Introduction: There is a demand for injectable, bioactive materials for the replacement, augmentation, or regeneration of bone. Gel aspiration-ejection (GAE) rapidly produces injectable dense collagen (IDC) gels with tunable collagen fibrillar densities (CFDs) and microstructures[1]. Through the application of pressure differentials, GAE initially draws prefabricated hydrogels into a needle, simultaneously imparting compaction and anisotropy, which are then controllably ejected to generate IDC gels. Herein, a GAE system was applied for the advanced production and delivery of IDC and IDC-Bioglass® (IDC-BG) hybrid gels for potential bone tissue engineering applications.
Materials and Methods: IDC [1] and IDC-BG (3:1 collagen:BG ratio) gels were produced via a GAE system (Fig. 1A-C) using needle gauge numbers 8G (3.4 mm diameter and 6 wt% CFD) and 14G (1.6 mm diameter and 14 wt% CFD). Collagen fibril orientation was investigated through second harmonic generation (SHG) imaging. Kokubo’s[2] protocol for SBF was used to investigate mineralization, in vitro. In vivo, the mineralization of, and host response to, acellular gels were investigated following subcutaneous injection in adult rats for up to day 21. Mineralization was assessed through SEM, ATR-FTIR, XRD and micro-CT analyses. Histomorphometry analyses included haematoxylin and eosin stain (H&E) for baseline, Toluidine blue-von Kossa (T&vK) for mineralization, and Goldner’s Trichrome (GT) for collagen fibrils and osteoid presence.
Results: SHG imaging of as-made gels revealed an increase in collagen fibril alignment with needle gauge number (Fig. 1).
In vitro mineralization of IDC-BG gels was confirmed where carbonated hydroxyapatite (CHA) was detected as early as day 1 in SBF, which progressively increased up to day 14. These results were confirmed in vivo, where subcutaneously implanted IDC-BG gels extensively mineralized (Fig. 2).
Relative to IDC alone, neovascularization and cell infiltration into the gels was enhanced by the addition of BG (Fig. 3). At day 21, there was evidence of remodelling of granulation tissue into woven bone-like tissue in IDC-BG. SHG imaging of injected gels indicated collagen fibril remodelling through cell infiltration and mineralization over time.
Discussion: While bone induction in an ectopic intramuscular site has been reported by BG-ceramic scaffolds[3], this is the first study to report BG-induced bone formation in a subcutaneous site without the addition of osteogenic cells. IDC-BG gels present macro- and micro-pores through folds, channels and pores allowing for cell infiltration. In addition, rapid CHA formation acted either as a trigger for osteogenic differentiation of local stem cells or included growth factors that drive the process. Furthermore, given that Bioglass® is proangiogenic[4], IDC-BG gels contributed to osteoinduction since angiogenesis and osteogenesis are closely related[5].
Conclusions: IDC-BG hybrid gels have osteoinductive properties and potentially offer a therapeutic approach for procedures requiring the injectable delivery of a malleable and dynamic bone graft that mineralizes under physiological conditions.
Funding of CIHR, NSERC, CFI, Quebec MEIE, and McGill University Faculty of Engineering Gerald Hatch Faculty Fellowship are gratefully acknowledged
References:
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