The Impact of Human Placental Tissue–Derived Xenograft on Bone Healing and Pain Behaviors in a Murine Segmental Bone Defect Model

Abstract

Introduction

Segmental bone defects (SBD), frequently resulting from high-energy trauma like military combat injuries and major accidents, represent a significant clinical challenge because of the loss of a large bone segment and associated soft-tissue injury. These injuries often occur in military settings, where blast and ballistic trauma cause complex injuries requiring advanced reconstructive strategies. Similar injuries also occur in the civilian population in car accidents, falls, and workplace injuries. Existing therapies (autografts, allografts, and biomaterials) have limitations, including donor site morbidity and graft rejection, highlighting the need for novel bone regenerative approaches. Human placental tissue–derived xenografts (connective tissue matrix [CTM]), rich in structural proteins and growth factors, are under investigation for enhancing bone repair in severe musculoskeletal injuries. This study investigates the efficacy of placental tissue xenografts in a murine SBD model.

Materials and methods

A murine SBD model with a 2 mm defect in the femoral diaphysis of male C57BL/6J mice was used. Mice with SBD were divided into 5 experimental groups: saline control, bone morphogenic protein (BMP-2), and 3 different formulations of CTM (CTM Paste, CTM Membrane, and CTM Membrane+Paste). Treatments were delivered at the defect site at the time of surgery and mice were euthanized 13 weeks postsurgery. Bone healing and pain behaviors were assessed including radiographic scoring, microcomputed tomography (µCT), biomechanical torsion testing, weightbearing, locomotion, and spontaneous nociceptive behaviors. Statistical analyses included one-way or two-way ANOVA and unpaired Student’s t-tests.

Results

BMP-2-treated groups showed significantly larger callus formation, however, lower bone volume/total volume (BV/TV) % compared to saline control was observed. In contrast, the CTM Membrane+Paste group exhibited significantly higher BV/TV%. Biomechanical tests revealed enhanced ultimate torque in BMP-2 and CTM Membrane+Paste groups versus saline controls. All treatment groups of SBD mice exhibited sustained pain behaviors after fracture, which did not return to baseline levels even when bone healing was complete.

Conclusion

The CTM Membrane+Paste treatment group exhibited improved bone healing parameters in a murine model of SBD. Specifically, BV/TV and some biomechanical properties were improved. Notably, CTM did not increase pain behaviors. Further studies are needed to optimize graft effects and assess therapeutic value for clinical translation. Overall, the results support continued investigation, including potential combination therapies and trials in larger animal models.

INTRODUCTION

Musculoskeletal conditions affect people of all ages, sex, and demographic groups, posing a major health concern. Bone fractures, comprising approximately 9% of all occupational injuries, represent a major source of disability across both civilian populations and military personnel.^1,2 Traumatic bone fractures are prevalent among military personnel, resulting from combat-related events such as explosions, ballistic wounds, and high-risk training activities.² A recent study reported explosions were the leading cause of injury (56.4% and 52.2% across 2 major military campaigns), with upper and lower extremity fractures accounting for 43.6% and 58.9% of injuries, respectively.³ Given the high occurrence rate and severity of these injuries, the resulting fractures often present significant health challenges. Because of these complex injuries, traumatic fractures frequently result in complications like nonunion or malunion, where the bone either fails to heal properly or heals in a misaligned position, leading to long-term functional impairment. These complications in healing increase medical and disability costs, limit redeployment, and can result in chronic pain or increased risk of secondary health ailments such as chronic pain syndromes, posttraumatic arthritis, muscle atrophy, osteoporosis, and psychological conditions, including depression and opioid dependence.^4,5 The murine segmental bone defect (SBD) model is a widely used experimental approach for studying bone regeneration and repair, especially injuries resulting from high energy trauma.^6-8 Effective bone regeneration involves a coordinated sequence of events, beginning with angiogenesis, followed by bone formation by osteoblasts and resorption by osteoclasts. Unlike simple fractures, SBDs involve substantial loss of bone and surrounding soft tissue, resulting in impaired vascularization, disrupted biological signaling, and reduced mechanical stability that together hinder spontaneous healing.⁶ Currently, local applications of bone morphogenic protein (BMP-2, BMP-7), or platelet-derived growth factor (PDGF) are the only FDA-approved biological therapies for treating nonunion fractures.⁹ However, BMP-2 therapy has been linked to complications such as local inflammation, ectopic bone formation, bone resorption, infection, pain and a potential increased risk of malignancy.^10-12

A novel class of therapeutics for bone regeneration that have been used experimentally in the clinic are placental allografts. These allografts (Connective Tissue Matrix [CTM], CTM Biomedical, Lake Worth, FL, United States) are derived from human amnion, chorion, and umbilical tissues. These grafts are processed from donor placental tissues using a proprietary method that retains key regenerative components such as PDGF, BMP-2, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), transforming growth factor (TGF), antiinflammatory factors, and extracellular matrix proteins factors known to support bone repair and tissue regeneration.¹³

The goal of this study was to evaluate the efficacy of human-derived CTM in promoting bone regeneration and modulating postfracture pain behaviors using a murine SBD model. Although some CTM-associated growth factors may enhance pain sensitivity, improved bone repair could, in turn, reduce nociceptive signaling. To assess pain in this model, we examined spontaneous pain behaviors in the mice. We hypothesize that CTM xenografts will improve bone healing in an SBD murine fracture model.

MATERIAL AND METHODS

Animals, Surgery, and Experimental Outline

Experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Indiana University School of Medicine, Indianapolis, Indiana [IACUC protocol number is 23136] and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

A schematic of the experimental design and timeline is shown in Figure 1. This study used 70, 12-week-old male C57BL/6J mice divided into 5 treatment groups: (1) saline control, (2) BMP-2, (3) CTM Paste, (4) CTM Membrane, and (5) CTM Membrane+Paste (Figure S1). The SBD surgical procedure was performed as previously described.¹⁴ Briefly, SBD was achieved by making a lateral skin incision over the right femur and creating a 2 mm defect and placing a scaffold.¹⁴ A 25G needle was passed through the synthetic graft and the greater trochanter to stabilize the defect. Polypropylene fumarate (PPF)/tricalcium phosphate (TCP) scaffolds were used as a biodegradable mechanical support (Figure 1).¹⁵ In this model, the scaffold is required to maintain the bone length, and if the healing is delayed, the scaffold may degrade/break before sufficient bone bridging occurs.^15,16 The treatments were initiated at the fracture site intraoperatively. RCM6 resorbable collagen membranes (ACE Surgical, Melville, New York, United States) were prepared by soaking in either saline (10 µl) (negative control) or in recombinant human BMP-2 (5 µg in 10 µl saline) solution (positive control), where the collagen membrane absorbed the BMP-2 before being wrapped around the scaffold at the defect site.^14,15 On the other hand, CTM-treated groups received the treatments directly at the fracture site on top of the scaffold, and collagen membrane was not used. CTM Paste group was treated by placing 500 µl of CTM Paste at the fracture site on the scaffold using a 1 ml syringe. The CTM Membrane group was treated by securing the membrane and wrapping it around the fracture site and scaffold. TheCTM Membrane+Paste group was treated by placing 500 µl of CTM Paste at the fracture site, as above, and then wrapping CTM Membrane around the Paste.

Figure 1.

Overview of the fracture study timeline and design. This schematic outlines the experimental setup and timeline used for SBD study and the subsequent evaluation of outcomes in this study.

Radiographic Imaging and Modified Radiographic Union Scoring for Tibial Fractures (mRUST) Scoring

Radiographic X-ray imaging was conducted weekly postsurgery for 4 weeks and then every other week until euthanasia. X-ray images were taken in both lateral and anterior-posterior views using a Kubtec XPERT80 (Kubtec Medical, Stratford, Connecticut, United States). The modified Radiographic Union Scale for Tibial fractures (mRUST) quantitatively evaluates cortical bone bridging and overall fracture healing progression.^17,18 To minimize bias, X-ray images were randomized and reviewed by an orthopedic surgeon who was blinded to both the experimental groups and timepoints.

Micro-Computed Tomography (µCT)

Following euthanasia, the femurs were stored at −20 °C. Before scanning, the bones were thawed and kept hydrated in phosphate-buffered saline (PBS) with calcium and magnesium (HiMedia Laboratories, Nashik, India) at 4 °C for ∼8 hours. The fractured femurs were scanned longitudinally using the SkyScan 1272 imaging system (SkyScan, Kontich, Germany). Scanning and calibration parameters were consistent with previous studies (voxel size of 10 µm, beam energy of 70 kV, tube current of 142 μA, frame averaging set to 3, and a 0.5 mm aluminum filter).¹³

Three-dimensional image reconstruction was performed using NRecon software (v1.7.3), applying a smoothing-factor of 2, beam-hardening correction set to 20%, and a ring-artifact reduction value of 5. Reconstructed scans were analyzed using CTan software to quantify callus area. The volume-of-interest (VOI) was manually selected to encompass the entire callus region, manually excluding the scaffold and cortical bone. The region-of-interest (ROI) was defined using a CTan-based algorithm to differentiate mineralized callus tissue from VOI and were analyzed for mineralized callus area (B.Ar), trabecular-separation (Tb.Sp), trabecular-thickness (Tb.Th), trabecular-number (Tb.N), bone volume (BV), total volume (TV), and the ratio of bone volume to total volume (BV/TV%), as described previously.^17,18 Additionally, a custom MATLAB script (R2023b) was used to calculate tissue mineral density (TMD) within the pre-determined ROI.¹⁹ CTVox software was used to ­create 3D renderings.

Biomechanical Torsion Testing

Torsion testing was carried out immediately after the µCT scanning to avoid multiple freeze-thaw cycles and retain bone integrity. Femurs were potted by securing both proximal and distal ends in 9 mm bullet casings using a fast-setting orthodontic acrylic resin (Ortho-Jet, Lang Dental, Wheeling, IL, United States). Bones were held vertically during curing (∼20 minutes), with hydration maintained using PBS-soaked gauze, leaving the fracture callus and the mid-diaphysis was left exposed. Torsional testing was conducted using a Mark-10 torque-testing apparatus (Mark-10, Copiague, NY, United States) equipped with a Model M5i torque indicator, as previously described.¹³ Biomechanical parameters were derived from the torque–twist curves. Ultimate torque was defined as the maximum torque value recorded before mechanical bone failure. Stiffness was calculated by averaging the 15-steepest slopes within the linear region before the ultimate torque. Toughness, representing the total energy absorbed by the bone until failure, was calculated as the area under the curve. Twist at failure was defined as the angular displacement corresponding to the point of ultimate torque. To account for inter-animal variability, all values from fractured femurs were normalized to the corresponding contralateral femur and are expressed as percentage.¹³

Pain-behavioral Analysis

Postfracture pain behaviors were evaluated using a battery of standardized pain assessments. Pain perception in animal models can be influenced by environmental and experimental factors; therefore, to enhance the reliability and reproducibility of the results, all assessments were conducted in a controlled, quiet environment by the same individual(s) at approximately the same time each day for longitudinal assessments. Behavioral experiments were performed at baseline and weekly post-surgery for the first 4 weeks, and biweekly thereafter until euthanasia. A detailed description of each behavioral assessment is provided below.

Static weightbearing

A constant load on one limb without movement is performed to assess bone’s ability to bear weight. Bilateral hind-paw weightbearing on the surgical leg was tested using an incapacitance device (Bioseb, Pinellas Park, FL, United States). The mice were positioned upright, with their body weight supported by their hind paws resting on 2 separate plates. We recorded 4 consecutive measurements, each lasting for 10 seconds, with a 10-second interval between each measurement. The mean of the 4 values was calculated to be the weightbearing values of the individual hind paws. The weightbearing data from the fractured paw was analyzed as the percent of weight borne on the right (fractured) hind limb, calculated by the ratio between the right (fractured) hind paw weight and the mean of the right and the left hind paw values: ((R/(R + L)) × 100%). A value of less than 50% was considered a reduction in the weightbearing on the fractured leg.²⁰

Locomotor activity

Mice were placed in a dark activity chamber measuring 30 cm × 30 cm × 35 cm (width × length × height) under red lights and allowed to freely explore the area for 8 minutes. Video recordings were captured using GoPro HERO 8 (GoPro Inc, San Mateo, California, United States) cameras and were analyzed using DeepLabCut (Mathis Laboratories of Adaptive Intelligence, EPFL, Lausanne, Switzerland)²¹ as previously described.¹³

Spontaneous paw withdrawal

Evaluation of spontaneous nociceptive pain was conducted through video surveillance, capturing spontaneous behaviors indicative of discomfort or pain. The mice were acclimated for 30 minutes before videos were recorded for 5 minutes using GoPro-Hero-8 cameras. The experimental conditions and the video analysis techniques were kept ­consistent with our previous work.¹³

Statistical Analyses

This study was designed to a priori evaluate the effects of CTM products on bone healing and pain behaviors compared to saline controls. Given the study’s design, which incorporated distinct treatment delivery modes—such as the use of BMP-2 as a positive control with a collagen membrane, compared to CTM treatment groups without a collagen membrane—and the primary objective of comparing 2 independent variables, multiple statistical tests were employed to analyze the data. Specifically, Student’s t-tests were used to assess pairwise relationships between treatment groups in addition to ANOVA to provide an overall assessment of variance. By performing both the Student’s t-test and one-way ANOVA with post hoc tests, we have increased statistical rigor, ensuring that pairwise comparisons align with exploration of group differences, and that the overall variance was thoroughly considered. The experimental design limits formal multi-group inference thus the results should not be interpreted as intergroup comparisons. Analyses for which time was included as a factor were analyzed using two-way ANOVA. P-values <.05 were considered significant. Outliers were identified and excluded using the ROUT method. Data are reported as mean ± standard error of the mean (SEM). GraphPad Prism software (version 8.0, GraphPad Software, La Jolla, California, United States), was used for all statistical analysis. A detailed description of the number of animals initially recruited, study schematic design, and the final sample numbers for each experimental outcome are provided in (Figure S1).

RESULTS

Enhanced Fracture Healing Observed with BMP-2 and CTM Treatments

To examine fracture healing, longitudinal X-ray images and terminal (84 days postfracture) 3D renderings from µCT of femurs from the different treatment groups were evaluated (Figure 2A and B). mRUST scoring, which evaluates the degree of fracture bridging,¹⁷ indicated that progression of fracture healing was improved in BMP-2, CTM Membrane, and CTM Paste-treated groups compared to saline control mice (Figure 2C). We did not observe any statistically significant differences between CTM Membrane+Paste treated mice and saline control mice.

Figure 2.

X-ray and μCT assessment of femoral SBD healing in treatment groups. (A) Representative X-ray images from each treatment group at 84 days post fracture. (B) Representative μCT images of femurs from each treatment group at 84 days post fracture. (C) mRUST score from each treatment group. Overall differences in the curves were observed for BMP-2 vs. saline control (A, ****P < .0001), CTM Membrane vs. saline control, (B, ****P < .0001), and CTM Paste vs. saline control, (C, *P = .05). (D) BMP-2 showed a significantly larger callus area, P = .003, although the CTM-treated groups exhibited no statistically significant differences. (E) BV/TV% representing mineralized callus fraction comparing all groups. BMP-2-treated group showed lower BV/TV% compared to saline control, P = .001. (F) Quantification of Tb.Th in all treatment groups showed no statistical significance. (G) Although the other groups showed no statistical significance, BMP-2 group showed increased Tb. Sp compared to controls, P = .01. (H) Saline control group exhibited higher Tb.N compared to BMP-2, P = .001, however, no significant differences were seen for the rest of the groups. (I) No statistically significant differences were observed for TMD comparing all treatment groups. All statistical analysis were performed by compared the treatment groups with respect to the saline control. Two-way ANOVA with Holm–Sidak post hoc test (C). One-way ANOVA with Holm-Sidak post hoc test (D-I). ^*P < .05, **P < .01, ***P < .001, ****P < .0001.

We observed significantly increased callus area in femurs from the BMP-2 treatment group compared to saline control (Figure 2D). BMP-2 also exhibited significant reduction in BV/TV% compared to saline control, suggesting the presence of low mineralization even though a large callus is present (Figure 2E). Using the predefined analytical approach comparing each treatment condition to control, CTM Membrane+Paste treated mice exhibited a significant increase in BV/TV% (Figure S2B). No significant increase was found in femur trabecular bone Tb.Th among any of the treatment groups (Figures 2F and S2C). Tb.Sp was increased in BMP-2-treated mice compared to saline control, although no differences were observed among any of the other treatment groups (Figures 2G and S2D). Tb.N was decreased in BMP-2-treated mice compared to saline control (Figure 2H) and no differences were observed for any of the CTM treatments compared to saline controls (Figure S2E). BMP-2-treated mice exhibited a decrease in TMD compared to saline control mice (Figure 2I) and no differences were observed in any of the CTM-treated groups compared to saline control (Figure S2F).

BMP-2 and CTM Membrane+Paste Exhibited Increased Ultimate Torque

To provide a direct functional assessment of bone repair quality, we performed biomechanical testing on femurs from each treatment group. Ultimate torque analysis of the femurs exhibited an increase in BMP-2 mice compared to saline control (Figure 3A). Using the predefined analytical approach comparing each treatment condition to control, no differences were observed for CTM Paste or CTM membrane-treated mice compared to saline controls, but there was a significant difference between CTM Membrane+Paste and saline-treated mice (Figure S3A). There were no differences observed for bone stiffness for any of the treatment groups (Figures 3B and S3B). No statistically significant differences were observed in toughness among any of the treatment groups (Figures 3C and S3C). Finally, BMP-2-treated mice exhibited a decrease in twist to failure compared to saline control mice (Figure S3D) whereas, no differences in twist to failure were observed for CTM-treated groups (Figure S3D).

Figure 3.

Biomechanical assessment of fractured femurs in a SBD murine model. (A) Ultimate torque was assessed for all treatment groups. There was an increase in ultimate torque in BMP-2, P = .02. (B) No significant difference was observed in twist to failure among any of the groups. (C) No difference in stiffness was observed in stiffness among any of the groups. (D) No significant difference was observed in toughness among any of the groups. All statistical analysis were performed by comparing the treatment groups to the saline control. All graphs are expressed as a percentage of the contralateral limb. One-way ANOVA with Holm–Sidak post hoc test. *P < .05.

Treatments Did Not Exacerbate Pain Behavior Postfracture

CTM xenografts were tested for their potential to influence postfracture pain behaviors in SBD mice. To quantify spontaneous, nonevoked pain and assess functional recovery, we performed static weight bearing (SWB), open field testing, and spontaneous paw lifts in mice from all treatment groups. SWB analysis showed a 40%-47% reduction in weight bearing on the fractured limb across all treatment groups (Figure 4A), which slowly started to improve, however did not return to baseline. Similarly, open field testing resulted in a 52%-58% decrease in locomotor activity postfracture across all treatment groups (Figure 4B), which failed to return to baseline during the course of this study. Spontaneous rapid paw withdrawal exhibited an increase in all treatment groups following fracture (Figure 4C). In each of these assays, there was an immediate increase in pain behaviors following surgery that did not fully recover during the 84 days of monitoring postfracture. Importantly, there was no additional increase in spontaneous pain behaviors in any of the treatment groups compared to saline-treated control.

Figure 4.

Behavioral assessment following femoral defect. (A) Static weight bearing analysis showed no significant differences among treatment groups over 84 days, though all groups exhibited reduced weight bearing from baseline post-surgery. (B) Total distance traveled did not differ between CTM treatment groups or BMP-2-treated mice and saline controls, but all groups showed a sustained reduction in locomotion postfracture. (C) Spontaneous paw withdrawal showed no significant group differences at any time point. All statistical assessments were performed by comparing the treatment groups to saline control, using Two-way ANOVA and Holm–Sidak post hoc test.

DISCUSSION

Managing SBD remains a challenging problem in orthopedic surgery, largely because of the complexity of achieving both mechanical stability and biological regeneration in critical-sized gaps.²² Bone grafts used to repair critical-sized defects include autografts, allografts, xenografts, and synthetic substitutes. Although autologous bone grafts remain the gold standard because of their combined osteogenic, osteoinductive, and osteoconductive properties; synthetic graft materials like hydroxyapatite, β-tricalcium phosphate, bioactive glass, and calcium phosphates are also used, providing osteoconduction, but these lack the complete replication of biological activity of autografts.²³

Animal models of SBD are commonly employed to assess bone graft substitutes.²² Using a 2mm SBD model in mice, we sought to assess the therapeutic potential of CTM products in promoting bone regeneration. This study focused on local delivery at the defect site. Since CTM xenografts are human tissue–derived, we also evaluated whether CTM led to a systemic immune response. We previously performed longitudinal complete blood count (CBC) analysis and observed no systemic hematologic changes.¹³ In the current study, CBC analysis was performed, but only at the terminal timepoint. Of note, the same mouse strain, sex, age, and treatments were used, only the bone healing model used, and time points post-surgery differed. As shown in Figure S4, only one parameter varied among the groups, neutrophils within the CTM Paste group, suggesting CTM does not cause major systemic hematologic alterations. In the current study, The CBC analysis was performed at the terminal timepoint,²⁴ showing a slight increase in the neutrophil cell counts for the CTM Paste group; this may be attributed to CTM Paste being the only group lacking any membrane at the fracture site. CBCs measured at baseline and euthanasia showed no differences because of CTM xenografts when using the same mouse strain, sex, and age (Figure S4). Fracture elevates systemic VEGF, FGF-2, BMP-2 and PDGF which support healing through both local angiogenesis and modulation of distant progenitor cells.^9,24 The CTM treatment formulations are proprietary, but placental-derived extracellular matrices have been reported to contain VEGF, PDGF, FGF-2, and BMP-2. These are therefore hypothesized to promote repair through similar mechanisms. Our prior work showed that CTM Membrane+Paste enhanced callus formation, increased BV/TV%, and reduced early pain behavior in a surgically induced fracture model, supporting its clinical use as a locally applied treatment and motivating our evaluation in the more challenging SBD model.¹³

Consistent with prior studies, BMP-2 used as a positive control improved fracture healing as measured by radiographic, μCT, and biomechanical outcomes, confirming osteoinductive activity.^9,25,26 BMP-2 treatment significantly enhanced fracture healing, as indicated by improved mRUST scores and increased callus formation. A recent study provides a direct temporal comparison of fracture healing between mice and humans, which enhances the clinical translatability of our findings.²⁷ Despite accelerated radiographic healing, increased callus area, and improved ultimate torque, BMP-2-treated defects exhibited reduced mineralization and mechanical integrity, demonstrated by lower BV/TV, TMD (Figures 2E and S2F), and twist to failure (Figure S3D) relative to saline controls. In contrast, CTM Membrane+Paste increased BV/TV and ultimate torque compared to control, despite no improvement in mRUST scores, likely because this 2D bridging-oriented metric, does not fully capture 3D volumetric or mechanical changes (Figures 2C, S2B, and S3A). The CTM Membrane and CTM Paste groups had very small sample sizes, limiting statistical power and increasing variability particularly with µCT and biomechanical interpretations. Indeed, no statistical significance was observed for stiffness or twist to failure. As would be expected, femurs from mice treated with either BMP-2 or CTM Membrane+Paste exhibited improved ultimate torques to controls, indicating improved mechanical strength. Overall, these findings show that although BMP-2 quickly promotes bone bridging, CTM products show promise as a biologically-derived alternative capable of improving the quality of bone healing. Notably, CTM Membrane+Paste enhanced callus formation in our previous fracture model¹³ but not in the more severe SBD model. The 2 models are not directly comparable, and because of the larger defect size, higher mechanical instability, and longer healing time, we saw different healing outcomes as would be expected. Further studies in larger animal models and at multiple time points will be necessary to clarify the durability, ­quality, and translational relevance of CTM-mediated bone repair.

In this study, we used PPF/TCP scaffolds¹⁵ as a biodegradable mechanical support for the femurs in the SBD model. Although these scaffolds are effective for treatment delivery of BMP-2, a limitation of this study was the occurrence of scaffold failure in a subset of samples. This failure may reflect the limited mechanical support of PPF/TCP composites under load-bearing conditions, particularly before sufficient callus formation and bridging occur. Additionally, if the scaffold degraded faster than new bone formation, structural integrity would be compromised, leaving the defect unsupported and increasing the probability of failure.²⁸ The scaffold failures limit our statistical power and are a limitation of our study, although we still determined important improvements because of CTM when scaffolds did not fail. Although not the focus of this study, and additional experiments would be required to make this conclusion, it is formally possible that saline scaffolds did not experience the same failure rate as those with CTM products, because some constituent(s) within the CTM products modify cellular responses which in turn could regulate scaffold integration and/or stability (Figure S1). Should this be the case, it would be an important consideration in clinical cases using CTM or similar products in conjunction with scaffolds. That said, all specimens with scaffold failure were excluded from the final analysis to ensure consistency in outcome measures.

CTM xenografts include a mix of growth factors and cytokines, some of which are known to encourage bone and wound healing.¹³ Notably, placental derived tissues like CTM may also contain NGF, which can increase pain sensitivity by increasing the density of sensory nerve endings at injury sites and by sensitizing neurons to enhance neurotransmitter release.^13,29 Despite this potential, our previous work demonstrated that CTM xenografts did not intensify pain responses in a standard surgical fracture model.^13,29 The SBD model examined here, however, is more severe and clinically relevant to injuries resembling high-energy fractures. To assess pain behaviors in clinically relevant SBD mouse model, we evaluated static weightbearing, open-field locomotion, and spontaneous pain behavior assays.²⁹ Static weightbearing measurements are particularly valuable for modeling pain, as they assess the discomfort experienced when a limb is loaded in the absence of active movement. We observed that mice with SBDs had decreased SWB consistent with increased pain, and that none of the treatment groups assessed altered SWB compared to saline controls (Figure 4A). SWB deficits peaked between 4-14 days post-surgery and then slowed returned towards baseline (equal distribution of weight), never recovering completely. We also observed that SBD surgery reduced the locomotion of mice and resulted in an increase of spontaneous pain behaviors. Again, no differences were observed between any of the treatment groups and saline control mice (Figure 4B and C). Of interest, decreases in mouse locomotion persisted well beyond the timepoint by which the bone had healed. An mRUST score of ≥10 represents healed bones.²⁷ Although BMP-2-treated mice achieved mRUST scores greater than 10 by 3 weeks post-surgery, pain behaviors remained similar to mice that never achieved complete healing from days 21 to 84 post-surgery (Figures 2C and 4). Despite prior reports of BMP-2 associated inflammation and pain^11,12 and our hypothesis of possible transient nociceptive effects because of growth factors in the CTM preparations, no increases in pain behaviors were observed in BMP-2 or CTM-treated mice compared to saline controls, but they clearly demonstrate continued nociceptive effects compared to baseline measures. Notably, we observed that fracture induced pain persisted, even after 13 weeks postfracture, despite clear improvements in bone healing, reflecting the well-recognized disconnect between structural recovery and pain resolution.³⁰ This is consistent with clinical observations whereby patients’ fractures are healed on x-ray but they continue to experience persistent pain (personal ­communication from orthopedic trauma surgeons Todd McKinley and Roman Natoli).

Overall, SBD mice-treated with either BMP-2 or CTM Membrane+Paste exhibited larger callus formation and enhanced mechanical strength. Some variation between our 2D and 3D healing outcomes was expected. The 2D mRUST score assesses cortical bridging, but provides limited information on callus structure or mineralization, whereas 3D microCT measures callus volume, BV/TV, and TMD, offering a more detailed assessment of bone quality. Because these methods capture different aspects of fracture repair, their results are not directly comparable. Future studies should include mice from both sexes as well as different age groups to capture sex- and age- differences in bone regeneration³¹ and pain responses.³² Although CTM Membrane+Paste treatment enhanced several healing parameters in C57BL/6J mice, the translational relevance may be limited, as murine models do not fully reflect the biological and clinical complexity of human fracture healing. Therefore, extending these investigations to more complex models or large animal systems would strengthen the translational bridge to human clinical care.

SUPPLEMENTARY MATERIAL

Supplementary material is available at Military Medicine online.

FUNDING

Funding for this study was supported by CTM Biomedical LLC, Lake Worth, FL. The CTM products used in the experiments were also supplied by CTM Biomedical LLC. Funding for this work was also provided in part by NIH AG060621, AR083130, DK106846, and CA082709. This work was also supported by the Small Animal Skeletal Phenotyping, which is funded in part by the Indiana Center for Musculoskeletal Health (ICMH). The ICMH also provided an Industry grant for this work. This study was also supported with resources and the use of facilities at the Richard L. Roudebush VA Medical Center, Indianapolis, IN, and through VA grants: VA Merit #BX006399, Research Career Scientist Award #RX004809, and VA ShEEP #IS1BX004801.

Supplement Sponsorship

This article appears as part of the supplement “Proceedings of the 2025 Military Health System Research Symposium,” sponsored by the Assistant Secretary of Defense for Health Affairs.

CONFLICT OF INTEREST STATEMENT

M.A.K. is a co-inventor on a patent for the use of thrombopoietic agents in bone healing. M.A.K. is the founder of OsteoFuse, LLC. For all other authors, no conflicts of interest exist.

DATA AVAILABILITY

The data underlying this article will be shared on reasonable request by the corresponding author.

INSTITUTIONAL REVIEW BOARD

Not Applicable.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

Experiments involving mice were performed in accordance with protocols approved by Indiana University’s IACUC (#23136).

INSTITUTIONAL CLEARANCE

Does not apply.

INDIVIDUAL AUTHOR CONTRIBUTION STATEMENT

UG collected, and analyzed data and drafted the original manuscript, T.J.M., W.A.V., M.K.N., R.S.P., H.X., A.J.M., A.W., K.Y., R.J.B., A.T., S.J.K., J.L., A.C., D.L.W. helped with data collection, data interpretation, and editing the manuscript. J.C.F. and M.A.K. conceived and designed the study, interpreted the data, and revised/edited the manuscript. All authors have reviewed the manuscript and accept responsibility for the content of the manuscript.