Decellularized Muscle Supports New Muscle Fibers and Improves Function Following Volumetric Injury.

Current strategies to treat volumetric muscle loss use primarily pedicle or free muscle transfers, but these grafts fail to adequately regenerate functional tissue. Decellularized soft tissue grafts possess physical and chemical cues to promote muscle regeneration, suggesting their potential for use in large muscle defects. In this study, we developed a decellularized muscle matrix (DMM) graft using rat gastrocnemius. Anisotropy and chemical components of the extracellular matrix were retained, including laminin, fibronectin, and collagen. We compared the ability of DMM, autologous muscle grafts (clinical standard), and type I collagen plugs (negative control) to support muscle regeneration. DMM supported regeneration over a 56-day period in 1 × 1 cm and 1.5 × 1 cm gastrocnemius defects in rats. Muscle function tests demonstrated improved muscle recovery in rats with DMM grafts when compared to collagen. Histological sections were assessed using morphometrics and immunostaining. DMM supported muscle regeneration with less fibrosis and more de novo neuromuscular receptors than either autograft or collagen. Overall, our results indicate that DMM may be used as a muscle replacement graft based on its ability to improve muscle function recovery, promote muscle regeneration, and support new neuromuscular junctions.

Hyaluronic acid stimulates neovascularization during the regeneration of bone marrow after ablation.

Restoration of vasculature is a critical component for successful integration of implants in musculoskeletal tissue. Sodium hyaluronate (NaHY) has been used as a carrier for demineralized bone matrix (DBM). DBM is osteoinductive and osteoconductive, but whether NaHY by itself has an effect is not known. NaHY has been reported to promote neovascularization, suggesting it may increase neovasculature when used with DBM as well. To test this, we used a rat tibial marrow ablation model to assess neovascularization during bone formation and regeneration of marrow with different combinations of NaHY alone and NaHY+DBM. To assess neovascularization during normal healing, animals were euthanized at 3-, 6-, 14-, 21-, and 28-days post-ablation, and the vasculature perfused using a radio-opaque contrast agent. Vascular morphology was assessed using µCT and histology. Peak vessel volume within the marrow cavity was observed on day-14 post-ablation. Test materials were injected into the ablated marrow space as follows: (A) empty defect controls; (B) high MW (700-800 kDa) NaHY + heat inactivated DBM; (C) DBM in PBS; (D) low MW NaHY (35 kDa) + DBM; (E) high MW NaHY + DBM; (F) D:E 50:50; (G) low MW NaHY; (H) high MW NaHY; and (I) G:H 50:50. Neovascularization varied with bone substitute formulation. µCT results revealed that addition of NaHY resulted in an increase in vessel number compared to empty defects. Total blood vessel volume in all NaHY only groups were similar to DBM alone. Histomorphometry of sagittal sections showed that all three formulations of NaHY increased blood vessel number within the marrow cavity, confirming that NaHY promotes neovascularization.

Osteoinductivity of demineralized bone matrix is independent of donor bisphosphonate use.

BACKGROUND: Demineralized bone matrix is commonly used as a bone graft substitute, either alone or to supplement an osteoconductive material, because of its osteoinductive properties. The aging of the population has led to an increase in the number of prospective donors of demineralized bone matrix who have taken bisphosphonates to prevent osteoclast-mediated bone resorption. The aim of this study was to determine whether oral bisphosphonate usage affects the osteoinductivity of demineralized bone matrix from donors. METHODS: Sex-matched and age-matched pairs of samples were provided by four tissue banks (three or four pairs per bank). Demineralized bone matrix donors without bisphosphonate treatment had a mean age (and standard deviation) of 69.1 ± 2.5 years, and donors with bisphosphonate treatment had a mean age of 68.9 ± 2.0 years. Each pair included one donor known to have taken bisphosphonates and one who had not taken bisphosphonates. Demineralized bone matrix previously confirmed as osteoinductive was the positive control, and heat-inactivated demineralized bone matrix was the negative control. Demineralized bone matrix incubated with 1 mL of phosphate-buffered saline solution containing 0, 0.002, 2.0, or 2000 ng/mL of alendronate was also tested. Gelatin capsules containing 15 mg of demineralized bone matrix were implanted bilaterally in the gastrocnemius muscle of male nude mice (eight implants per group). The mice were killed thirty-five days after implantation, and hind limbs were recovered and processed for histological analysis. Osteoinductivity was measured with use of a qualitative score and by histomorphometry. RESULTS: Nine of fifteen samples from donors who had had bisphosphonate treatment and ten of fifteen samples from patients who had not had bisphosphonate treatment were osteoinductive. Qualitative mean scores were comparable (1.7 ± 0.4 for those without bisphosphonates and 1.9 ± 0.7 for those with bisphosphonates). Osteoinductive demineralized bone matrix samples produced ossicles of comparable size, regardless of bisphosphonate usage. Histomorphometric measurements of the area of new bone formation and residual demineralized bone matrix were also comparable. The addition of alendronate to control demineralized bone matrix did not affect its osteoinductivity. CONCLUSIONS: Demineralized bone matrix samples from donors treated with bisphosphonates and donors not treated with bisphosphonates have the same ability to induce bone formation. However, it is not known if the quality of the new bone is affected, with subsequent consequences affecting bone remodeling.

The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness.

The cell response to an implant is regulated by the implant's surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn approximately 750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA-DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-beta1, and VEGF-A) were assessed. Cells exhibited a more differentiated phenotype on the PEGDMA-DEGDMA copolymers compared to the 2HEMA-PEGDMA copolymers. On the PEGDMA-DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA-PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry.

Osteoinductive ability of human allograft formulations.

BACKGROUND: Bone graft materials are needed in periodontics that are osteoinductive, have good handling characteristics, and have physical properties that provide appropriate stiffness for the treatment site. Demineralized freeze-dried bone allograft (DFDBA), also called demineralized bone matrix (DBM), is osteoinductive but requires a carrier to meet the other clinical objectives, thereby decreasing the DBM content per volume of the bone graft material. The present study determined whether the DBM content of a carrier formulation is an important variable with respect to its effectiveness as an osteoinductive material. METHODS: The immunocompromised Nu/Nu mouse-muscle implantation assay of osteoinductivity was used to test human DBM formulated with hyaluronic acid (HY) and cancellous and cortical bone granules from the same donor: DBM alone (11 mg); DBM (11 mg):HY, 55:45, weight/weight (wt/wt); DBM (6.4 mg):HY, 32:68, wt/wt; DBM mixed with cortical and cancellous bone chips 1:4 (DBMC) (11 mg total, of which 2.2 mg was DBM); DBMC (11 mg):HY, 55:45, wt/wt; heat-treated DBM (11 mg); HY alone; and positive-control DBM (11 mg). Osteoinduction was scored using a qualitative scale and by histomorphometry. RESULTS: Results showed that all DBM was osteoinductive and the addition of HY did not change this as long as the amount of DBM used was held constant. The reduction in the absolute amount of DBM resulted in a reduced osteoinduction score, reduced ossicle area, and reduced new bone formation. The addition of HY also caused a decrease in the amount of residual non-vital bone particles, particularly when DBMC was implanted. Results were donor dependent. CONCLUSION: This study showed the importance of DBM content and donor variability in osteoinductivity of DBM formulations with improved handling and stiffness characteristics.