THE EVALUATION OF OSTEOGEMC POTENTIAL OF CULTURED PERIOSTEUM-DERIVED CELLS

骨膜具有成骨潜能，作为骨和关节缺损区的移植材料已引起广泛关注。最近，培养的骨膜细胞具有成骨潜能的报道。我们报道了培养的大鼠胫骨骨膜来源的细胞诱导骨形成的研究结果。将骨膜置于含10%胎牛血清的培养液中培养14天。骨膜细胞达到融合后，用025%胰酶/EDTA重新悬浮，然后在胶原海绵上三维重新培养7d。将骨膜细胞/胶原复合体移植到大鼠下颌下肌和含有小鼠BMP-4(pMiw-BMP-4，由东北大学Brigid I.M.Hogan博士和Kishimoto博士提供)的质粒DNA溶液中。注射入研磨组织，通过成对的针电极施加电脉冲。注射生理盐水作为对照质粒。免疫抑制剂FK506 1.0 mg/kg/d肌肉注射。分别于移植后14、21、35d摘除移植组织，并与对照组进行组织学和放射学比较。实验组移植后14d骨膜细胞已增殖分化为成骨细胞。细胞增殖标记物增殖细胞核抗原阳性细胞数较对照组明显增多。移植后第21天，新骨形成明显。在这一阶段，与对照组相比，VEGF和BMP-4阳性细胞显著增加。在移植后35天，观察到骨生长，并在缺损处发现新的钙化。实验组新骨形成明显，总钙化面积大于对照组。在大鼠骨膜细胞移植模型的早期阶段，经BMP-4基因处理的培养骨膜来源细胞具有较高的成骨能力。

Periosteum has osteogenic potential and considerable attention has been focused on its use as a grafting material for the repair of bone and joint defects. Recently, the osteogenic potential of cultured periosteal cells has been reported. We present findings of bone formation induced from cultured periosteum-derived cells of rats tibiae. Periosteum was placed in culture medium with 10% Fetal Bovine Serum for 14 days. After reaching confluence, periosteal cells were resuspended with 025% trypsin/EDTA and then re-cultured three dimensionally on a collagen sponge for 7 days. Periosteal cell/collagen complex was grafted into rat submandibular muscle and A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP-4, kindly provided by Dr.Brigid I.M. Hogan and Dr.Kishimoto, Tohoku Univ.) was injected into grfated tissue and electric pulses were applied through paired-needle electrodes. As a control plasmid, Normal saline solution was injected. Immunosuppressant (FK506, 1.0 mg/kg/day) was administered intramuscularly. At 14, 21 and 35 days post grafting, grafted tissue was extirpated and compared histologically and radiographically with control groups. In the experimental group, periosteal cells had proliferated and differentiated into osteogenic cells by 14 days post grafting. Cell proliferation marker PCNA positive cells significantly increased compared with control groups. At 21 days post grafting, new bone formation was evident. At this stage, VEGF and BMP-4 positive cells significantly increased compared with control groups. By 35 days post grafting, bone growth was observed and new calcification was detected radiographically in the defect. New bone formation in experimental group developed and total calcified area was larger than control group. Cultured periosteum-derived cells treated with BMP-4 gene showed higher osteogenic potential in the early stage of periosteal cell graft model of rat.