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Collagen scaffolds with in situ grown calcium phosphate for osteogenic differentiation of wharton’s jelly and menstrual blood stem cells

Karadaş, Özge
The importance of developing new techniques for the treatment of bone and joint diseases is increasing continuosly together with the increase of human population and the average life span. Especially bone fractures as a result of osteoporosis are often seen in humans older than 50 years old. The expenses of bone and joint disease operations are very high and the duration of recovery is long. Because of these reasons World Health Organization, The United Nations and 37 countries announced that the years 2000-2010 is the Bone and Joint Decade. Tissue engineering is an alternative approach to clinically applied methods. In this study collagen scaffolds crosslinked with genipin, to improve the stability of foams in culture media, were prepared by lyophilization. To mimic the natural bone structure calcium phosphate mineral phase in the foam was formed by wet chemical precipitation. Collagen concentration (0.75% and 1%, w/v), freezing temperature (-20 oC and -80 oC) of the collagen solution before lyophilization and immersion duration (2x4 h and 2x48 h) of the foams in calcium and phosphate solutions for wet chemical precipitation were changed as process v parameters of foam production. Pore size distribution and porosity analysis as well as compression test were performed for characterization of the scaffolds. The foam with 1% w/v collagen concentration, frozen at -20 oC before lyophilization and immersed for 2x4 h in calcium and phosphate solution was chosen for in vitro cell culture studies. The defined foam had 70% porosity and pore sizes varying between 50 and 200 μm. The elastic modulus and compressive strength of the foam was calculated as 127.1 kPa and 234.5 kPa, respectively. Stem cells isolated from Wharton’s jelly (WJ) and menstrual blood (MB) were seeded to foams to compare their osteogenic differentiation. Both cells are isolated from discarded tissues and used in this study as an alternative to the commonly used cells which are isolated by invasive techniques such as bone marrow stem cells. Cells were seeded to collagen foams with and without calcium phosphate (CaP). It was observed that WJ cells proliferated during 21 days on collagen foams without CaP, but MB cell number decreased after day 14. Collagen foams with CaP supported the alkaline phosphate (ALP) activity compared to tissue culture polystyrene (TCPS) and foams without CaP. Contrarily lower cell numbers achieved on CaP containing collagen foams, possibly because of the calcium and phosphate concentration changes in the medium and as the result of osteogenic differentiation. ALP activity of both cell types increased almost 10 times and specific ALP activity (activity per cell) increased 40 times and 150 times for WJ and MB cells, respectively on the CaP containing foams compared to TCPS. Therefore, in this study it was shown that in situ CaP formed collagen foams induce osteogenic differentiation of WJ and MB cells, and these cells isolated from discarded tissues can be used as alternative cell sources in bone tissue engineering applications.