Osteochondral progenitor cells in acute and chronic canine nonunions

This study examined the ability of cells isolated from early healing segmen tal defects and from tissue from chronic nonunions to support bone and cart ilage formation In vivo and their response to transforming growth factor-be tal in vitro. Ostectomies (3 mm) were created in the radial diaphysis of fo ur dogs. The dogs were splinted 3-5 days postoperatively and then allowed t o bear full weight. At 7 days, tissue in the defect was removed and any per iosteum was discarded; cells in the defect tissue were released by enzymati c digestion. The dogs were splinted again and allowed to bear full weight f or 12 weeks. Radiographs confirmed a persistent nonunion in each dog. Defec t tissue was again removed, any periosteum was discarded, and cells were is olated. Cells were also obtained from the defect tissue by nonenzymatic mea ns with use of explant cultures. One-half of the tissue and one-half of any preconfluent, first-passage cultures were shipped to Cleveland by overnigh t carrier. At second passage, cells were loaded into ceramic cubes and impl anted into immunocompromised mice for 3 or 6 weeks. Harvested cubes were ex amined histologically for cartilage and bone with use of a semiquantitative scoring system. Confluent fourth-passage cultures of 7 and 84-day defect t issue cells were cultured with 0.03-0.88 ng/ml transforming growth factor-b etal for 24 hours, and [H-3]thymi dine incorporation and alkaline phosphata se specific activity were determined. Donor-dependent differences were note d in the rate at which defect cells achieved confluence; in general, cells from 7-day tissue divided most rapidly. Seven-day defect cells formed less bone and at a slower rate than was seen in the ceramic cubes containing sam ples from day 84. Cells derived enzymatically behaved similarly to those fr om explant cultures. Ceramic cubes contained fibrous connective tissue, car tilage, bone, and fat, indicating that multipotent cells were present, Stim ulation of [H-3]thymidine incorporation in response to transforming growth factor-betal was donor dependent and variable; only two of six separate iso lates of cells exposed to it had measurable alkaline phosphatase activity ( which was relatively low), and none of the cultures exhibited an increase i n response to transforming growth factor-betal for 24 hours. This indicates that mesenchymal progenitor cells are present in the healing defect tissue at 7 and 84 days and that the relative proportion of osteochondroprogenito r cells is greater at the later time. The response to transforming growth f actor-betal is typical of multipotent mesenchymal cells but not of committe d chondrocytes or osteoblasts, indicating that these committed and differen tiated cells are not present in early stages of healing and suggesting that their differentiation is inhibited in chronic nonunion.