BACKGROUND: Bone defects are commonly accompanied by disuse-induced osteoporosis, which in turn exacerbates the difficulty of bone regeneration. Biomimetic bone scaffolds represent one of the most promising strategies for bone defect repair.
METHODS: In this study, we constructed poly(lactic-co-glycolic acid) (PLGA) microspheres with sustained release of melatonin (M-PLGA), an anti-osteoporosis hormone that simultaneously promotes osteogenesis and inhibits osteoclastogenesis. The microspheres were then integrated into a silk fibroin (SF)/nanohydroxyapatite (nHA) scaffold. To improve the cell inoculation rate and survival rate, a methacrylated gelatine (GelMA) hydrogel was laden with bone marrow mesenchymal stem cells (BMSCs, G-B). The gel acted as the cell carrier and was then inlaid into the M-PLGA/SF/nHA scaffold via negative pressure inhalation. The biomimetic scaffold was then photocrosslinked to yield the M-PLGA/SF/nHA/B-G scaffold. The microstructure, mechanical properties, pore diameters and sustained release of melatonin from the scaffold were evaluated. In vitro, the cytocompatibilities of the scaffolds were assessed using a CCK-8 assay, live/dead staining and phalloidin staining, and the osteogenesis-inducing abilities of the scaffolds were assessed by alkaline phosphatase, Alizarin Red S, and von Kossa staining, as well as immunohistochemical staining for collagen type 1. In vivo, a rat femoral distal bone defect was constructed and then immobilized by tail suspension to imitate disuse osteoporotic bone defects. Once the model was established, different scaffolds were implanted within the bone defect regions. Six weeks after implantation with different scaffolds, microcomputed tomography and histological staining were conducted to evaluate new bone formation within the bone defects in vivo.
RESULTS: In vitro, live/dead staining revealed that almost all the cells inoculated into the three types of scaffolds remained viable. Specifically, on day seven, the number of living cells in the M-PLGA/SF/nHA/G-B group was 621.7 ± 8.1% greater than that in the SF/nHA group. Phalloidin staining revealed that all the scaffolds were suitable for BMSC attachment, among which the M-PLGA/SF/nHA/G-B scaffolds were most conducive to BMSC distribution. Alizarin Red S staining and Von Kossa staining revealed more mineralized nodules in the M-PLGA/SF/nHA/G-B scaffolds following osteogenic induction, and ALP staining and collagen type 1 immunohistochemistry analysis confirmed that the M-PLGA/SF/nHA/G-B scaffolds were more conducive to osteogenic differentiation. In vivo, analysis by micro-CT and histological staining showed more new bone formation in the osteoporotic bone defect in the M-PLGA/SF/nHA/G-B group than in the SF/nHA or M-PLGA/SF/nHA group six weeks after implantation.
CONCLUSIONS: The novel multifunctional composite scaffold holds great promise as a bone repair biomaterial for future clinical translation.
