The effect of different isolation techniques on human osteoblast-like cell growth

Surgical treatment of post-traumatic or neoplastic bone defects often represents a problem in orthopaedic routine. Autologous tissue always stands for the first choice material. The recent therapeutic approaches for tissue repair 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 for 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 from 22 bone specimens obtained from donors were established in two different ways and their proliferation was compared. An enzymatic procedure to extract human osteoblasts (hOBcol) 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 phosphatase activity (ALP) detected in the cell cultures. 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 harvesting. Cells from under 65-year old donors were growing significantly faster by the hOBcol method as compared to hOBsog 20.57 +/- 2.99 days vs. 30.00 +/- 4.36. Cells harvested from donors older than 65 years of age needed 23.88 +/- 2.95 in the hOBcol compared to 34.25 +/- 4.27 days in the other group. 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 hOBsog group found on days 8, 9 and 10 of cultivation. Immunohistochemical staining as well as ALP activity were similar in both groups. In conclusion this study evaluated an important step for a tissue engineering approach to the repair of bone defects, which may have clinical applications in post-traumatic orthopaedic surgery.