Evaluating the usage of human placental tissue-derived xenograft in a surgically induced murine fracture model

Abstract

Impaired bone fracture healing can lead to chronic pain, loss of function, or life-long complications which can lead to limb-amputation. This study evaluated the effectiveness of human placental tissue-derived xenograft preparations (Connective Tissue Matrix, [CTM Biomedical^®]) in promoting bone healing and reducing post-fracture pain behaviors using a preclinical, surgically induced, murine fracture model. CTM is thought to contain structural proteins and growth factors important for fracture healing. An intramedullary nail was used to stabilize the femur, and a mid-shaft femoral fracture was induced in 12-week-old male C57BL/6J mice. Mice were divided into four groups (Saline Control, CTM Membrane, CTM Paste, and CTM Membrane + Paste). Complete blood count (CBC) and pain behavioral assessments were performed weekly. Modified radiographic union for tibial fracture (mRUST) scoring was used to assess healing from bi-weekly X-rays. Mice were euthanized 23 days post-fracture (dpf), and femurs were collected for μCT analysis and biomechanical or histomorphometric analyses. No significant changes in CBC were observed in any group. Compared to Saline Control, CTM Membrane (p=0.002) and CTM Membrane + Paste (p=0.04) treated groups exhibited a reduced pain score 4 dpf (grimace scores). μCT and histomorphometry showed that CTM Membrane + Paste group had significantly higher callus area (p=0.01) and % bone (p=0.001) compared to Saline Controls. mRUST scores, endomucin staining, and biomechanical outcomes between groups were not different. Preclinical findings suggest that CTM Membrane + Paste exhibits potential in enhancing bone healing. CTM products also do not increase pain behaviors in the surgical fracture model.

1. Introduction

Annually, approximately 6.3 million fractures occur in the United States and about 178 million occur globally [1]. Despite advances in treatment, nonunion of long bone fractures continues to be a challenge. Previous studies in humans have shown that 5–10% of femur fractures result in nonunion, depending on the degree of injury and treatment approaches [2, 3]. Fracture nonunion and inadequate bone repair can impair function, prolong pain, lead to associated nerve injury, and increase healthcare costs. Post-surgery pain management often involves opioids, which can be effective but can hinder bone healing, induce hyperalgesia, and pose serious threats like opioid addiction [4, 5].

Current fracture treatments can be broadly classified into surgical and non-surgical methods, with distinct advantages and disadvantages for each approach. Non-surgical approaches, such as casting, can be used for stable fractures; however, improper application of casts can cause impaired healing. Additionally, long-term castings can also result in skin irritation, joint stiffness, muscle atrophy, or complex regional pain syndrome (CRPS) [6, 7]. Surgical methods may involve the introduction of biological implants like autografts, allografts, and xenografts, but introduce risks for donor-site morbidity or graft rejection [8, 9]. Non-biological implants, such as metal or polymer-based devices, provide structural support but can corrode or induce hypersensitivity reactions to metal implants [10]. Internal fixation techniques, including intramedullary nailing, are effective but may cause infections, while external fixation stabilizes fractures with risks of non-union and neurovascular injury [11]. The limitations of current therapies necessitate the urgent need to seek additional therapeutic approaches that enhance bone healing to improve patient care.

Bone regenerative therapies and biological implants serve as potential additive therapies to current fracture treatments to improve fracture recovery and patient outcomes. Bone morphogenetic proteins (BMPs), particularly BMP-2, have gained attention for their ability to stimulate osteoblast proliferation and enhance bone formation, making them a promising option for accelerating fracture healing and improving bone regeneration. However, the use of BMP-2 is also associated with adverse clinical effects like inflammation, nerve root compression, ectopic bone formation, bone resorption, infection, and increased risk of cancer [12, 13]. Thus, challenges remain in optimizing the safety, efficacy, and clinical application of fracture healing treatments.

The primary objective of this study was to evaluate the efficacy of a human placental tissue-derived xenograft in a preclinical mouse femur fracture model. These xenografts, manufactured by Connective Tissue Matrix Biomedical (CTM Biomedical^®, Lake Worth, FL), are derived from human amnion, chorion, and umbilical tissue components. CTM xenografts are available in various formulations, including CTM Membrane, CTM Paste, and CTM Flow [14]. In this study, we used CTM Paste for packing, filling, and spreading within the fracture site, and CTM Membrane for wrapping the fracture site. These xenografts are derived from human donor tissues and developed using a proprietary process to enrich for growth factors, like platelet-derived growth factor (PDGF), BMP-2, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), anti-inflammatory mediators like transforming growth factors (TGFs) and extracellular matrix (ECM) components that may enhance fracture healing. Human placental tissue is rich in growth factors and cytokines that contributes to its regenerative potential [15]. The primary focus of this study was to assess the efficacy of CTM xenografts in promoting bone healing in a surgically induced femoral fracture model. We also evaluated the impact of CTM formulations on post-fracture pain behaviors, as some of the growth factors that comprise the formulations have been shown to increase pain [16]. Alternatively, an increase in bone healing might decrease the levels of nociception in the mice following fracture; therefore, we used a battery of nociceptive behavioral tests to assess both spontaneous and stimulus-induced pain behaviors in the mice. We hypothesize that CTM xenografts will enhance bone healing in a preclinical murine model.

2. Materials and Methods

2.1. Animals, Surgery, and Experimental Design

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee at Indiana University School of Medicine, Indianapolis, IN and were completed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed in group cages in a light-controlled room with food and water available ad libitum. An experimental design schematic and timeline are shown in Figure 1. The study used a total of 105, 12-week-old male C57BL/6J mice (Supplemental Figure 1). A 25G pin was inserted through the intramedullary canal to stabilize the femur prior to inducing a mid-shaft surgical fracture of the right femur as previously described by Alentado et.al [2]. After inducing a mid-shaft fracture in right femur, the following treatments were introduced into the surgical fracture site: Saline Control, CTM Paste, CTM Membrane, or a combination of CTM Membrane + Paste. The Saline Control group was treated with 10 µl of saline applied to the fracture site, while CTM Paste group received 500 µl of CTM Paste directly at the fracture site. In our studies we are using saline as a negative control due the number of different CTM delivery formulations and due to the proprietary nature of the CTM manufacturing process and unavailability of membrane and paste negative controls. In the CTM Membrane group, CTM Thick Membranes measuring 0.5 × 1.0 cm were wrapped around the fracture site and were sealed with 10 µl of saline. Finally, the combination group of CTM Membrane + Paste received both treatments where 500 µl of CTM Paste was applied to the fracture site, followed by wrapping with CTM Thick Membranes (0.5 × 1.0 cm), which were then sealed with 10 µl of saline. At the time of surgery mice were administered buprenorphine extended release at 1.0 mg/kg subcutaneous injection (Ethica XR, Fidelis Animal Health, New Brunswick, NJ). After the surgery, the mice were transferred to clean cages positioned so that a portion of the cage was set on a heating pad at 37°C. Mice were returned to the colony after fully recovering from anesthesia. Post-surgery, the mice were observed daily for 4 days to check wound site, gait, and any visible signs of distress or pain. Due to the implantation of human tissue, these studies were considered Biosafety Level 2 (BSL-2) and appropriate safety procedures as determined by the Institutional Biosafety Committee (IBC) were followed.

Figure 1: Timeline and schematic for fracture study.

Illustration of the experimental design and timeline for the surgically induced femoral fracture and outcomes assessment for this study.

2.2. Blood Collection and Complete Blood Count (CBC) Analysis

Saphenous blood collection was performed on the contralateral leg at baseline and at 8, 15, and 22 days post fracture (dpf). Briefly, an 18G x 1 ½ (1.2mm x 40mm) needle was used to puncture the saphenous vein of the contralateral leg, and approximately 50 µl was collected directly into ethylenediaminetetraacetic acid (EDTA) coated tubes (Greiner Bio-One GmbH, Kremsmunster, Austria). A CBC was completed using an Element HT5 machine (Heska, Loveland, Colorado).

2.3. Rodent Radiographic Analysis and modified Radiographic Union Scoring for Tibial Fractures (mRUST)

Biweekly radiographic X-ray imaging was performed in both anterior-posterior and lateral positions at 8, 11, 15, 18, and 22 dpf using Kubtec XPERT 80 (Kubtec Medical, Stratford, Connecticut). mRUST scoring allows for quantitative assessment of cortical bridging and healing of the fracture site over time. Radiographic imaging and mRUST scoring were performed as previously described by Alentado et. al and Knox et. al [2, 17]. To ensure unbiased scoring, orthopaedic surgeons were presented with the X-ray images in a randomized order, blinding them to both treatment group and time point post-fracture.

2.4. Micro-Computed Tomography Analysis of Femoral Fractures

μCT imaging and subsequent analysis was conducted on the fractured femurs. One batch of collected femurs was initially fixed using neutral buffer formalin (NBF) for 48 hours then stored in 70% ethanol and scanned using a desktop Skyscan 1172 imaging system (SkyScan, Kontich, Germany). The scanning parameters were kept consistent with our previously published μCT scanning techniques [2]. A second batch of femurs, stored in −20°C for biomechanics, was scanned using SkyScan 1272 CMOS imaging system. During the scanning, the femur was aligned longitudinally along the axis and scanned using 10mm voxel size with beam voltage at 70kV, current at 142µA, frame averaging of 3, and a 0.5mm Al filter. Scans of two hydroxyapatite-mimicking phantoms 2mm in diameter with concentrations of 0.25 g/cm³ and 0.75 g/cm³, were used for calibration, and bone was differentiated from non-bone using a grayscale threshold value of 60.

Image reconstructions were completed using NRecon v.1.7.3. software and a smoothing value of 2, beam hardening of 20%, and a ring artifact reduction value of 5. The reconstructed images were analyzed for callus parameters using CTan software. The volume of interest (VOI) was manually chosen to include the entire callus. The region of interest (ROI) was determined using a CTan code to separate callus from cortical bone. Using CTan, the scanned images were analyzed for the following parameters: callus area (B.Ar) trabecular separation (Tb.Sp), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), mineralized bone volume (BV), tissue volume (TV), and bone volume / tissue volume (BV/TV) as previously described [2, 17]. A custom script was used in MATLAB R2023b to determine the tissue mineral density (TMD) of the ROI [18]. Representative 3D renderings were created using CTVox.

2.5. Biomechanical Torsion Testing

The second batch of femurs (described above) were placed at −20°C until the day of μCT scanning and biomechanics. Bones were thawed and remained hydrated in ice cold (4°C) 1X Dulbecco’s Phosphate Buffer Saline (PBS) with calcium and magnesium (HiMedia Laboratories, Nashik, India) for ~8 hours prior to scanning. To minimize repeated freeze-thaw cycles and preserve the structural integrity of the femora, biomechanical torsion analysis was performed immediately following μCT scanning.

After μCT scanning, the femurs were secured on both the proximal and distal sides within 9 mm bullet casings by potting the bones in a fast-curing orthodontic acrylic resin (Ortho-Jet^™, Lang Dental, Wheeling, Illinois). Femurs were placed vertically straight after the resin was poured into the bullet casing and bones were kept hydrated with PBS-soaked gauze during the approximately 20-minute curing period of the resin. Fractured femurs were potted such that the callus was visible above the resin and contralateral femurs were potted so that the mid-shaft was visible above the resin. A Mark-10 torque-testing system (Mark-10, Copiague, NY) with a Model M5i torque indicator was used to apply torsional stress at a rate of 1 degree per second until fracture occurred. Ultimate torque, stiffness, toughness, and twist at failure were determined from the resulting torque-twist curves. Ultimate torque was the maximum torque reached by the bone. Stiffness was determined by the slope of the linear growth region, as determined by averaging the maximum 15 slopes calculated before the ultimate torque. Toughness was defined as the total area under the torque-twist curve. Twist at failure was determined as the angle of displacement at the time of ultimate torque. All fractured femur values were normalized and expressed as a percentage of the corresponding value from the contralateral femur of the same animal [2].

2.6. Histomorphometry Analysis

Following the μCT analysis, the surgical femurs reserved for histological assessments were decalcified in Immunocal Decalcifier (StatLab Medical Products Inc., McKinney, Texas) at pH 7.2–7.4 for 48 hours. The samples were then embedded in paraffin and 5 µm thick longitudinal sections were prepared. The medial shaft of the paraffin-embedded femurs was sectioned and stained with picrosirius red and alcian blue to assess the trabecular bone and cartilage formation. Endomucin staining by immunohistochemistry was performed to assess angiogenesis [19]. The slides were imaged using a Leica DM2700M microscope, where brightfield and polarized images were observed. The histological images were analyzed using Osteomeasure software (Osteometrics Inc., Decatur, GA) to determine total callus area, percentage of bone, percentage of cartilage, total blood vessel area and total number of blood vessels [17] within the callus area.

2.7. Pain-Behavioral Analysis

Pain behavioral assessments were conducted on the mice to assess post-fracture pain outcomes. Pain behaviors can be influenced by experimental conditions; therefore, all experiments were performed by the same individual at approximately the same time of the day to ensure consistency and unbiased analysis. In addition, experiments were done in a quiet room to avoid triggering spontaneous fear behaviors. Each of the behavioral assessments are described in detail below.

2.7.1. Dynamic Weightbearing Assay

Dynamic weightbearing (DWB) measures the weight borne on each limb individually by a freely moving mouse. DWB was measured at baseline and at 4, 7, 14, and 21 dpf using Bioseb DWB 2.0 (Bioseb, Vitrolles, France). Each mouse was placed inside the chamber and recorded using the top-mounted camera for 5 minutes preceded by an initial acclimation period of 2 minutes. Data were analyzed by Bioseb DWB2 software and validated by an analyst blinded to the treatment groups to avoid bias [20]. Data are expressed as a percent of weight borne on the surgically fractured right hindlimb: ((R / (R + L)) × 100 [21].

2.7.2. Open Field Locomotion Assessment

Locomotion assessment was performed using open field testing at baseline and at 4, 7, 14, and 21 dpf, to assess the total distance traveled by a mouse. The mice were individually allowed to explore in a 30cm x 30cm x 35cm (wall height) activity cage for 8 minutes and video recordings were taken using GoPro Hero 8 (GoPro Inc, San Mateo, California) cameras. The video recordings were processed using DeepLabCut (Mathis Laboratories of Adaptive Intelligence, EPFL, Lausanne, Switzerland) [22] to extract positional data, resulting in tracked body part coordinates (x,y). Subsequent data were then filtered using a butterworth low pass filter to smooth trajectories and visualized through time-encoded line plots, as well as density heat maps. These allowed for quantification of total distance traveled.

2.7.3. Spontaneous Behavior Analysis

To assess spontaneous fracture nociceptive pain, researchers observed mice at baseline and at 4, 7, 14, and 21 dpf. Recording spontaneous behavior via video surveillance [20, 23] was used to assess pain. Mice were placed on a mesh wire grid and allowed to acclimatize for 30 minutes before starting the video recording using a GoPro HERO 8 camera. The cameras were mounted at 45 degrees to cover the entire floor area. During the video analysis, rapid lifting of the hindpaws was counted by an analyst blinded to the treatment groups to avoid bias.

2.7.4. Mouse Grimace Scoring (MGS)

MGS is a pain behavioral assessment that is conducted by observing facial expressions in a murine model. Five facial expressions that are considered potential indices of pain are orbital tightening, nose bulging, whisker positioning, cheek bulging, and ear positioning. To complete grimace scoring, mice were placed in a clear plastic enclosure and recorded for 5 minutes with a GoPro Hero 8 camera after a 30-minute acclimation. Videos were taken at baseline and subsequently at 4, 7, 14, and 21 dpf. At each time point, mice were scored on a scale of 0 to 2 for five different facial features with 0 being absent, 1 being moderately visible, and 2 being severe. All scoring was done by one analyst who was blinded to the treatment group. The mean of the 5 scores was calculated to give the MGS [24].

2.7.5. von Frey Mechanical Sensitivity

von Frey testing determines hypersensitivity to a mechanical stimulus, and in this case we evaluated the sensitivity of the plantar hindpaw [25]. The mice were placed in plexiglass dividers on a metal screen with circular holes 5 mm in diameter. The holes allow full access to the plantar aspect of the hindpaws. The testing was conducted prior to surgery (baseline) and weekly post-surgery at approximately the same time of the day. 30–45 minute of behavioral acclimation time was allowed to accommodate cage exploration and grooming. The mid-plantar left hindpaw was touched with 1 of a series of 8 standard von Frey filaments starting at 1.4gms. The von Frey filament was presented perpendicularly against the middle of the paw pad causing the filament to slightly bend and was held for approximately 5 seconds. The filament was presented several times at intervals of a few seconds, giving the mice some recovery time from the previous stimuli. Presentation of the stimuli was done in a manner that avoided startling the mouse to prevent instinctive fear behavior. A sharp withdrawal of the paw or immediate flinching upon touching the paw with the von Frey filament was noted as a positive response. The von Frey filaments were capped at 6 gms, as a stimulus higher than that lifts the hindpaw of the mouse. 50% threshold was calculated using Chaplan’s modeling with an updated algorithm to account for the lower thresholds for withdrawal observed in mice [26, 27].

2.8. Sample Size Calculations and Statistical Analyses

Statistical analyses were performed using GraphPad Prism (version 8.0, GraphPad Software, La Jolla, CA). The results are presented as mean±SEM. This study aimed to evaluate the effects of CTM treatments compared to Saline Control. Assessment of results were performed using one-way or two-way ANOVA analysis, with Holm Sidak post hoc analysis to provide an overall assessment of variance between groups wherever applicable. P-values < 0.05 were considered significant. The ROUT Outlier test was performed to determine and exclude outliers from the dataset.

Power calculations were used to determine sample sizes. We selected biomechanical properties as our primary outcome as it is the gold standard for bone healing. Based on the variance seen in previous mouse Einhorn fracture models (similar to our surgically induced fracture model) for both biomechanical and µCT parameters we found that for two-tailed comparisons 7 animals would be needed per group for an alpha significance of 0.05, and a power of 0.80 [28]. We also confirmed that this N would be sufficient to determine differences in radiographic bone healing outcomes, based on power analysis on previous rat bone healing studies with radiographic assessments [29]. Due to different experimental outcomes and the numbers of mice needed for each, and to account for animals that do not survive surgery or were excluded due to complications, we increased the numbers of mice recruited into the study. Supplemental Figure 1 shows the number of mice used for the study, the general experimental design, and the final number of specimens available for each outcome.

3. Results

3.1. Complete Blood Count Analysis Post-Fracture

As CTM xenografts are human tissue derived, we first assessed the potential for an immune response that could confound our analysis. Complete blood count (CBC) was performed and compared to reference ranges for white blood cells (WBC), lymphocytes, neutrophils, monocytes, and platelets. A minor increase in blood cell numbers was noted when compared to baseline as would be expected post-fracture. However, no statistically significant differences were observed at any time point among the treatment groups when compared to the Saline Control, as shown in Figure 2.

Figure 2: Complete blood count (CBC) analysis.

Saphenous blood was collected and analyzed on day 0 (baseline) and at the days indicated post-surgery. CBC was measured and compared to their reference ranges shown graphically as dashed lines. A) WBC (reference 0.8 – 10.6 ×10³ cells/μl); B) lymphocytes (reference 0.6–8.9 ×10³ cells/μl); C) neutrophils (reference 0.23 – 3.6 ×10³ cells/μl); D) monocytes (reference 0.04 – 1.4 ×10³ cells/μl); and E) platelets (reference 400 – 1600 ×10³ cells/μl). No statistically significant differences were observed among the treatment groups by Two-way ANOVA.

3.2. Radiographic and μCT Imaging Assessing Fracture Healing and Callus Formation

Radiographic imaging was performed to monitor the progression of healing. Figure 3A shows representative X-ray images from the different treatment groups 3 weeks post-fracture. μCT was also conducted after euthanasia to assess bone healing outcomes, and representative reconstructions are shown in Figure 3B. No significant differences in mRUST scores were observed between the Saline Control and CTM-treated groups, indicating that CTM treatments did not alter the time to union (Figure 3C). μCT assessment revealed a significant increase in callus area in the CTM Membrane + Paste group compared to the Saline Control (p=0.01) (Figure 3D). However, no significant changes were detected in BV/TV, TMD, Tb.Th, Tb.Sp, or Tb.N, in any of the treatment groups compared to Saline Control (Figures 3E–I).

Figure 3: Radiographic and µCT Imaging Analysis of Fracture Healing Progression in Murine Models.

A) Radiographic images depict fracture healing progression across treatment groups in the surgically induced femoral fracture model. B) Representative µCT three-dimensional reconstructions illustrate fracture callus formation from Saline Control and CTM-treated groups at 23 days post-fracture. C) mRUST analysis based on X-ray images for fracture healing progression/time to union. No significant differences were observed between the Saline Control and CTM-treated groups, as analyzed using a Two-way ANOVA. D-I) Quantitative µCT analysis of fracture healing parameters, including callus area, mineralized callus volume (BV/TV), tissue mineralization density (TMD), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). The CTM Membrane + Paste group exhibited a significant increase in callus area compared to the Saline Control (*p=0.01). No other significant differences were detected in the remaining parameters, as analyzed by One-way ANOVA with Holm-Sidak post hoc analysis.

3.3. Evaluating Fracture Strength Using Biomechanical Torsion Testing

Biomechanical testing provides a direct measure of bone strength and its ability to support movement, which makes it essential for evaluating the functional outcome of fracture healing. No significant differences among the groups were observed in any of the biomechanics parameters analyzed (ultimate torque, twist to failure, stiffness, toughness) (Figure 4A–D). A minor, non-significant increase in stiffness for the CTM Membrane group was observed compared to Saline Controls (Figure 4C). Toughness and twist to failure also showed non-significant increases for the CTM Membrane and CTM Membrane + Paste groups compared to Saline Controls (Figure 4B & 4D).

Figure 4: Biomechanical assessment of surgically induced femoral fractures 23 days post-fracture.

The above graphs describe biomechanical properties which are expressed as percentage of contralateral limb (%) on the Y axis while the treatment groups are specified on the X axis. A-D) Ultimate torque, twist to failure, torsional stiffness, and toughness for the surgically induced femoral fracture model. No significant differences were observed among the treatment groups, based on One-way ANOVA.

3.4. Histomorphometric Analysis of the Fracture Callus

Fractured femurs were sectioned and stained with alcian blue (cartilage) and picrosirius red (bone) to analyze several fracture callus parameters. Representative microscopic images are shown in Figure 5A. A significant increase in percent bone for CTM Membrane + Paste was observed (Figure 5B) compared to Saline Control (p=0.03). However, no statistical significance was observed in the callus area or percent cartilage (Figure 5C and D). We also stained callus sections with endomucin by immunohistochemistry to identify the blood vessels (Figure 5E). No statistical significance was observed for the total blood vessel area and number of blood vessels present in the callus area, among the CTM treated groups when compared to Saline Control (Figure 5F and G).

Figure 5: Histomorphometric analysis of surgically induced femur fractures 23 days post-surgery.

A) Femurs were sectioned longitudinally and stained for histomorphometric analysis. Alcian blue and picrosirius red staining of femurs for indicated groups. Black dotted lines demarcate the callus region. Scale bar: 1000 μm. B) CTM Membrane + Paste group exhibited significant increase in % bone compared to Saline Control (*p=0.03). C-D) Quantitation of callus area and % cartilage comparing all groups. E) Representative image of endomucin staining for blood vessels within the callus. Images are at 40X magnification. F) Quantification of total blood vessel area showed no significant differences between treatment groups. G) The number of blood vessels was not altered across groups. All statistical comparisons were performed using One-way ANOVA.

3.5. Longitudinal Study of Mouse Pain Behavioral Outcomes Post-Fracture

CTM xenografts were developed from human donor tissue using proprietary methods and are designed to contain a combination of growth factors. Hence, to investigate whether growth factors present in CTM products aggravate post-fracture pain in mice, we used multiple behavioral assessments to validate pain responses. DWB analysis demonstrated a significant and persistent decline in the percentage of weight borne on the fractured limb (Figure 6A) with approximately a 43% reduction compared to baseline consistent across all groups. Similarly, open field analysis revealed about a 34% decline in locomotory activity post-fracture with no significant group-wise variation observed (Figure 6B). This indicated that fracture caused a decline in weightbearing and locomotion in mice, with no statistically significant difference over time among the treatment groups compared to Saline Control. Similarly, spontaneous pain responses increased following fracture, with a peak at day 14 post-fracture and some resolution of pain responses at day 21; however, there were no differences observed between treatment groups (Figure 6C). MGS was increased in all treatment groups post-fracture and returned to baseline by day 21 post-fracture. We observed lower MGS for the CTM Membrane (p=0.002) and CTM Membrane + Paste (p=0.04) groups on day 4 post-fracture compared to Saline Controls (Figure 6D). Significantly lower MGS score not only indicated that CTM xenografts did not trigger additional pain response, but it also reduced pain behavior. This finding is particularly notable given the potential presence of growth factors such as CNTF and NGF in the CTM xenografts, which are traditionally associated with pro-nociceptive roles; yet CTM did not exacerbate pain and in at least one assessment, demonstrated a reduction in pain behavior. von Frey testing exhibited increased pain sensitivity in the ipsilateral limb post-fracture, while the contralateral limb remained unchanged. No significant differences were observed between treatment groups for the ipsilateral limb when compared to Saline Control, indicating no significant impact of CTM xenografts (Figure 6E).

Figure 6: Assessment of pain behavior outcomes during fracture healing.

A) Dynamic weightbearing analysis and B) locomotion analysis showed fracture-induced decreases from baseline, but no significant differences observed among CTM-treated groups and Saline Control. C) Spontaneous pain behavior analysis showed a fracture-induced increase from baseline with no significant differences among treatment groups at any time point. D) Mouse grimace score (MGS) increased post-fracture in all groups but returned to baseline by day 21. CTM Membrane (**^ap=0.002) and CTM Membrane + Paste (*bp=0.04) groups had significantly lower MGS compared to the Saline Controls on 4 days post-fracture. E) von Frey assessment indicated higher mechanical paw sensitivity in the ipsilateral limb (IL, solid lines) post-fracture, while the contralateral limb (CL, dashed lines) remained at baseline levels, with no significant differences among treatment groups observed when compared to Saline Control. Statistical analysis was performed using Two-way ANOVA with Holm-Sidak post hoc comparisons.

4. Discussion

Biological and medical implants are designed to mimic natural structures and are implanted in the human body to replace or support damaged tissues. Biological implants are now becoming a crucial part of clinical practice and are broadly classified as autografts or allografts [30]. CTM products are acellular implant allografts designed to be used for wound healing indications. These human donor tissue-derived products are already in use by orthopaedic surgeons, as some promising anecdotal results showing acceleration of bone healing have been observed (MAK personal communications). CTM products are proprietary formulations that have been reported to contain several osteoblast, angiogenesis and nerve stimulating factors such as PDGF, BMP-2, VEGF, FGF, and NGF, which make this biomaterial highly favorable for bone regeneration [14, 31]. A limitation of this study is that the composition of the CTM products is not available due to the proprietary manufacturing process. Therefore, it is not possible to determine the underlying mechanism impacting fracture healing or pain behaviors. In our studies we are using saline as a negative control due the number of different CTM delivery formulations and due to the proprietary nature of the CTM manufacturing process and unavailability of membrane and paste negative controls. For these studies, CTM products were used as xenografts in surgically induced femur fractures in mice.

Using xenografts in preclinical research offers the significant benefit of developing clinical treatment options; however, there are risks involved, such as the potential for graft rejection or adverse immune reactions. Typically, studies with human tissue-derived xenografts in mice have been completed using immunocompromised mice or mouse models with humanized immune components [32]. For these studies, we chose to use the CTM products in an immune competent C57BL/6J mouse model, as the immune system (i.e. macrophages, T cells) plays a significant role in bone healing [33] and the use of an immunocompromised mice would confound the interpretation of our findings. As observed in Figure 2, the introduction of CTM products at the time of surgery did not appear to result in any adverse reaction or cause alterations in white blood cells or platelets when compared to Saline Control group, suggesting that this acellular preparation did not elicit an innate or adaptive immune response. However, one limitation of this study is having one single terminal time point which limits our ability to observe the changes in the immune responses during the early stages of healing process. Earlier time points would have allowed better assessment of immune cell population within the local fracture microenvironment rather than relying completely on systemic assessments.

The surgically induced femoral fracture model involves creating a controlled fracture in the femur of a mouse and is widely used to study the natural healing process and the effects of various treatments on fracture repair [3]. The surgical fracture model is a simple model for fracture healing and heals on its own over time without any clinical intervention. Our experimental findings of mRUST scores indicate that there was no impact on the time course of healing for the CTM-treated groups when compared to the Saline Control group. However, findings from our µCT analysis and histomorphometry analyses demonstrate that the CTM Membrane + Paste group showed improvements in some fracture healing parameters such as a larger callus area when compared to the Saline Control group, as seen in Figure 3D, and a significantly higher percent bone was observed within the fracture callus compared to Saline Control (Figure 5B). The differences observed between histomorphometry and μCT outcomes may be attributed to the fact that histology assesses a single two-dimensional longitudinal plane of the callus, whereas μCT provides a three-dimensional assessment of the entire callus.

Angiogenesis plays a critical role in fracture healing, by ensuring the delivery of oxygen, nutrients and other essential components for callus formation and bone regeneration, so we also examined endomucin expression using immunohistochemical staining and subsequent analysis. Specifically, we evaluated total blood vessel area, and the number of blood vessels present. No significant differences among the blood vessel numbers or area were observed when compared to Saline Controls. In this study the femurs were demineralized using Immunocal to preserve antigenicity for immunostaining. While Immunocal is effective for immunohistochemistry, it is not compatible with tartrate-resistant acid phosphatase (TRAP) staining, which limits our ability to assess the presence of osteoclast.

With these histological and µCT findings, we predicted that the CTM Membrane + Paste group would have better biomechanical properties compared to the Saline group. Unexpectedly, the biomechanical torsion testing did not show any statistical significance among the groups, although there was a trending increase in toughness for the CTM Membrane + Paste group compared to that observed in the Saline Controls. This trend, even though not statistically significant, indicates there might be a potential benefit of using CTM treatment, that warrants further investigation. In this study the day 23 time point was chosen for the biomechanical torsion testing to differentiate between the impacts of treatment groups on bone healing. To more effectively evaluate the regenerative potential of CTM xenografts, it would be important to test their efficacy in a more severe bone healing model such as a critical sized defect model.

Fracture healing outcomes and pain trajectories are closely linked and, impaired fracture healing can cause long-term lingering pain. One of the biggest predictors of fracture-induced chronic pain is the intensity and duration of the pain experienced in the acute and subacute phase of fracture healing [20]; therefore, mechanisms or treatments that alter acute pain could modify the development of chronic pain. CTM xenografts are comprised of a proprietary blend of growth factors and cytokines, which are intended to promote wound and bone healing but may also contribute to the sensitization of sensory neurons, leading to pain [34–36]. Among the growth factors present in the CTM products, NGF poses the highest risk, as it is well known to cause increased pain hypersensitivity by increasing sensory neuronal terminal density at the fracture or injury site [37, 38] and by sensitizing sensory neurons to release more neurotransmitters [37]. On the flip side, an increase in NGF could drive sensory reinnervation of the fracture callus, which could promote angiogenesis [39–41], modulate the immune response within the fracture microenvironment [42], and shift the balance between osteoblast and osteoclast activity to drive bone formation [43, 44]. This led us to examine whether the presence of this combination of growth factors within CTM products, including NGF, may result in increased pain behaviors in mice undergoing fracture surgery and repair or whether we would see a protective effect of the CTM products.

In this study, five different types of pain behavior assessments were completed to comprehensively investigate spontaneous and evoked pain outcomes. Weightbearing is one of the most clinically relevant outcomes following fracture: the sooner full weightbearing is achieved, the sooner a patient can resume normal activities. Further, weightbearing has the added benefit of improving fracture healing via mechanical loading, stimulating osteogenesis and bone remodeling [45]. Fracture induces sudden pain and inflammation which causes a reduction in weightbearing. Our experimental outcomes validate this in the mouse, as we observed a significant fracture-induced reduction in weightbearing that did not recover during the time-course of this study (Figure 6A). Similar behavioral outcomes were seen in the case of locomotion assessment, where fracture significantly reduced locomotion, which also did not recover prior to the end of the experiment. No statistically significant differences were observed between treatment groups, suggests that CTM products do not alter ambulation deficiencies following fracture. Spontaneous pain induced by fracture, was evaluated by assessing videotapes of paw movements and facial grimace. Limb pain was assessed by counting the rapid lifting of hindpaw, a well-established nociceptive response [46]. As expected, we observed a significant increase in paw lifts following fracture compared to the baseline in spontaneous behavioral assessment, which did not return to baseline during this study. When facial grimace was evaluated, we observed a sharp increase in grimacing that peaked at the first instance of assessment (4 days post fracture). Day 4 post-fracture was intentionally selected for behavioral analysis, as it is a critical time point to assess the acute inflammation at its peak and early fracture healing process such as immune cell infiltration and active tissue remodeling. Additionally, day 4 allows the evaluation of pain without the interference of analgesic whose effect is up to approximately 72 hours from the time of drug (buprenorphine extended release, Ethica XR) administration during surgery. As far as we know, this is the first instance using the MGS to evaluate spontaneous pain behaviors following long bone fracture [47]. Aligning with reports of other injury models without neuropathy [48], we found that the facial grimace resolved by 21 days post injury. Using facial grimace as an endpoint [24], we observed a significant reduction in pain response at day 4 in both the CTM Membrane and CTM Membrane + Paste groups, when compared to Saline Controls. Previous studies have shown that implants containing growth factors, such as BMP-2 and VEGF, can promote early tissue regeneration and reduce local inflammation, which may explain the observed decreases in MGS scores [49]. We did not see corroboration between the two different assays for spontaneous pain. The grimace scores peaked earlier than hindpaw spontaneous pain, which might be a result of the hindpaw responses being a product of referred pain, which could develop later. In addition, the CTM effects were only observed in the grimace outcomes, suggesting that differential signaling pathways might mediate bone pain and the hindpaw pain behaviors associated with fracture, which has been demonstrated previously [50]. Finally, we assessed evoked hindpaw responses to mechanical stimulation, a commonly used methodology to study mechanical allodynia related to fracture pain [26]. Mechanical withdrawal thresholds for the contralateral hind paws did not deviate from baseline levels throughout the study, whereas ipsilateral hind paw scores decreased, indicating fracture-induced hindpaw allodynia (Figure 6E). No significant differences were observed among the treatment groups when compared to Saline Control, which is consistent with the observations that we made with the hindpaw spontaneous pain behaviors.

A limitation to our experimental design was that we did not introduce CTM products in the absence of fracture, so we cannot definitively state that these products are not nociceptive; however, CTM xenografts did not exacerbate overt pain behaviors induced by fracture. In addition, it is possible that the CTM membrane could have impacted callus formation. However, we do not have data to describe the fate per se of the CTM xenograft during our experiments. Although, at the time of euthanasia there was no evidence of residual CTM membrane. Conversely, the CTM products had some modest benefit to decrease spontaneous fracture pain behaviors, as indicated by the grimace scores. The notion that the CTM products had an effect so quickly following fracture (4d) may suggest an immune-modulatory effect; however, further research is necessary to confirm this potential mechanism and determine their long-term effects on pain reduction.

Overall, our data suggest that the use of CTM Membrane in conjunction with CTM Paste supports improvements in callus area and percent bone warranting further investigation. This study initially focused on the compatibility and utility of CTM xenografts in a murine model using male C57BL/6J mice to minimize variability. Incorporating mice from both sexes and mice from different age groups in future studies would offer a more comprehensive understanding of potential sex-based and age associated differences in bone healing and pain responses. Even though CTM treatments showed enhancement of some bone healing parameters in C57BL/6J mice, these results may not fully replicate human responses to xenografts/allografts or account for the complexity of human bone healing, as physiological and injury variations will play a role. However, a key limitation for this study remains, having one single terminal time point which restricts our ability to assess the early fracture healing milestones. Incorporating an earlier post-fracture time point in the future studies would strengthen our ability to understand how CTM treatments can influence early phases of fracture healing and pain modulation. An important next step would be to evaluate bone healing using CTM products in more complex bone healing models and/or large animal models, which could better inform clinical translation. Our data strongly support the continued investigation of CTM products for clinical applications, including the gold standard of randomized controlled trials in human patients for weight-bearing long bone fracture indications.

Highlights of the study:

• In this study we used human placental tissue-derived xenografts (Connective Tissue Matrix, CTM [CTM Biomedical]) to assess bone healing and pain behaviors in a murine femoral fracture model.

• CTM Membrane + Paste, two formulations of CTM xenografts, significantly increased callus area and percent bone at the fracture site compared to the saline control group.

• No significant differences were observed in systemic immune response measured by complete blood count.

• CTM treatment did not exacerbate post-fracture pain; in fact, mouse grimace scoring showed significant improvement at day 4 post-fracture indicating a potential analgesic effect, providing additional rationale for usage of CTM to mitigate pain associated with fracture.

• mRUST scores, endomucin staining, and biomechanical outcomes were similar across all groups, and did not show any significant differences.

• These findings suggest that CTM Membrane + Paste allows for equivalent or better fracture repair without adverse effects on pain or systemic immune response.