9. Nanoparticulate Mineralized Collagen Glycosaminoglycan in Conjunction With Chitosan And B-glycerophosphate Hydrogel Improves In Vivo Skull Regeneration

PURPOSE: Our group has been studying nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) for over a decade, showing that it can promote skull regeneration up to 50% of the mineralization and biomechanical properties of adjacent native calvarium without growth factors or ex vivo progenitor cell seeding. These data suggest that MC-GAG is a potentially translatable material, provided that additional efficacy can be attained. One of the mechanisms by which MC-GAG induced pro-anabolic and anti-catabolic effects in bone homeostasis is the temporospatial elution of phosphate ions from the intrinsic nanoparticulate mineral phase within MC-GAG. Specifically, within the first nine days of culture, the increase in local phosphate ion concentrations induces osteogenic differentiation via the solute carrier 20A1 (SLC20A1/PiT-1) and SLC20A1/PiT-2 sodium phosphate symporters. Simultaneously, MC-GAG also induces the expression of anti-osteoclastogenic soluble factors via phosphate elution via SLC20A1/PiT-1 homodimers. Thus, we hypothesize that extending the elution of phosphate ions by adding a hydrogel phosphate depot may improve the efficacy of MC-GAG. METHODS: Hydrogels were synthesized using chitosan (CS) and b-glycerophosphate (β-GP) disodium salt with varying ratios of β-GP. The CS/β-GP hydrogels were characterized by degradation rate, gelation time, and phosphate release. MC-GAG scaffolds were formed in a calcium salt-containing solution, and mineralization was achieved by crosslinking 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide. Composite materials of MC-GAG scaffold and CS/β-GP hydrogels were assessed with live/dead assays for primary human mesenchymal stem cell (hMSC) viability and osteogenic differentiation. For in vivo studies, a 14-mm full-thickness, extradural defect was created in each New Zealand white rabbit. Animals were divided into three groups: 1) defect only, 2) MC-GAG scaffold only, and 3) Composite material. Bone healing was evaluated at three-month intervals using in vivo microcomputed tomography (microCT) and ex vivo indentation at six months. RESULTS: CS/β-GP hydrogels were first characterized in isolation. The panel of hydrogels with varying concentrations of β-GP correlated inversely by β-GP concentration to gelation and degradation rates. CS/β-GP hydrogels were also found to extend phosphate release. Next, we assessed composite MC-GAG and CS/β-GP materials, finding that the composite material sustained phosphate release. Additionally, we investigated the composite materials impact on hMSC cell proliferation, viability, and differentiation. No differences were found among the control (MC-GAG) and the test composite materials. In a rabbit model, three months after implantation of the materials, regenerated bone mass, detected by in vivo microCT, was greater in the composite MC-GAG/hydrogel materials compared to the negative control (defect only) or MC-GAG control. Similarly, at six months, composite MC-GAG/hydrogel showed the highest bone mass, followed by MC-GAG scaffold only. Explanted skulls underwent mechanical property testing via indentation at six months, which showed that the strength and stiffness of the regenerated bone were highest in the composite MC-GAG/hydrogel group, closely comparable to that of native bone. CONCLUSION: These results show that a cranial defect with a composite MC-GAG scaffold and CS/β-GP hydrogel material significantly enhanced bone regeneration compared to the MC-GAG scaffold alone.