The stiffness variability of a silk fibroin scaffold during bone cell proliferation.

Complex assessment of gradual changes in scaffold morphology and stiffness is an essential step in bone filler development. Current approach, however, does not reflect long term cell proliferation effect as the mechanical tests are usually conducted on pristine materials without cells or cell influence on material stiffness is evaluated after one time period only. Here, biocompatible silk fibroin (SF) porous scaffolds envisioned for bone defect filling were prepared by dissolving of fibroin fibers, followed by dialysis, freeze-drying and final stabilization. Particular attention was devoted to the influence of bone cell proliferation up to 2 months on the stiffness of the material. The morphology of the material was studied in terms of its inner structure and the overall changes in the surface characteristics due to proliferation of MG 63 bone cell line. The SF scaffold stiffness significantly increased during first month followed by its decline during second month due to bone cell seeding. After 2 months, the SF scaffold was completely colonized, which resulted in a gradual decay of its structure. The length of degradation due to bone cell proliferation and mechanical behavior corresponded to the requirements set for reasonable filler material indicating that porous SF scaffolds comprise a promising biomaterial for bone regeneration.

Crayfish hemocyanin on chitin bone substitute scaffolds promotes the proliferation and osteogenic differentiation of human mesenchymal stem cells.

Crustacean chitin-hemocyanin-calcium mineral complexes were designed as bone biomimetics, with emphasis on their ability to bind or release calcium ions. Chitin scaffolds were prepared by dissolving chitin flakes in LiCl/dimethylacetamide, followed by gel formation and freeze-drying. Some of these scaffolds were modified by incorporation of CaCO3 . In some of the chitin-CaCO3 scaffolds, macroporosity was introduced by HCl treatment. Hemocyanin from the crayfish Cherax quadricarinatus was used to further modify the chitin scaffolds by dip coating. Cytocompatibility, cellular adherence and proliferation of human mesenchymal stem cells (hMSCs) were evaluated in terms of cell number as reflected in lactate dehydrogenase activity. The chitin, chitin-CaCO3 , and porous chitin-CaCO3 scaffolds were all found to facilitate cell attachment. Hemocyanin dip-coating of these scaffolds led to increased initial cell adhesion, enhanced proliferation, and osteogenic differentiation. Since the hemocyanin loading of the scaffolds was impaired by sterilization by gamma-irradiation (as required for biomedical applications), the hemocyanin loading was performed on previously sterilized scaffolds. All scaffolds facilitated osteogenic differentiation of osteoblasts, with the highest cell ALP-activity being found on hemocyanin-modified porous chitin-CaCO3 scaffolds. Thus, chitin-hemocyanin scaffolds enhanced the initial stages of bone cell development and could serve as promising biomaterials for bone regeneration.