Systemic Cisplatin Does Not Affect the Bone Regeneration Process in a Critical Size Defect Murine Model

Abstract

Osteosarcoma (OS) is the most common primary malignant bone tumor, and the current standard of care for OS includes neoadjuvant chemotherapy, followed by an R0 surgical resection of the primary tumor, and then postsurgical adjuvant chemotherapy. Bone reconstruction following OS resection is particularly challenging due to the size of the bone voids and because patients are treated with adjuvant and neoadjuvant systemic chemotherapy, which theoretically could impact bone formation. We hypothesized that an osteogenic material could be used in order to induce bone regeneration when adjuvant or neoadjuvant chemotherapy is given. We utilized a biomimetic, biodegradable magnesium-doped hydroxyapatite/type I collagen composite material (MHA/Coll) to promote bone regeneration in the presence of systemic chemotherapy in a murine critical size defect model. We found that in the presence of neoadjuvant or adjuvant chemotherapy, MHA/Coll is able to enhance and increase bone formation in a murine critical size defect model (11.16 ± 2.55 or 13.80 ± 3.18 versus 8.70 ± 0.81 mm³) for pre-op cisplatin + MHA/Coll (p-value = 0.1639) and MHA/Coll + post-op cisplatin (p-value = 0.1538), respectively, at 12 weeks. These findings indicate that neoadjuvant and adjuvant chemotherapy will not affect the ability of a biomimetic scaffold to regenerate bone to repair bone voids in OS patients. This preliminary data demonstrates that bone regeneration can occur in the presence of chemotherapy, suggesting that there may not be a necessity to modify the current standard of care concerning neoadjuvant and adjuvant chemotherapy for the treatment of metastatic sites or micrometastases.

Introduction

Osteosarcoma (OS) is the most common primary malignant bone tumor, representing 56% of bone malignancies in patients less than 20 years old.¹⁻⁴ Current standard of care is 2–3 cycles of neoadjuvant chemotherapy, followed by an R0 surgical resection (specimen margins free of tumor) of the primary tumor.⁵⁻⁸ OS extremity resection usually includes limb salvage and reconstruction because it most frequently occurs in the metaphysis of long bones (60% in distal femur or proximal tibia/fibula).⁹ Allografts, metal endoprostheses, or a magnetic extendible implant (Stanmore)¹⁰⁻¹⁴ are commonly used to reconstruct bone voids.¹⁵⁻²³ Though bone allografts have been utilized since the early 1900s, their effectiveness and durability continue to be debated. Up to 15% of allografts fail, and the rate of positive outcomes has been reported to range from 29–45%.²¹⁻²³ Similarly, though prosthetic materials have been utilized in the clinic for years, they can cause incomplete and extended healing time, prolonged nonweight-bearing periods, and increased risk of fractures, infections, degenerative arthritis, and joint instability.^{21,22,24−30} The durability and lifespan of implants are critical features when dealing with pediatric patients affected by OS, who have not yet achieved their adult height; 40–80% of prosthetic implants lasts <10 years, therefore requiring implant replacement is required within the adolescent population.^11−13,31 Moreover, surgical resection in children leads to poor quality of life.^{1,7,10−13,22,24,25,32−34}

Following surgery, adjuvant chemotherapy is administered to complete a total of one year of chemotherapy. This regimen has a 5-year survival of 60–70%.⁵⁻⁸ The standard protocol for OS includes induction chemotherapy for 10 weeks prior to surgery and then 17 additional weeks of chemotherapy after surgery. The four most common chemotherapeutics utilized include methotrexate, doxorubicin, cisplatin, and ifosfamide.³⁵ Neoadjuvant and adjuvant chemotherapy is given to treat microscopic subclinical metastases that are present at the time of diagnosis. In addition, it has not been definitively established that neoadjuvant chemotherapy, resulting in a delay in surgical resection of the primary tumor, impacts outcomes or survival. However, neoadjuvant chemotherapy does allow for improved quality of obtaining negative surgical margins and allowing for less complex surgical excision.^36,37 Adjuvant chemotherapy can result in complications, such as myelosuppression, hypomagnesemia, and different types of cells involved in bone formation. Though there may be an effect on these types of cells, the effect of chemotherapy on these cells has been shown to be temporary, as chemotherapy has a less significant impact on bone-forming cells, such as osteoblasts and MSCs compared to osteoclasts.^34,38,39

MSCs have been studied for their ability to mitigate the side effects of chemotherapy; therefore, some studies suggest that MSCs can protect normal cells from the toxic effects of chemotherapy drugs.

Also, MSCs can differentiate into osteoblastic cells under appropriate conditions, and this capability makes them a potential source for bone regeneration and repair. Since osteoblastic cells play a crucial role in bone formation and repair, chemotherapy drugs can have toxic effects on these cells, leading to bone loss and impaired bone healing. MSC application in bone tissue engineering and regeneration can potentially counteract the bone loss caused by chemotherapy.

Research is ongoing to explore how MSCs can be utilized to protect and repair bone tissues damaged by chemotherapy. Scientists are investigating methods to enhance the osteogenic differentiation of MSCs, which could be beneficial for patients undergoing chemotherapy and experiencing bone-related complications.

Because chemotherapy is considered the standard of care regardless of the disease stage at clinical presentation, it is important to determine the effect of chemotherapy on mesenchymal stem cells (MSCs) and bone differentiation. Studies have demonstrated that MSCs have resistance to multiple chemotherapeutics^40,41 through the activation of STAT3 and IL-6^42,43 via the promotion of MDR-1 (multidrug resistance gene 1) expression and MRP (multidrug resistance-associated protein).⁴¹ Summed together, this suggests that the differentiation of MSCs into osteoblasts will be unaffected by chemotherapeutics.⁴⁴

Our lab previously developed a biomimetic magnesium-doped hydroxyapatite/type I collagen-based material (MHA/Coll) synthesized through a biologically inspired method, which recapitulates the bone biomineralization process.^45,46 The material resembles the main components of the bone’s extracellular matrix, such as type I collagen and hydroxyapatite. Type I collagen allows for structural and morphological control over growth of apatite crystals, resulting in a nano- and microscopic structure lattice similar to human trabecular bone.⁴⁵ This results in a true composite material, rather than a mix of apatite and collagen fibers, which gives the scaffold unique chemical, morphological, and mechanical cues, which is crucial to guide cell recruitment, migration, and differentiation. The hydroxyapatite is doped with magnesium, an ion naturally present within the natural apatite structure of young bone.^45,47 In addition, in young growing bone, magnesium is higher than in adults; therefore, the magnesium in MHA/Coll mimics the magnesium prevalence found in the growing bone. In vitro, MHA/Coll induces MSCs to differentiate into bone faster than osteogenic media under the two-dimensional (2D) condition. In addition, due to its composition, MHA/Coll recruits endogenous MSCs, allowing for bone regeneration.⁴⁸ Moreover, MHA/Coll is unique and superior to other osteogenic scaffolds because it does not rely on the release of growth factors or stimulating bioactive molecules, therefore decreasing the chance of tumor recurrence and undesired side effects. Our lab has successfully applied this material for regeneration of several bony defects due to traumatic injuries in different animal models, such as rats, rabbits, and sheep,^45,49,50 and has shown that MHA/Coll is able to accelerate osteogenesis and promote bone formation in ectopic and orthotopic sites.^45,49,50 This results in regenerated bone that matches the structural and histological features of native bone. Moreover, as early as 4 weeks after implantation, MHA/Coll was able to promote bone remodeling from trabecular to cortical bone, allowing regenerated bone to be less porous and more mechanically stable.⁵¹

However, our material has not been used to repair bone defects in the presence of systemic chemotherapy. As the current standard of care for OS includes neoadjuvant and adjuvant systemic chemotherapy,⁵² it is vital to determine if bone regeneration is equivalent if chemotherapy (neoadjuvant or adjuvant) is given compared to bone regeneration in a critical size defect without chemotherapy. This study will examine if our highly osteogenic material promotes bone regeneration in the presence of neoadjuvant or adjuvant chemotherapy, allowing for a bone volume and postoperative gait that are comparable to outcomes achieved without chemotherapy.

Methods

All methods described were carried out in accordance with protocols approved by the Houston Methodist Institutional Animal Care and Use Committee (IACUC) to ensure the rights and welfare of animals established by the Houston Methodist Research Institute with identification numbers IS00005186.

Porous Mg-Doped Type I Collagen/Hydroxyapatite (MHA/Coll) Membrane Fabrication

MHA/Coll-functionalized membrane was fabricated from bovine tendon extracted type I collagen using a freeze-drying method.⁵³ 200 g of type I collagen in acetic acid (5% w/v; Nitta Casings Inc., NJ) was dissolved in 1 L of deionized water at a final concentration of 10 mg/mL in an aqueous acetic buffer solution at pH 3.5. Briefly, 40 mM aqueous solution of H₃PO₄ was added to 100 g of the acetic collagen gel and dropped in a solution of Ca(OH)₂ (40 mM) and MgCl₂·6H₂O (2 mM) of deionized water. The material underwent cross-linking in an aqueous solution of 1,4-butanediol diglycidyl ether (BDDGE) (2.5 mM) at 4 °C for 24 h. After cross-linking, the slurry was washed once with distilled water.

Casting of MHA/Coll Membrane

After rinsing with water, the pH of the final slurry was adjusted by adding 50 mL of acetate buffer, rewashed, and then resuspended in 100 mL of H₂0. 500 μL of glacial acetic acid was added to bring the final pH to 4.5. Then, 70 mL of slurry was cast in a lid and placed under a tissue culture hood to dry for 72 h by solvent casting.

Scanning Electron Microscopy (SEM)

The morphology of the scaffold was characterized by scanning electron microscopy (SEM). Scaffolds were coated with 7 nm of Pt/Pl for scanning electron microscope (SEM; Nova NanoSEM 230, FEI, Hillsboro, OR, http://www.fei.com) examination.

Compression Testing

Scaffolds of 0.5 cm thickness were soaked in PBS and loaded onto the UniVert Mechanical Test System. A load cell of 10 N was calibrated and used to perform a compression test with a stretch magnitude of 35%, a stretch duration of 60 s, and a relaxation time of 60 s. A minimum of 3 technical replicates was performed and recorded for each condition.

Fourier Transform Infrared Spectroscopy (FTIR)

The samples were analyzed in transmission mode at resolution 4, 64 points, over the range of 500–4000 cm^–1 using a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com). The FTIR spectra were determined after background subtraction, baseline correction, and normalization on amide I within the range of 500–1800 cm^–1 and reported on the graph.

Mechanical Testing of MHA/Coll

The mechanical properties of MHA/Coll were collected by using the UniVert CellScale testing apparatus (CellScale Biomaterials Testing). Briefly, the system used two clamps to stretch a membrane sample to quantify the tensile force and displacement experienced by each sample. Areas of light and heavy mineral deposition were identified on a source membrane 92 mm × 77 mm (Figure 2A) in size. Six membrane samples of dimensions 40 mm × 5 mm were created from the source membrane. Three samples were created from areas of light mineralization and three samples were created from areas of heavy mineralization. Each sample was marked with a permanent marker for visual identification and then photographed for gross analysis. Prior to mechanical testing, each sample was submerged in 1× PBS (Thermo Fisher Scientific) for 5 min to mimic implantation conditions. The UniVert load cells were calibrated using test weights, and the clamps were aligned. Each sample was then inserted into the grip of the UniVert apparatus. The apparatus was programmed to stretch each membrane sample at 15% of the apparatus’s maximum tensile force for a duration of 150 s. These testing conditions were chosen to collect data on membrane failure conditions. Data on the tensile force and clamp displacement were collected at a sampling rate of 15 Hz. Three metrics were chosen to analyze the mechanical properties of the membrane samples. Ultimate tensile strength quantified the maximum stress the sample could withstand before complete failure. Young’s modulus, defined as the ratio of tensile stress to tensile strain, quantified the membrane samples’ resistance to deformation.⁵⁴ Maximum tension is defined as the tensile force recorded at the point of complete failure.

Figure 2.

MHA/Coll membrane mechanical testing. (A) Representative image of MHA/Coll membrane sample source. Lightly and heavily mineralized areas are labeled. Stress–strain curve for (B) a lightly mineralized sample and for (C) a heavily mineralized sample with failure points indicated. (D) Stress–strain curve displaying both lightly and heavily mineralized sample data. (E) Comparison of maximum tension, Young’s modulus, and ultimate tensile strength for lightly and heavily mineralized samples. Mechanical testing was performed using the CellScale UniVert apparatus. Values are depicted as mean ± SD. Statistical analysis by unpaired t-test with Welch’s correction; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fabrication of Intramedullary Pins and Assembly of the Intramedullary Pin and MHA/Coll Membrane

Pins were fabricated as previously described in the protocol established by Clough et al.⁵⁵ Briefly, 22 G stainless steel hypodermic tubing was cut to a length of 9 mm (shaft) and 19 G stainless steel hypodermic tubing was cut to a length of 3 mm (collar). A 3 mm collar was passed over the 9 mm shaft and secured via laser. The pin was sterilized with an autoclave on a dry cycle at 120 °C at 15 psi. The MHA/Coll membrane was sterilized under UV light for 24 h and then cut to a strip of 2 cm × 0.5 cm, dipped in gelatin (bovine), and wrapped around the 3 mm collar and left to dry overnight (Figure 3).

Figure 3.

Intramedullary pin: Images of (A) schematic representation of how intramedullary pin is inserted into murine femur following critical size defect surgery. (B) Intramedullary pin (bottom) and collar (top). (C) Final assembled intramedullary pin. (D) Intramedullary pin with the MHA/Coll membrane wrapped around the collar.

Critical Size Defect Surgical Technique

The critical size defect was performed in male C57BL/6 mice at 4 weeks of age (Charles River). Prior to surgery, the mice were given carprofen wafers (5 mg/kg PO) 3 days prior to surgery, and 30 min prior to surgery, mice received 0.1 mg/kg buprenorphine subcutaneously for pain control. A sanitized heating pad was placed and covered with disposable drapes, and a sterile field was set up with an induction chamber and a nose cone assembly. Mice were anesthetized using an induction chamber set to an output of 2 L/min oxygen and an isoflurane concentration of 2.5% (v/v). Fur from the left hind leg was removed using hair removal cream, sterilized with an ethanol prep pad, and sterile drapes were placed over the body except for the entire right hind limb.

The proximal and distal ends of the femur were located, and a 3–5 mm incision was made on the longitudinal axis parallel to the femur using a #15 scalpel. Using a lateral approach to the femur, the muscles and fascia were dissected along the intermuscular boundary until the femur was visible using a mayo scissor. Using a fine drill fitted with a rodent dental diamond disc (8 mm × 0.1 mm) (iM3), the femur was cut with the periosteal elevator placed underneath the femur in order to protect the tissue below. The second cut was made about 3–4 mm away from the first cut while holding the extremity of the diaphysis with forceps. The medullary cavities of the femur were carefully reamed using a 23 G needle. The intramedullary pin alone or the intramedullary pin with the membrane was placed within the proximal end of the femur first and then inserted into the distal end to re-establish the original length of the femur. The muscle and peripheral tissue were repositioned over the pin and closed with a continuous absorbable 5–0 suture. The skin was closed using a continuous 6–0 proline suture.

Postoperatively, 0.1 mg/kg buprenorphine was administered subcutaneously 8–12 h, 2 days, and 4 days after surgery. Carprofen wafers (5 mg/kg of PO) were given for 3 days after surgery. Daily postoperative monitoring was performed according to institutional policies. At postoperative day 7, sutures were removed.

Surgical Treatment Groups

The first surgical group compared mice with only the intramedullary pin (no MHA/Coll membrane) to mice with the intramedullary pin with the MHA/Coll membrane to determine the ability of MHA/Coll to regenerate bone in a critical size defect murine model. These mice were followed until 16 weeks postoperatively (Table 1).

Table 1. Experimental Group Organization.

procedure 1	recovery time	procedure 2	time point evaluation
critical size defect surgery MHA/Coll and pin			16 weeks end point, gait analysis μ-CT analysis, and quantification histology validation
critical size defect surgery, pin only			16 weeks end point gait analysis μ-CT analysis, and quantification histology validation
critical size defect surgery, pin only			4, 8,12 weeks, gait analysis, μ-CT analysis, and quantification histology validation
critical size defect surgery, pin only	10 days recovery	postsurgical cisplatin administration	4, 8,12 weeks, gait analysis, μ-CT analysis, and quantification histology validation
presurgical cisplatin administration	10 days recovery	critical size defect surgery, pin only	4, 8,12 weeks, gait analysis, μ-CT analysis, and quantification histology validation
critical size defect surgery MHA/Coll and pin			4, 8,12 weeks, gait analysis, μ-CT analysis, and quantification histology validation
critical size defect surgery MHA/Coll and pin	10 days recovery	postsurgical cisplatin administration	4, 8, 12 weeks, gait analysis, μ-CT analysis, and quantification histology validation
presurgical cisplatin administration	10 days recovery	critical size defect surgery, pin only	4, 8,12 weeks, gait analysis, μ-CT analysis, and quantification histology validation

The second surgical group compared mice in 3 different groups to determine if systemic chemotherapy had an effect on bone regeneration in a critical size defect. The three groups were (1) critical size defect surgery with the intramedullary pin and MHA/Coll membrane, (2) preoperative cisplatin (0.5 mg/kg IP twice per week for 4 doses), followed by 10 days recovery, and then critical size defect surgery with the intramedullary pin and MHA/Coll membrane (neoadjuvant chemotherapy), and (3) critical size defect surgery with the intramedullary pin and MHA/Coll membrane, 10 days of recovery postoperatively, and then postoperative cisplatin (1 mg/kg IP q48 h for 4 doses; adjuvant chemotherapy). These mice were followed for 4, 8, and 12 weeks postoperatively (Table 1). Each experimental group had a control group that had the intramedullary pin without MHA/Coll implanted during critical size defect surgery. Each experimental group and control group had 4 mice.

Bone Regeneration

CT scan (Hounsfield units) was used to assess the bone volume at 4, 8, and 12 weeks (or 16 weeks) postoperatively. Mice were euthanized at respective time points, and the left hind leg was isolated and placed in 10% neutral buffered formalin solution. Each specimen was scanned using a North Star Imaging (NSI) micro-CT machine (Rogers, MN) at 95 kV, 315 μA, 1 fps, and 1000 projections with a 20 μm focal spot and 0.2 mm copper filter. The micro-CT data were reconstructed with NSI software at approximately 23 μm and segmented using a 3D Slicer (Cambridge, MA).

Bone Histology

Following CT scan, the bone morphology was assessed and compared to the native bone in the contralateral femur (H&E). The following steps were followed to create slides.

Dehydration and Infiltration

Samples underwent dehydration and infiltration for about 6 weeks. During the dehydration process, samples were submerged in increasing concentrations of 100% alcohol to completely remove all water within the sample. Once fully dehydrated, they were immersed in a Technovit solution, sealed in a vacuum tight capsule, and stored at 4 °C. Each sample was left for a minimum of 24 h in each solution as follows: (1) 24 h periods in a 30/70 Technovit 7200 and 100% alcohol solution, (2) 24 h periods in 50/50 Technovit 7200 and 100% alcohol, (3) 24 h periods in a 70/30 Technovit 7200 and 100% alcohol solution, (4) 24 h in a 90/10 Technovit 7200 and 100% alcohol solution, (5) 24 h in a 100% Technovit 7200 solution, and (6) a final solution of Technovit 7200 and benzoyl peroxide for a maximum of 6 days.

Polymerization

Once the samples were fully infiltrated with Technovit 7200, they were polymerized within a block of plastic by exposing the sample to UV light. Specifically, the sample was placed in a light polymerization unit (LPU) and surrounded by a thin layer of water to cool the sample as it polymerized. The sample was polymerized for 5 h in white UV light and 10 h in blue UV light.

H&E Staining of Plastic Embedded Samples

Samples were sectioned at a thickness of 4 μm and placed in a 2% formic acid bath for 2 min, followed by a 50% alcohol solution for 5 min. Slides were exposed to heat and hematoxylin for 30 min, allowed to rest in Scott’s tap water for 8 min, and then rinsed with water. A 50/50 mix of Eosin Y and Phloxine B was pipetted over the sample and left on a hot plate for 15 min. The samples were dunked 30 times in a 50% alcohol solution that contained 3 drops of glacial acetic acid per 50 mL and then rinsed with water once.

Organ Histology

Organs were also collected, fixed using 10% natural buffer formalin, and embedded in paraffin. Samples were sectioned at a thickness of 4 μm, and hematoxylin and eosin (H&E) staining was performed. The slides were imaged using a Keyence BZ-X810 microscope.

Gait Analysis

Video footage of mouse gait was collected using the DigiGait Imaging System (Mouse Specifics, Inc.). Briefly, the system uses a high-speed ventral camera and LED backlit panels to create digital paw prints. Each mouse was introduced to the DigiGait apparatus and trained for gait analysis three times prior to recording gait (parameters: speed of 20 cm/s at an incline of 0°). Baseline gait metrics were obtained prior to surgery. Gait was obtained 4, 8, and 12 weeks postoperatively (or 16 weeks only) to determine the functional recovery following bone regeneration. Recorded gait videos were processed by using DigiGait Analysis software (Mouse Specifics, Inc.). Contrast parameters were determined from a set of still images, and digital paw prints were created based on this input. Manual corrections were made to plots of the paw area over time to remove erroneous step recordings. Of the metrics obtained, eight variables were chosen to measure the temporal, spatial, weight distribution, and limb loading aspects of gait. To assess overall gait, two composite metrics were used, gait symmetry and hind limb stance factor.⁵⁶ The temporal measurements included the stance duration and % stance; spatial measurements included the stride length and stride frequency. Peak paw area and hind limb stance width were used to determine the weight distribution. Minimum and maximum paw area rates of change were used to assess limb loading and unloading. Multiple different assessments of gait were analyzed in order to evaluate the weight-bearing status and mechanical load that the regenerated bone could bear compared with the contralateral native bone. Temporal and spatial measurements provide indirect data on the mechanical load and functional outcome of the regenerated bone.

Statistics

To obtain a power of 80% and p-value of 5%, 4 mice were required per group. Statistics were calculated with Prism GraphPad software. Statistics were performed using a two-way ANOVA followed by Tukey’s multiple comparison test. In all cases, an * indicates p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All data were presented as mean ± SD.

Results

MHA/Coll Membrane Characterization

After the synthesis, the morphology of the MHA/Coll membrane was characterized by scanning electron microscopy (SEM). The micrographs demonstrate a mineralized layer with an average pore size of 2000 μm that looked interconnected as shown at lower magnification (Figure 1A). TGA analysis was performed on the MHA/Coll membrane to determine the amount of the mineral phase that was nucleated on type I collagen. Figure 1B demonstrates that the mineral content of the MHA/Coll membrane was 50–57 wt %, which was comparable to human trabecular bone (53 wt %). The FTIR spectra (Figure 1C) showed the characteristic collagen peaks at amide I (1700–1600 cm^–1) and amide II (1600–1500 cm^–1), related to the stretching vibration of C=O bonds and to C–N stretching and N–H bending vibration, respectively. The sample contained C=O, C–N, and N–H bonds. The amide III region (approximately 1200–1300 cm^–1) is related to the C–N and C–C stretching, N–H bonds, and CH₂ wagging from the glycine backbone and proline side chain. In addition, the peak at 900–1000 cm^–1 demonstrates that the collagen was mineralized.

Figure 1.

MHA/Coll membrane characterization: (A) Representative SEM micrographs of the MHA/Coll membrane at 5×, 10,000×, and 50,000×, (B) TGA analysis on the mineralized collagen scaffold and membrane with a heating ramp of 10 °C/min. Temperature ramp from 25 to 1100 °C, (C) attenuated total reflection (ATR)-FTIR (Fourier transform infrared) spectroscopy to record the absorption IR spectrum of the MHA/Coll scaffold and membrane. Infrared spectra were recorded in the range of 2000–500 cm^–1. 64 scans were performed with a resolution of 4 (H₂O and CO₂ correction applied). Samples are normalized on amide I peak. Scale bar = 20, 10, 2 μm.

MHA/Coll Mechanical Testing

When the MHA/Coll membrane was fabricated by solvent casting, various areas of highly or lightly mineralized areas were observed (Figure 2A). To determine if there was a difference in the mechanical properties of the light and heavily mineralized regions of the MHA/Coll membrane, mechanical testing was performed. In both the lightly mineralized and heavily mineralized sample groups, stress increased roughly linearly with strain over the initial elastic region. Then, the samples entered the plastic region and began to deform. After reaching ultimate tensile strength, the samples partially failed and then completely failed (Figure 2B,C). Failure generally occurred at a higher stress in the heavily mineralized sample group rather than in the lightly mineralized sample group (Figure 2D). Moistening the samples prior to mechanical testing led to more gradual and more ductile material failures. Despite the heavily mineralized samples failing at higher stress values, no significant difference in maximum tension was observed between the heavily and lightly mineralized samples (Figure 2E, p-value = 0.31). Additionally, the ultimate tensile strength was not found to differ significantly between the two sample groups (Figure 2E, p-value = 0.29). Young’s modulus, however, was significantly larger in the heavily mineralized sample group (Figure 2E, p-value = 0.043), indicating that the more heavily mineralized samples were significantly stiffer and better able to resist linear deformation for a given stress when compared to the lightly mineralized samples. Therefore, increased mineralization on MHA/Coll produces a less elastic membrane sample. The intramedullary pin was custom-made to create controlled, uniform critical size defects in our experimental model. Moreover, in our surgical approach, the intermedullary pin has been employed to provide structural support and stability of the implant in the mice femur (Figure 3). The pin is a thin, elongated cylindrical structure resembling a small rod. It has a smooth surface to minimize tissue irritation and facilitates insertion into the medullary canal of the long bone. The pin is made of stainless steel, hollow in the middle (to allow for movement of MSCs between the proximal and distal ends of the femur), has an internal diameter of 19 G, and is 9 mm long. The membrane was cut in 0.5 cm × 3 cm sections, wrapped around the intramedullary pin, and adhered to the pin with gelatin (Figure 3).

Cisplatin Treatment

Cisplatin was utilized as systemic chemotherapy, given either as a neoadjuvant or adjuvant therapy. First, we determined the tolerated dose and toxicity of cisplatin in mice, comparing intravenous (IV) tail and intraperitoneal (IP) injections. Mice were injected with decreasing doses of cisplatin (2, 1, and 0.5 mg/kg). Mice were initially injected with 2 mg/kg cisplatin IV q24 h for a total of 4 doses. Major side effects were observed after the fourth dose, including >20% weight loss. We then compared IP injections of various cisplatin doses (2 mg/kg, 1 mg/kg) every 48 h for a total of 4 doses. As shown in Supporting Figure 1, 1 mg/kg IP q48 h for 4 doses was tolerated in 50% of the mice. Therefore, to achieve survival, the dose was lowered to 0.5 mg/kg IP twice a week for 4 doses.

Regenerated Bone

To determine if the MHA/Coll membrane had an effect on bone generation, we implanted a pin with and without the membrane in mice femur (Table 1, green) and observed bone regeneration at 16 weeks after surgery. Mice with the MHA/Coll membrane implanted showed significantly greater regenerated bone compared to mice with only the intramedullary pin, as demonstrated by three-dimensional (3D) reconstruction (Figure 4A,C, 6.97 ± 1.72 versus 4.36 ± 0.98 mm³ for MHA/Coll and pin only, respectively, p-value = 0.0317, Figure 4E) and by CT (Figure 4B,D, regenerated bone = red arrow, intramedullary pin and collar = white arrow). We then checked bone regeneration over time by comparing (1) the intramedullary pin alone, (2) the intramedullary pin with the membrane without chemotherapy, and (3) the intramedullary pin with the membrane with either neoadjuvant cisplatin or adjuvant cisplatin. Animals were euthanized at 4, 8, and 12 weeks after implantation and new bone formation was observed by CT and 3D reconstruction, as shown in Figure 5A,B and Supporting Figure 2A,B images. When quantifying the volume of new bone formation (Figure 6A), significantly more bone was regenerated at 8 weeks (3.79 vs 10.20, p-value < 0.01) and 12 weeks (3.11 vs 8.70, p-value < 0.01) among mice that had the intramedullary pin and MHA/Coll membrane implanted compared to the intramedullary pin alone.

Figure 4.

3D reconstruction, CT of bone regeneration, and bone quantification induced by the MHA/Coll membrane: (A, C) Representative 3D reconstruction (intramedullary pin in gray, new regenerated bone in taupe) of bone regeneration of the intramedullary pin group (A) and (C) MHA/Coll membrane with the intramedullary pin at 16 weeks postoperatively. (B, D) Representative CT scan of the intramedullary pin control group (B) and (D) intramedullary pin with the MHA/Coll membrane (regenerated bone = red arrow, intramedullary pin and collar = white arrow) at 16 weeks postoperatively. (E) New bone growth volume average in the MHA/Coll membrane with intramedullary pin versus intramedullary pin p at 16 weeks (p-value = 0.0317). Values are depicted as mean ± SD. Statistical analysis by unpaired t-test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.

3D reconstruction and CT of bone regeneration induced by the MHA/Coll membrane with preoperative or postoperative cisplatin. Representative 3D reconstruction (gray = intramedullary pin, newly regenerated bone in taupe) and representative CT scan (regenerated bone = red arrow, intramedullary pin and collar = white arrow): (A) MHA/Coll and (B) pre-op cisplatin and (C) post-op cisplatin at 4, 8, and 12 weeks postoperatively.

Figure 6.

Bone quantification induced by the MHA/Coll membrane with neoadjuvant vs adjuvant cisplatin: (A) New bone growth volume average in the MHA/Coll membrane with the intramedullary pin versus intramedullary pin control group at 4, 8, and 12 weeks postoperatively. (B) New bone growth volume average in mice given pre-op cisplatin induced by the MHA/Coll membrane with the intramedullary pin group versus intramedullary pin control group at 4, 8, and 12 weeks postoperatively. (C) New bone growth volume average in mice given post-op cisplatin induced by the MHA/Coll membrane with the intramedullary pin group versus intramedullary pin control group at 4, 8, and 12 weeks postoperatively. (D) Values are reported as mean ± SD (n = 5). Statistical analysis by unpaired t-test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To further investigate the effect of systemic chemotherapy on bone regeneration, we compared mice to whom cisplatin was given before (neoadjuvant) or after (adjuvant) surgery (Table 1, yellow). When mice were given adjuvant cisplatin, MHA/Coll induced a greater volume of bone compared to the control mice (no treatment) 8 weeks after surgery, as shown in CT and 3D reconstruction images in Figure 5A,B. The same effect was observed when mice were given neoadjuvant cisplatin; MHA/Coll induced a greater volume of bone compared to the control mice 8 weeks after surgery, as shown in CT and 3D reconstruction images in Figure 5A–C. In particular, at 8 weeks postoperatively, the average bone volume was 13.82 ± 1.67 versus 10.2 ± 0.68 mm³ for MHA/Coll + post-op cisplatin and MHA/Coll, respectively (Figure 6C,D, p-value = 0.0098, Table 1). Similarly, at 8 weeks postoperatively, the average bone volume was 17.26 ± 1.98 versus 10.2 ± 0.68 mm³ for pre-op cisplatin + MHA/Coll and MHA/Coll, respectively (Figure 6B–D, p-value = 0.0292, Table). A similar effect was seen at 12 weeks, though not statistically significant (Figure 7A,B): 11.16 ± 2.55 or 13.80 ± 3.18 versus 8.70 ± 0.81 mm³ for pre-op cisplatin + MHA/Coll, MHA/Coll + post-op cisplatin, and MHA/Coll, respectively (Figure 6B–D, p-value = 0.1639 and 0.1538 for pre-op cisplatin and MHA/Coll and MHA/Coll and post-op cisplatin, respectively).

Figure 7.

Pathology at 12 weeks in representative mouse with MHA/Coll implanted: (A) H&E stain of axial section, (B) magnification of the new bone formation with a circumferential zone of osseous proliferation (O*) around the implant and fibrovascular connective tissue (F*), (C) H&E stain of longitudinal section, and (D) magnification of the new bone formation with a circumferential zone of osseous proliferation (O*) around the implant and fibrovascular connective tissue (F*)

To further validate the effect of systemic chemotherapy on bone regeneration, we used control mice that were treated with neoadjuvant or adjuvant cisplatin in the presence of only an intramedullary pin (no MHA/Coll membrane). Mice from all 3 groups (pin alone, pin with neoadjuvant cisplatin, pin with adjuvant cisplatin) had similar regenerated bone volume at 4 weeks (6.85 ± 2.11, 7.47 ± 1.73, 10.61 ± 1.8 mm³), 8 weeks (3.79 ± 0.53, 1.95 ± 0.78, 6.89 ± 0.55 mm³), and 12 weeks (3.11 ± 0.96, 2.88 ± 1.39, and 2.64 ± 0.39 mm³) for intramedullary pin with no cisplatin, pre-op cisplatin + intramedullary pin, and intramedullary pin + post-op cisplatin, respectively (Supporting Figure 3). However, there was only a statistical difference at 8 weeks (Supporting Figure 3, p-value 0.05: pin vs pre-op CIS, p-value < 0.001: pin vs post-op CIS).

When the new bone volume induced by the MHA/Coll membrane was compared, if mice were given neoadjuvant or adjuvant cisplatin, MHA/Coll induced greater bone formation. At 8 and 12 weeks postoperatively, mice with the MHA/Coll membrane implanted had significantly more bone volume compared to mice with only the intramedullary pin (Figure 6A, p-value < 0.0001). At 4, 8, and 12 weeks postoperatively, mice given neoadjuvant (pre-op) cisplatin and the MHA/Coll membrane implanted had significantly more bone volume compared to mice with only the intramedullary pin (Figure 6B, p-value < 0.0001). A similar effect was seen in mice given adjuvant (post-op) cisplatin at 8 and 12 weeks; mice given post-op (adjuvant) cisplatin and the MHA/Coll membrane implanted had significantly more bone volume compared to mice with only the intramedullary pin (Figure 6C, p-value < 0.0001).

Bone Formation Evaluation via Histology

A partially dissected mouse hind limb was processed for histology (Figure 7). Figure 7 illustrates H&E stains revealing a small quantity of shapeless to mildly granular debris, accompanied by sporadic, minimal clusters of diverse inflammatory cells (Figure 7A,B). In a cross-sectional cut, fibrovascular connective tissue that extends to a circumferential zone of osseous proliferation is seen (Figure 7C). In the longitudinal section, the osseous cortex is viewed (Figure 7D); no inflammation, bone remodeling, or fibroplasia was observed in any transection. At 4, 8, and 12 weeks, histological analyses of the explanted femur demonstrated regions of new cortical bone from the existing cortical bone, as well as fibrovascular connective tissue within the woven bone (Figure 8). Finally, H&E stains revealed a normal organ tissue architecture with no significant inflammation or inflammatory cell infiltration between the three experimental groups (MHA/Coll, pre-op CIS + MHA/Coll, and MHA/Coll + post-op CIS) in the heart, lung, liver, kidney, or spleen (Supporting Figure 4).

Figure 8.

Histology: Transverse and longitudinal cut at 4, 8, and 12 weeks demonstrating new bone growth from the existing cortical bone and fibrovascular connective tissue within the woven bone.

Gait Analysis

No significant differences were observed between the MHA/Coll group, the Pin Ctrl group (pin implant without an MHA/Coll membrane), or preoperative values for any of the following metrics: gait symmetry index (Figure 9A, MHA/Coll p-value = 0.4175, Pin Ctrl p-value = 0.4126), and stride length and stride frequency (Supporting Figure 5). Composite metrics for treatment groups generally did not change significantly from the preoperative baseline. The minimal difference of the gait symmetry index observed at 4 and 8 weeks (Figure 10A,B) was not further noticeable by 12 weeks postoperatively. No significant difference from the preoperative gait symmetry index had developed in any treatment group (Figure 10C, pre-op cisplatin + MHA/Coll p-value = 0.7071, post-op cisplatin + MHA/Coll p-value = 0.9883, MHA/Coll p-value = 0.4175). At 8 weeks, the hind limb stance factor was significantly lower than the baseline for the pre-op cisplatin + MHA/Coll mice (Figure 10B, p-value = 0.0054), and at 12 weeks, the hind limb stance factor was significantly below the preoperative value for the MHA/Coll group (Figure 10C, p-value = 0.0149) as well as the pre-op cisplatin + MHA/Coll mice (Figure 10C, p-value = 0.0334).

Figure 9.

Comparison of composite gait metrics in MHA/Coll vs pin at 16 weeks. (A) Gait symmetry and (B) hind limb stance factor. Gait was recorded using the Digigait Imaging System and analyzed in a Digigait Analyzer. Gait symmetry was calculated as the ratio of right step frequency to left step frequency. Stance factor was calculated as the ratio of right hind limb stance duration to left hind limb stance duration. No significant differences in gait symmetry or stance factor were observed between the MHA/Coll and pin control groups, and only the difference between the Memb Ctrl hind limb stance factor and the preoperative value was significant. Values are depicted as mean ± SD. Statistical analysis by unpaired t-test with Welch’s correction; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. LH, left hind limb; RH, right hind limb. N = 5 mice per group.

Figure 10.

Postoperative composite gait metrics of treated mice. (A) Gait symmetry and hind limb stance factor at 4 weeks (A), at 8 weeks (B), and at 12 weeks (C). Gait was recorded using the Digigait Imaging System and analyzed in a Digigait Analyzer. Gait symmetry was calculated as the ratio of right step frequency to left step frequency. Stance factor was calculated as the ratio of right hind limb stance duration to left hind limb stance duration. Values are depicted as mean ± SD. Treatment groups were compared to the baseline and the MHA/Coll membrane control group. Statistical analysis by unpaired t-test with Welch’s correction; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. LH, left hind limb; RH, right hind limb. N = 5 mice per group.

In comparing the baseline gait to the MHA/Coll group and the Pin Ctrl, the most significant differences were observed in the paw area statistics. At 16 weeks postoperatively, the minimum and maximum paw area rate of change was significantly more negative in the MHA/Coll group (Supporting Figure 6AB, p < 0.05) and Pin Ctrl group (Supporting Figure 6B, p-value < 0.05) compared to the mice prior to surgery. Additionally, the RH peak paw area was significantly larger than the LH peak paw area for the MHA/Coll group (Supporting Figure 6C, p-value < 0.01).

The critical size defect surgery significantly affected temporal aspects of gait in the mice that received cisplatin prior to surgery (pre-op cisplatin + MHA/Coll), received cisplatin after surgery (post-op cisplatin + MHA/Coll), and did not receive chemotherapy (MHA/Coll). At 4 weeks postoperatively, a significantly lower % stance was seen in the pre-op cisplatin + MHA/Coll (Supporting Figure 7A, p-value < 0.01) compared to the mice prior to surgery. At 12 weeks, % stance was significantly lower than the preoperative % stance for the pre-op cisplatin + MHA/Coll (Supporting Figure 7C, p-value < 0.01) and post-op cisplatin + MHA/Coll (Supporting Figure 7C, p-value < 0.05) treatment groups. Hind limb loading and unloading were also significantly impacted, with unloading impacted more than loading. At 4 weeks, the right hind (RH) minimum paw area rate of change was significantly more negative than the baseline value for the pre-op cisplatin + MHA/Coll mice (Supporting Figure 8A, p-value < 0.05) and the MHA/Coll mice (Supporting Figure 8A, p-value < 0.01); the decrease in the paw rate of change corresponds to a more rapid unloading of weight from the limb. At 8 weeks, the pre-op cisplatin had a significantly larger RH maximum paw area rate of change (Supporting Figure 8B, p-value < 0.01), corresponding to a more rapid loading of the limb. At 12 weeks, the RH minimum paw area rate of change was significantly more negative than the baseline value for the MHA/Coll mice (Supporting Figure 8C, p-value < 0.05), post-op cisplatin + MHA/Coll group (Supporting Figure 8C, p-value < 0.01), and pre-op cisplatin + MHA/Coll groups (Supporting Figure 8C, p-value < 0.05). Weight distribution was only marginally affected in all treatment groups. At 4, 8, and 12 weeks, the left hind (LH) paw area was lower than the RH paw area across all treatment groups, but this difference was only significant for the MHA/Coll mice at 12 weeks (Supporting Figure 8C, p-value < 0.01). Spatial measures of gait were slightly affected as well. Although LH and RH stride lengths both decreased relative to the preoperative values in all treatment groups, the difference was only statistically significant for the pre-op cisplatin + MHA/Coll group at 4 weeks (Supporting Figure 8A, p-value < 0.01) and the post-op cisplatin + MHA/Coll group at 8 weeks (Supporting Figure 8B, p-value < 0.01).

Discussion

Although OS can initiate in nearly any bone within the human skeleton, the metaphyseal growth plate in long bones is the most common site. Forty-two percent of OS occurs in the distal femur, 19% in the proximal tibia, and 10% in the proximal humerus.⁵⁷⁻⁵⁹ During the development of OS, a ball-like mass develops in a radial pattern that eventually penetrates the cortical bone, and as growth continues, it compresses surrounding muscles.⁶⁰⁻⁶² During surgical resection, the primary goal is to achieve the complete removal of the tumor with wide excisions. There are two main surgeries to accomplish this: limb salvage surgery and amputation.^63,64 Limb salvage surgery often involves the use of prosthetics, but prosthetic materials cause incomplete and extended time to heal, prolonged non-weight-bearing periods, and increased risk of fractures, infections, degenerative arthritis, and joint instability.^{11−13,21,22,24−31} Therefore, the inadequacy of current therapies urges the development of novel treatment options for regeneration of bone loss to repair bone voids. Regenerative strategies for bone regeneration after OS resection have been explored to some extent, but there are several reasons that they may not be widely implemented or fully established as standard practice. OS is a complex and aggressive form of bone cancer that often requires a multimodal treatment approach. The primary treatment for OS involves surgical resection of the tumor followed by chemotherapy. The priority in these cases is to completely remove the cancer and prevent its spread, which may take precedence over immediate regenerative strategies. In addition, OS has a relatively high recurrence rate, particularly in the vicinity of the original tumor site. This can make it challenging to implement regenerative strategies, as there is a risk that the regenerated tissue could potentially become a site for cancer recurrence.⁶⁵ Therefore, extensive research and caution are necessary to ensure that regenerative approaches are safe and do not promote cancer recurrence.

Finally, the surgical resection of OS requires specialized skills and expertise.⁶⁶ Implementing regenerative strategies add complexity to the surgical procedure, requiring the availability of experienced surgeons and dedicated facilities equipped with regenerative techniques. The limited availability of such resources may contribute to the slower adoption of regenerative strategies. Although these limitations are discussed in the current literature, we explored the hypothesis to develop a material that can facilitate bone regeneration after OS resection. This study is the first step toward exploring the ability to regenerate bone using regenerative tissue strategies while also assessing if a biomimetic osteogenic material retains its osteogenic properties with chemotherapy administration, specifically cisplatin.

Cisplatin is commonly used as a chemotherapy drug in the treatment of OS. It is part of a multidrug chemotherapy regimen known as MAP (methotrexate, doxorubicin, and cisplatin), which is considered the standard of care for OS first intervention.⁶⁷ Though bone is innately one of the most regenerative tissues due to its rich population of MSCs in niches, such as bone marrow and the periosteum, certain conditions involving large bone defects, such as trauma or OS resection, compromise these niches, making it difficult for the bone to repair on its own.⁶⁸⁻⁷⁰ Due to this limitation, we previously developed a biomimetic scaffold (MHA/Coll) that recapitulates the bone biomineralization process.^45,46 In vivo, we have demonstrated that MHA/Coll is able to accelerate osteogenesis in ectopic and orthotopic sites.^49,50,71 However, our material has not yet been used to repair bone defects in the presence of chemotherapy. Chemotherapy drugs exert both direct and indirect impacts on bone regeneration. While chemotherapeutics primarily target rapidly dividing cancer cells, these drugs can also affect the cells responsible for bone regeneration (i.e., MSCs, osteoblasts, etc). Consequently, chemotherapeutics could potentially suppress bone cell proliferation and differentiation, interfering with the natural processes of bone healing and regeneration. By disrupting the delicate balance of bone remodeling (which involves both bone resorption and formation), chemotherapy drugs could potentially contribute to reduced bone density and a compromised healing capacity. Furthermore, chemotherapy treatments can occasionally result in delayed wound healing, thereby prolonging the recovery period and affecting the expected timeline for bone regeneration following surgical procedures.

In this study, we were able to demonstrate that MHA/Coll induces significantly more bone regeneration following the implantation of MHA/Coll when mice are exposed to neoadjuvant or adjuvant cisplatin compared to mice that did not receive chemotherapy. Moreover, when comparing mice with the intramedullary pin versus intramedullary pin + MHA/Coll in all chemotherapy arms (no cisplatin, neoadjuvant cisplatin, adjuvant cisplatin), all mice with the MHA/Coll implanted had increased bone volume at 8 and 12 weeks postoperatively (Figure 6). In mice having only an intramedullary pin implanted in the critical size defect surgery, we were still able to see an increase in bone volume at 4, and 8 weeks postoperatively. At 8 weeks, mice given adjuvant or neoadjuvant cisplatin had a higher bone volume compared to mice given no chemotherapy (Table 1, Supporting Figure 4). At 12 weeks, mice in all 3 groups had a similar regenerated bone volume for intramedullary pin with no cisplatin, pre-op cisplatin + intramedullary pin, and intramedullary pin + post-op cisplatin groups (Table 1, Supporting Figure 4). However, the decrease in statistical significance could be due to the remodeling process as trabecular bone becomes remodeled into cortical bone. Overall, the increase in bone regenerated in the control mice when cisplatin is given provided additional evidence that bone formation induced by MHA/Coll is indeed enhanced by cisplatin. Future experiments should evaluate if oncologic safety remains in vivo and if the effect of systemic chemotherapy on induced bone regeneration by MHA/Coll is affected in an environment that previously housed OS. Because OS will recruit a variety of immune cells, our results suggest that the effect of bone regeneration will be further enhanced.

Our study has some limitations due to the murine model. The size of the murine femur allowed us to only be able to assess the total bone volume, rather than the volume of cortical versus trabecular bone at 4, 8, 12, and 16 weeks postoperatively. In the MHA/Coll control group, it is evident that remodeling takes place at the site of MHA/Coll implantation because the standard deviation decreases from 4 weeks to 12 weeks. The decrease in variability occurs because the bone becomes more compact and homogeneous at 12 weeks compared to 4 weeks postoperatively. Moreover, bone formation does not follow a linear increase over time; at 12 weeks, we showed a higher proportion of cortical versus trabecular bone compared to the ratio observed at 4 weeks. This provides additional evidence that bone modeling occurred postoperatively.

To evaluate the return to function, postoperative gait was analyzed and compared to preoperative gait measurements. Overall, gait results were characterized by high standard deviations, which were likely due to differential wound healing, mouse size, and adaptation to injury. Furthermore, the low numbers of mice in the treatment groups and the unsuitability of some videos for gait analysis reduced the sample sizes, contributing to large variances. Though there were some differences in gait postoperatively, we believe that these differences were due to the remodeling process that was occurring. In addition, differences in gait were not consistent at various time points when stratified by the treatment group. We believe that if the gait was evaluated at a longer time point when the remodeling process will be completed, such differences would not be observed. However, gait results demonstrate that although mice did not bear as much weight on the limb that had MHA/Coll implanted, bone regeneration still occurred, even in the absence of ideal mechanical stimulation. Multiple studies have demonstrated that mechanical stimulation and load bearing are necessary for bone regeneration.⁷²⁻⁷⁷ However, MHA/Coll was able to induce bone formation even without complete or nonideal load bearing of the affected limb.

Overall, the findings in this study provide important data that biomaterials such as MHA/Coll can be utilized to repair bone voids in patients given chemotherapy. This study provides additional evidence that rather than using the current standard of care, amputation or conventional implants,⁷⁰ a biomimetic material could be used to repair bone voids. Importantly, because systemic chemotherapy is considered standard of care, it is important to note that neither neoadjuvant or adjuvant systemic cisplatin does not negatively affect bone regeneration but in fact enhances bone regeneration. We were able to demonstrate this effect even without the use of a biomimetic osteogenic scaffold. We believe that the use of a biomimetic scaffold is essential for bone formation in the setting of chemotherapy because the scaffold compositions create an ideal environment to support the differentiation and formation of new bone. Magnesium, hydroxyapatite, and collagen type 1 are the main components of the bone. Magnesium is an essential mineral that plays a significant role in bone health. It is a cofactor for many enzymes involved in bone formation and can also enhance osteoblast activity, promoting bone formation. Hydroxyapatite is a natural mineral form of calcium apatite, the main component of bones. It provides structural support and promotes osteoconduction. Incorporating hydroxyapatite into the composition membrane can enhance bone regeneration and improve the integration of the membrane with surrounding bone tissue. Type I collagen is specifically found in bones and provides structural support to bone tissue. When used in a composition membrane, collagen can mimic the natural environment of bone tissue, promoting cell adhesion, proliferation, and differentiation. It can also facilitate the recruitment of osteoprogenitor cells to the site of bone repair, supporting the osteogenic process.

While cisplatin treatment negatively affects bone cells, using a composition membrane containing magnesium, hydroxyapatite, and collagen type 1 can create a conducive environment for osteogenesis. By providing the necessary structural support, promoting osteoblast activity, and enhancing bone regeneration, this membrane can potentially offset the adverse effects of cisplatin on bone health.

We hypothesize that chemotherapeutics such as cisplatin activate the immune system, releasing various cytokines that prime MSCs, macrophages, and T -cells, creating an environment that supports tissue regeneration, leading to increased bone formation. Moreover, this finding suggests that MHA/Coll could potentially be modified to release chemotherapeutics over an extended period of time during the bone regeneration and remodeling process in order to further prevent recurrence and micrometastases after OS resection. In addition, higher levels of chemotherapeutics at the site of bone repair could help to further enhance and accelerate bone formation at the defect. This has the potential to greatly improve long-term outcomes and morbidity in the pediatric population.

Conclusions

This study demonstrated that MHA/Coll is able to increase bone formation in the presence of systemic neoadjuvant or adjuvant chemotherapy compared to MHA/Coll alone in a murine critical size defect model. The potential to utilize MHA/Coll to not only regenerate bone but also increase bone regeneration following surgical resection of OS is a novel finding. This approach has several advantages over the current standard of care, including (a) fewer pediatric patients would require limb amputation, (b) multiple surgeries to repair bone defects in growing children could be prevented (saving costs and improving life quality), (c) faster recovery and increased bone volume and strength would occur, (d) life-long remodeling of new bone as it integrates with surrounding native skeletal structures would occur, and (e) recurrence and micrometastases would be avoided. The possibility to repair the bone defect in the presence of a neoadjuvant or adjuvant could lead to improved outcomes and fewer complications for patients affected by OS. Moreover, the ability to regenerate bone utilizing MHA/Coll when chemotherapy is given provides additional evidence that MHA/Coll could be used safely to repair bone voids following OS excision.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomaterials.3c01266.

• Kaplan–Meier survival of mice treated with CIS (Supporting Figure 1); 3D& CT reconstruction of bone regeneration with only intramedullary pin with preoperative or postoperative cisplatin (Supporting Figure 2); bone quantification induced by systemic chemotherapy (Supporting Figure 3); organ H&E 12 weeks postoperative (Supporting Figure 4); comparison of temporal gait metrics in MHA/Coll vs pin at 16 weeks (Supporting Figure 5); postoperative temporal gait metrics of treated mice (Supporting Figure 6); postoperative hind limb loading/unloading and weight distribution in MHA/Coll vs pin at 16 weeks (Supporting Figure 7); post-operative hind limb loading/unloading and weight distribution of treated mice (Supporting Figure 8) (PDF)

Author Contributions

A.A.B.: Conception & design, analysis and interpretation of data, drafting of article, collection and assembly of data. S.L.: Conception & design, critical revision of article for important intellectual content, collection and assembly of data. F.P., S.S., C.B.: Collection and assembly of data. P.M.: Conception & design, provision of study materials or patients. B.W.: Conception & design, critical revision of article for important intellectual content. F.T.: Conception & design, analysis and interpretation of data, critical revision of article for important intellectual content.

The authors declare no competing financial interest.