The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL.

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Healing of contaminated/infected bone defects is a significant clinical challenge. Prevalence of multi-antibiotic resistant organisms has renewed interest in the use of antiseptic silver as an effective, but less toxic antimicrobial with decreased potential for bacterial resistance. In this study, we demonstrated that metallic nanosilver particles (with a size of 20-40 nm)-poly(lactic-coglycolic acid) (PLGA) composite grafts have strong antibacterial properties. In addition, nanosilver particles-PLGA composite grafts did not inhibit adherence, proliferation, alkaline phosphatase activity, or mineralization of ongrowth MC3T3-E1 pre-osteoblasts compared to PLGA controls. Furthermore, nanosilver particles did not affect the osteoinductivity of bone morphogenetic protein 2 (BMP-2). Infected femoral defects implanted with BMP-2 coupled 2.0% nanosilver particles-PLGA composite grafts healed in 12 weeks without evidence of residual bacteria. In contrast, BMP-2 coupled PLGA control grafts failed to heal in the presence of continued bacterial colonies. Our results indicate that nanosilver of defined particle size is bactericidal without discernable in vitro and in vivo cytotoxicity or negative effects on BMP-2 osteoinductivity, making it an ideal antimicrobial for bone regeneration in infected wounds.

The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation.

Repair of cartilage due to joint trauma remains challenging due to the poor healing capacity of cartilage and adverse effects related to current growth factor-based strategies. NELL-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]), a protein first characterized in the context of premature cranial suture fusion, is believed to accelerate differentiation along the osteochondral lineage. We previously demonstrated the ability of NELL-1 protein to maintain the cartilaginous phenotype of explanted rabbit chondrocytes in vitro. Our objective in the current study is to determine whether NELL-1 can affect endogenous chondrocytes in an in vivo cartilage defect model. To generate the implant, NELL-1 was incorporated into chitosan nanoparticles and embedded into alginate hydrogels. These implants were press fit into 3-mm circular osteochondral defects created in the femoral condylar cartilage of 3-month-old New Zealand White rabbits (n=10). Controls included unfilled defects (n=8) and defects filled with phosphate-buffered saline-loaded chitosan nanoparticles embedded in alginate hydrogels (n=8). Rabbits were sacrificed 3 months postimplantation for histological analysis. Defects filled with alginate containing NELL-1 demonstrated significantly improved cartilage regeneration. Remarkably, histology of NELL-1-treated defects closely resembled that of native cartilage, including stronger Alcian blue and Safranin-O staining and increased deposition of type II collagen and absence of the bone markers type I collagen and Runt-related transcription factor 2 (Runx2) as demonstrated by immunohistochemistry. Our results suggest that NELL-1 may produce functional cartilage with properties similar to native cartilage, and is an exciting candidate for tissue engineering-based approaches for treating diverse pathologies of cartilage defects and degeneration.