合成聚合物-明膠複合體作為骨成形蛋白載體於骨再生研究中之評估

Abstract

Background: Tissue engineering for bone regeneration relies on the presence of proper growth factors inside a suitable delivery system. Bone morphogenetic proteins (BMPs) are well-known important growth factors capable of inducing ectopic bone formation. However, an effective carrier/delivery system is critical for BMPs to accomplish this result. The objective of this study was to test the in vitro and ex vivo potential of the novel composite scaffold (synthetic polymers/gelatin) as a carrier for rhBMP-2. Materials and methods: We prepared several scaffolds for carring rhBMP-2. The synthetic polymers of poly(DL-lactide) was fabricated with gelatin to be a test composite scaffold for carrying rhBMP-2. The gelatin sponge and PDLLA scaffold (PDLLA) were also prepared for carring rhBMP-2 as controls. The structure, pore sizes, and cell affinity of test and control scaffolds were studied by scanning electron micrograph (SEM). The compression strength between test and some control scaffold was compared. Compositional change between PDLLA and gelatin in composite scaffold were evaluated Fourier Transform Infrared Spectroscopy (FTIR) analysis. In ex vivo assay, Composite, PDLLA, and gelatin loaded with 30μg rhBMP-2 ( rhBMP-2-Composite; rhBMP-2-PDLLA; rh-BMP-2-gelatin) and control scaffolds were implanted into calf muscle of 24 male Wistar rats (4-5 weeks-old) to explore the ectopic bone formation at 3, 7, 14, and 28 days. Tissue blocks were harvested and ectopic bone formation was examined by histology, Masson’s trichrome staining, Goldner’s staining, and immunohistochemistry of key bone matrix proteins (OPN and BSP). RT-PCR analysis was performed for the expression of osteoblastic gene such as core binding factor 1 (Cbfa1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN). Results: Under SEM observation, PDLLA scaffolds showed a dense non-porous outer surface skin and a mean pore size ＜ 10μm and poor pores, interconnection cross-sectionally.. The composite scaffolds (test) exhibited porous outer surface and a porosity (pores size 50-60μm) inside the scaffold than those of control PDLLA scaffolds. Cell affinity of the test scaffolds were also better than that of PDLLA. Composite scaffolds showed better compression strength than galatin one. FTIR analysis showed that compositional profile of the test scaffold is a mixture of PDLLA and gelatin. In ex vivo study, all implants showed good biocompatibility without severe inflammatory responses and foreign body reaction. The PDLLA-rhBMP-2 and control PDLLA scaffolds failed to induce bone formation throughout the study. Initial events of ectopic bone formation were found in Gelatin-rhBMP-2 and Composite-rhBMP-2- at 7 and 14 days, respectively. The results of RT-PCR analysis indicated that consistent gene expression of Cbfa1, ALP and OPN are found in tissue specimens implanted harvested from Composit-rhBMP-2 and Gelatin- rhBMP-2. Conclusion: Novel composite scaffolds displayed a structure with proper micro-pore structure, good tissue biocompatibility and mechanical strength. The incorporation of rhBMP-2 into composite scaffolds could induce ectopic bone formation as shown by histology, immunohistochemistry and gene expression within 2 weeks. The results suggest that the novel composite scaffolds can be a promising scaffold for carrying rhBMP-2 in tissue engineering of bone.