Integrated design and fabrication strategies for biomechanically and biologically functional PLA/ß-TCP nanofiber reinforced GelMA scaffold for tissue engineering applications.

We present an integrated design and fabrication strategy for the development of hierarchically structured biomechanically and biologically functional tissue scaffold. An integration of ß-TCP incorporated fluffy type nanofibers and biodegradable interpenetrating gelatin-hydrogel networks (IGN) result in biomimetic tissue engineered constructs with fully tunable properties that can match specific tissue requirements. FESEM images showed that nanofibers were efficiently assembled into an orientation of IGN without disturbing its pore architecture. The pore architecture, compressive stiffness and modulus, swelling, and the biological properties of the composite constructs can be tailored by adjusting the composition of nanofiber content with respect to IGN. Experimental results of cell proliferation assay and confocal microscopy imaging showed that the as-fabricated composite constructs exhibit excellent ability for MC3T3-E1 cell proliferation, infiltration and growth. Furthermore, ß-TCP incorporated functionalized nanofiber enhanced the biomimetic mineralization, cell infiltration and cell proliferation. Within two weeks of cell-seeding, the composite construct exhibited enhanced osteogenic performance (Runx2, osterix and ALP gene expression) compared to pristine IGN hydrogel scaffold. Our integrated design and fabrication approach enables the assembly of nanofiber within IGN architecture, laying the foundation for biomimetic scaffold.

Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/ß-Catenin Signaling Pathway, Leading to Bone Formation.

The Pacific oyster, Crassostrea gigas, is well-known as a nutritious food. Recently, we revealed that fermented extract of C. gigas (FO) inhibited ovariectomy-induced osteoporosis, resulting from suppression of osteoclastogenesis. However, since the beneficial effect of FO on osteogenesis is poorly understood, it was examined in mouse preosteoblast MC3T3-E1 cells, human osteosarcoma MG-63 osteoblast-like cells, and zebrafish larvae in this study. We found that FO increased mitochondrial activity from days 1 to 7; however, total cell number of MC3T3-E1 cells gradually decreased without any change in cell viability, which suggests that FO stimulates the differentiation of MC3T3-E1 cells. FO also promoted the expression of osteoblast marker genes, including runt-related transcription factor 2 (mRUNX2), alkaline phosphatase (mALP), collagen type I α1 (mCol1α1), osteocalcin (mOCN), osterix (mOSX), bone morphogenetic protein 2 (mBMP2), and mBMP4 in MC3T3-E1 cells accompanied by a significant increase in ALP activity. FO also increased nuclear translocation of RUNX2 and OSX transcription factors, ALP activity, and calcification in vitro along with the upregulated expression of osteoblast-specific marker proteins such as RUNX2, ALP, Col1α1, OCN, OSX, and BMP4. Additionally, FO enhanced bone mineralization (calcein intensity) in zebrafish larvae at 9 days post-fertilization comparable to that in the ß-glycerophosphate (GP)-treated group. All the tested osteoblast marker genes, including zRUNX2a, zRUNX2b, zALP, zCol1a1, zOCN, zBMP2, and zBMP4, were also remarkably upregulated in the zebrafish larvae in response to FO. It also promoted tail fin regeneration in adult zebrafish as same as the GP-treated groups. Furthermore, not only FO positively regulate ß-catenin expression and Wnt/ß-catenin luciferase activity, but pretreatment with a Wnt/ß-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/ß-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.