    While the primary objective of this study was to assess the ability of hypertrophic chondrocytes to promote bone formation in the posterolateral spine, we also observed by μCT analysis successful bony union of the laminae/transverse processes as well as a longitudinal bony bridge across vertebrae, indicative of fusion in a subset of animals from both the FCCG and ICBG group (Video S1). Taken together, these results support the hypothesis that FCCG can promote bone formation to comparable to ICBG, and therefore warrants further investigation as a potential graft alternative in spinal fusion.

| DISCUSSION
To our knowledge, this is the first proof-of-concept study investigat- Although the findings of the current study provide a foundation for advancement in bone graft biologics, they are not without limitation. Primarily, the murine model utilized here is not directly applicable to human anatomy and/or physiology, and thus, directly extrapolating these results to human spinal fusion is not plausible.
Rather, the findings here serve to establish the proof-of-concept foundation for potential transition to small and larger animal studies.
Secondarily, use of a murine model precludes the use of pedicle screws or surgical stabilization to immobilized vertebral segments; yet even in an unstabilized setting, we did observe cortical bridging between vertebrae. Therefore, future studies in larger rodent and animal models, where pedicle screws/surgical stabilization can be employed, are warranted and should investigate the optimal mechanical stimulation of hypertrophic chondrocyte to promote maturation and ossification. Lastly, while this study establishes that hypertrophic chondrocytes may effectively augment bone formation in the posterolateral spine, soft-tissue fracture calluses are not a feasible harvest source in clinical practice. Thus, this study provides the foundation for future work aimed at producing clinically practical sources of hypertrophic chondrocytes. One such promising avenue may be isolating pluripotent cells from circulation or periosteum and differentiating these cellular populations into hypertrophic chondrocytes ex vivo. Despite these limitations, we believe that the findings presented here provide proof-of-concept and establish a new paradigm in bone graft alternatives that will drive future research in larger animal models, and ultimately if successful, humans.

| CONCLUSIONS
The findings of this proof-of-concept study, along with recent progress in tissue engineering, support the concept that hypertrophic   Figure S1). Points represent mean score between 3 reviewers per mouse AESD. N = 10 mice per experimental group. *Statistical significance between ICBG or FCCG and sham. **P < 0.01, ***P < 0.001, ****P < 0.0001. Alpha = 0.05. No statistical difference between experimental groups was detected at any time point. (C) in vivo fluorescent imaging of bone deposition using Osteosense 800 with or without mechanical stability. If these results are recapitulated, investigations into the therapeutic potential of hypertrophic chondrocytes to augment bone formation clinically may be warranted.