Isolation of human osteoblasts and in vitro amplification for tissue engineering and subsequent bone repair

Traumatic injuries, cancer treatment and congenital disorders with abnormal bone shape or segmental bone loss requires replacement of missing bone. This may be accomplished by implantation of bone substitute material or by surgical transfer of natural tissue from an uninjured location elsewhere in the body. However, these procedures are limited due to different disadvantages. One strategy to overcome these problems is to develop living substitutes based on tissue engineering. As first step we have investigated the possibility to establish osteoblast cultures from facial bone. After 14 days of culture the first cells grew out of the bone explants, after another 2-3 weeks the floor of the culture flask was covered by a subconfluent monolayer. During subculturing procedures, the period to establish first and secondary passages was 14 days, further subculturing resulted in a further shortened culture period of only 5 to 7 days. The morphometric assessment of the AP and Coll positive cells over the culture periods showed a maximal expression for both markers in the second passage. In the second passage in average 72 % of all cells stained for AP whereas 25 % resp. 42 % of all cells expressed AP in the first resp. in the third passage. From cortico-cancellous bone chips of the maxilla cultures of human osteoblast like cells can be established. The amplification of these cells in subculture is easy to facilitate. A maximal expression of osteoblast differentiation markers like alkaline phosphatase and collagen I could be detected in the second and third passage. The demonstration of culturing sufficient differentiated osteoblast material originating from the human maxilla is a crucial step in respect to tissue engineering of bone which will find its application in cranio-maxillofacial surgery. Parole chiave: tissue engineering, bone, skull base