Porous scaffolds were prepared using regenerated Bombyx mori silk fibroin dissolved in water or hexafluoroisopropanol (HFIP). The effects of these two preparations on the formation and growth of new bone on implantation into the rabbit femoral epicondyle was examined. The aqueous-based fibroin (A-F) scaffold exhibited significantly greater osteoconductivity as judged by bone volume, bone mineral content, and bone mineral density at the implant site than the HFIP-based fibroin (HFIP-F) scaffold. Micro-CT analyses showed that the morphology of the newly formed bone differed significantly in the two types of silk fibroin scaffold. After 4 weeks of implantation, new trabecular bone was seen inside the pores of the A-F scaffold implant while the HFIP-F scaffold only contained necrotic cells. No trabecular bone was seen within the pores of the latter scaffolds, although the pores of these did contain giant cells and granulation tissue.

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Existing scaffolds cannot adequately satisfy the simultaneous requirements for the regeneration of bone. The challenge remains of how to improve the integration of newly formed bone with the surrounding tissues. The purpose of this study was to investigate the effects of two silk fibroin scaffolds, a hexafluoro isopropanol-based silk fibroin (HFIP-F) and an aqueous-based silk fibroin (A-F), for their osteoinductive potentials in large critical size bone defects in vivo. β-tricalcium phosphate (β-TCP) was used as a positive control. After implantation into defects created in the knee joints of rabbits for 1 and 2 weeks, hematoxylin and eosin (H-E) and Azan staining revealed that the A-F scaffold as well as β-TCP had stronger osteoinductive ability than the HFIP-F scaffold. The A-F scaffold exhibited prominent areas of neo-tissue containing bone-like nodules. Furthermore, induced osteointegration was observed between native and neo-tissue within the osteo defects in the knee joints of rabbits. Immunohistochemical staining showed the highest expression of Runx2, BMP-2, BMP-7, Smad1/5/9 and Phospho-Smad in the A-F scaffold implants. Osteoinduction of the porous A-F scaffold might be influenced by the amount of BMP signaling present in the local microenvironment in the implants. This study opens a new avenue to use A-F silk fibroin scaffolds for the regeneration of bone defects.

: In this study, effects from pore size of the tube on the bone ingrowth and on the calcification of new bone permeated into the pore tube were examined through implantation of porous hydroxyapatite (HAp) in the lateral epicondyle in rabbits.The bone integration speed was strongly dependent on the pore size of the porous HAp. The 150-µm HAp provided a faster bone integration speed into the cylindrical tube than the 375-µm. However, when the pore size of the cylindrical tube in porous HAp was reduced to 20 µm, the bone integration speed into the cylindrical tube became slow. However, when the implanted period was prolonged to 8 weeks, the bone contact ratio into the 20-µm cylindrical tube dramatically increased and the bone density reached to74.0%. However, most new bones were remained as an osteoid bone. In contrast, the new bones formed inside of the 150-µm and 375-µm sized cylindrical tubes were calcified within the tube. It was concluded that the osteointegration into for the porous HAps and the calcification of osteoid formed were different with the pore size of the cylindrical tube. The optimum size of cylindrical tube was 150 µm.

This study characterized the molecular mechanisms of the effects of protamine-hydrolysate peptides (p-h peptides) on skin wound healing in rats by analyzing the transforming growth factor (TGF)-β signaling pathway. TGF-β was expressed in experimentally wounded skin tissues in fibroblasts and in keratinocytes. In p-h peptides-treated animals, the skin wounds exhibited an increased expression of TGF-β and of TGF-β target genes compared with control saline-treated skin wounds. Treatment with p-h peptides accelerated wound epithelialization and induced protein expression of TGF-β, CTGF and VEGF. The expression of tumor necrosis factor (TNF)-α was decreased in fibroblasts of p-h peptides-treated skin wounds. In addition, treatment with ph peptides significantly enhanced the phosphorylation of Smad3 and Smad4 in fibroblasts and also elevated the phosphorylation of Stat3 in skin wound tissues. In conclusion, treatment with p-h peptides activated the Smaddependent TGF-β signaling pathway, enhanced the differentiation of myofibroblasts and accelerated skin wound closure.