Enhanced bone formation by transforming growth factor-beta1-releasing collagen/chitosan microgranules

Collagen/chitosan composite microgranules were fabricated as bone substitutes for the purpose of obtaining high bone-forming efficacy. The microgranules have the flexibility to fill various types of defect sites with closer packing. The interconnected pores formed spaces between the microgranules, which allowed new bone ingrowth and vascularization. In addition, the transforming growth factor-beta 1 (TGF-beta1) was incorporated into the microgranules in order to improve bone-healing efficacy. The collagen/chitosan microgranules were fabricated by dropping a mixed solution into a NaOH/ethanol solution. TGF-beta1 was loaded into the collagen/chitosan microgranules by soaking the microgranules in a TGF-beta1 solution. Scanning electron microscopy (SEM) observations and experiments examining the release of TGF-beta1 from chitosan and the collagen/chitosan microgranules were performed. SEM was used to examine the cell morphologies on the microgranules and cell proliferation was evaluated using a dimethylthiazole tetrazolium bromide assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALPase) activity as well as detecting an osteocalcin assay. The in vivo bone-regeneration experiments were performed using a rabbit calvarial defect model. TGF-beta1 was released from the collagen/chitosan microgranules at a therapeutic concentration for 4 weeks. SEM indicated that the seeded osteoblastic cells were firmly attached to the microgranules and proliferated in a multilayer manner. The proliferation of the osteoblasts on the TGF-beta1-loaded microgranules was the highest among the different types of microgranules tested. The ALPase activity and osteocalcin level of all the samples increased during the culture period, and the TGF-beta1-loaded microgranules had a significantly higher ALPase activity and osteocalcin content than the other microgranules. The TGF-beta1-loaded microgranules demonstrated a higher bone-regenerative capacity in the rabbit calvarial defects after 4 weeks than the TGF-beta1-unloaded microgranules.