Yi-Chau Huang

The influence on gene-expression profiling of osteoblasts behavior following treatment with the ionic products of sintered β-dicalcium pyrophosphate dissolution

Sintered dicalcium pyrophosphate (SDCP) is biocompatible to bone tissue both in the in vivo and in vitro model. However, the molecular mechanisms that mediated these processes have yet to be identified. In this study, we investigated the influence of SDCP ions on in vitro osteoblasts behavior. The powder of sintered beta-dicalcium pyrophosphate (SDCP) was dissolved by HCl and then diluted into different concentration of solutions by culture medium used in the osteoblast cell culture. The effects of various concentration of SDCP on bone cell activities were evaluated by using MTT assay. For the differentiation of osteoblasts, alkaline phosphatase (AP) staining, von Kossa stain for mineralized nodules and bone markers messenger ribonucleic acid (mRNA) isolation and identification were performed at 3h, days 1, 3, 7 and 14. In the presence of 10(-8)M SDCP for 14 days, the osteoblasts population was still significantly higher than that of control. In the qualitative analysis for the formation of AP staining colonies and mineralization nodules formation were not affected by SDCP ions. When osteoblasts cultured in the presence of 10(-8)M SDCP ions, the osteocalcin mRNA expression was up-regulated; while the collagen, osteonectin and osteopontin mRNA expression were down-regulated. In this study, we demonstrated that the elevated concentration of calcium and pyrophosphate ions can activate genes of the bone cells. This study will contribute to a better understanding of cell/biomaterial interactions and mechanisms that SDCP affect the bone cells.

EFFECT OF CALCIUM ION CONCENTRATION ON KERATINOCYTE BEHAVIORS IN THE DEFINED MEDIA

Calcium ion concentration is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These actions are mediated by Ca2+ influx and inhibition of cell proliferation. To define Ca2+ action in the keratinocyte we investigated its effects on the proliferation and differentiation using the primary keratinocytes model. Primary keratinocytes were incubated in DMEM (containing 1.2mM calcium ion concentration) or DK11 medium (containing 0.4 mM calcium ion concentration) or K medium (containing 0.03mM calcium ion concentration). Cell viability was assessed with the MTT assay. Crystal violet assay was evaluated the proliferation rate and colony formation size of keratinocyte. Real-time PCR used to determine the terminal differentiated keratinocyte which expressed Caspase-14. Proliferation assays and real°Vtime PCR were correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes. High Ca2+ concentration was inhibited the cell viability and proliferation rate of keratinocyte. Ca2+ also increased caspases-14 expression, and inhibited cell viability, and cell colony forming efficiency. These results are consistent with Ca2+ induction of the keratinocyte differentiation. Thus, the overall Ca2+ actions connote protective functions for the epidermis that appear to include the triggering or acceleration of the differentiation.

PREPARATION AND EVALUATION OF GAG-INCORPORATED SKIN SUBSTITUTE: AN IN VITRO STUDY

A bi-layered gelatin-C6S-HA membrane with different pore sizes was prepared by freeze-drying at different temperatures - 20°C and -196°C, respectively Glycosaminoglycans (GAGs) were incorporated within the gelatin matrices to mimic the dermal composition and to create an appropriate environment for cell growth. The gelatin-C6S-HA membrane was cross-linked by 1-ethyl-3(3-dimethylaminopropryl) carbodiimide (EDC) to resist rapidly biodegradation by matrix enzymes. In this study, the lower layer of the sponge was inoculated with dermal fibroblasts for dermis development and as the feeder layer for epidermal keratinocytes. The upper layer was seeded with keratinocytes for epidermalization. After cultured for a period of time in air-liquid interface, the upper layer was developed into an epidermis structure with stratified epidermal layers. The lower part was developed into dermis-like structure synthesized by dermal fibroblasts surrounding with its own secreted extracellular matrix. In brief, the bi-layered skin equivalent with biological dermal analog and epidermal analog would be a suitable tool for autologous skin equivalent tissue engineering.