Injectable and in situ crosslinkable gelatin microribbon hydrogels for stem cell delivery and bone regeneration in vivo

Rationale: Injectable matrices are highly desirable for stem cell delivery. Previous research has highlighted the benefit of scaffold macroporosity in enhancing stem cell survival and bone regeneration in vivo. However, there remains a lack of injectable and in situ crosslinkable macroporous matrices for stem cell delivery to achieve fast bone regeneration in immunocompetent animal model. The goal of this study is to develop an injectable gelatin-based mu RB hydrogel supporting direct cell encapsulation that is available in clinics as macroporous matrices to enhance adipose-derived stromal cell (ASC) survival, engraftment and accelerate bone formation in craniofacial defect mouse. Methods: Injectable and in situ crosslinkable gelatin microribbon (mu RB)-based macroporous hydrogels were developed by wet-spinning. Injectability was optimized by varying concentration of glutaraldehyde for intracrosslinking of mu RB shape, and fibrinogen coating. The efficacy of injectable mu RBs to support ASCs delivery and bone regeneration were further assessed in vivo using an immunocompetent mouse cranial defect model. ASCs survival was evaluated by bioluminescent imaging and bone regeneration was assessed by micro-CT. The degradation and biocompatibility were determined by histological analysis. Results: We first optimized injectability by varying concentration of glutaraldehyde used to fix gelatin mu RBs. The injectable mu RB formulation were subsequently coated with fibrinogen, which allows in situ crosslinking by thrombin. Fluorescence imaging and histology showed majority of mu RBs degraded by the end of 3 weeks. Injectable mu RBs supported comparable level of ASC proliferation and bone regeneration as implantable prefabricated mu RB controls. Adding low dosage of BMP2 (100 ng per scaffold) with ASCs substantially accelerated the speed of mineralized bone regeneration, with 90% of the bone defect refilled by week 8. Immunostaining showed M1 (pro-inflammatory) macrophages were recruited to the defect at day 3, and was replaced by M2 (anti-inflammatory) macrophages by week 2. Adding mu RBs or BMP2 did not alter macrophage response. Injectable mu RBs supported vascularization, and BMP-2 further enhanced vascularization. Conclusions: Our results demonstrated that mu RB-based scaffolds enhanced ASC survival and accelerated bone regeneration after injection into critical sized cranial defect mouse. Such injectable mu RB-based scaffold can provide a versatile biomaterial for delivering various stem cell types and enhancing tissue regeneration.