The effect of different isolation techniques on human osteoblast-like cellgrowth

Surgical treatment of post-traumatic or neoplastic bone defects often repre sents a problem in orthopaedic routine. Autologous tissue always stands for the first choice material. The recent therapeutic approaches for tissue re pair of bone defects attempt to mimic the natural process of bone repair by delivering a source of cells capable of differentiating into osteoblasts. In this study two different types of human osteoblast cell harvesting were compared in primary cell culture to evaluate the best way to obtain cells f or clinical use. Numerous articles describe the characteristics of each one of these systems, but there is no report comparing the influence of these different isolation techniques on cell growth. Cultures fi om 22 bone speci mens obtained fi om donors were established in two different ways and their proliferation was compared. An enzymatic procedure to extract human osteob lasts (hOB(col)) was used in one group; spontaneous cells outgrowth, human osteoblasts (hOBsog) was expected in the other group. Cells of both groups were characterised as osteoblasts by immunohistochemical staining with Bone Morphogenetic Protein-2,4 (BMP-2,4), expression of collagen type I as well as the amount of alkaline phophatase activity (ALP) detected in the cell c ultures. We found that the time needed in primary cultures till confluence was dependent on the age of the donors as well as on the method of cell har vesting. Cells from under 65-year old donors were growing significantly fas ter by the hOB(col) method as compared to hOB(sog) 20.57+/-2.99 days vs. 30 .00+/-4.36. Cells harvested from donors older than 65 yeats of age needed 2 3.88+/-2.95 in the hOB(col) compared to 34.25+/-4.27 days in the other grou p. In the experimental cultures, after one passage with trypsin/EDTA, there was a significant difference between the two groups. There was an improved cell growth in the hOB(sog) group found on days 8, 9 and 10 of cultivation . Immuno-histochemical staining as well as ALP activity were similar in bot h groups. In conclusion this study evaluated an important step for a tissue engineering approach to the repair of bone defects, which may have clinica l applications in post-traumatic orthopaedic surgery.