Fujii H, Kitazawa R, Maeda S, Mizuno K, Kitazawa S
Second Department of Pathology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan,
Histochem Cell Biol. 1999 Aug;112(2):131-8. doi: 10.1007/s004180050399.
Platelet-derived growth factor (PDGF), abundant in bone tissue, has been reported to stimulate mesenchymal cell proliferation and migration. To elucidate the functional roles of PDGF during fracture healing, we investigated the expression of PDGF-A and -B chain proteins and receptor alpha and beta mRNAs in fractured mouse tibiae. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibia. On days 2, 4, 7, 10, 14, 21, and 28 after the operation, the fractured tibiae were excised, fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin to prepare 7-microm sections. Immunohistochemistry using polyclonal antibodies against human PDGF-A and -B chains was carried out by the avidin-biotin-peroxidase method. For in situ hybridization, we used digoxigenin-labeled single-stranded DNA probes specific for mouse PDGF receptors alpha and beta generated by unidirectional polymerase chain reaction. In the inflammatory phase on days 2-4 after the fracture, mesenchymal cells gathering at the fracture site expressed the PDGF-B chain and beta receptor mRNA. At the stage of cartilaginous callus formation on day 7, the immunoreactivity for PDGF-A and -B chains on proliferating and hypertrophic chondrocytes and the signals of alpha and beta receptor mRNAs on proliferating chondrocytes became manifest. At the stage of bony callus and bone remodeling on days 14-21, the predominant expression of the PDGF-B chain and beta receptor was observed on both osteoclasts and osteoblasts. On day 28, signals for PDGF ligand proteins and receptor mRNAs diminished. The coincidental localization of PDGF ligands and their receptors implies a paracrine and autocrine mechanism. Our data suggested that PDGF contributed in part to the promotion of the chondrogenic and osteogenic changes of mesenchymal cells from the early to the midphase of fracture healing; the functions mediated by the beta receptor, including cell migration, might be prerequisites to the recruitment of mesenchymal cells in the initial step and to the interaction between osteoclasts and osteoblasts in the bone remodeling phase.
血小板衍生生长因子（PDGF）在骨组织中含量丰富，据报道可刺激间充质细胞增殖和迁移。为了阐明PDGF在骨折愈合过程中的功能作用，我们研究了骨折小鼠胫骨中PDGF - A和 - B链蛋白以及受体α和β mRNA的表达。12周龄雄性BALB / c小鼠接受手术，在胫骨近端造成闭合性骨折。术后第2、4、7、10、14、21和28天，切除骨折的胫骨，用4％多聚甲醛固定，用20％乙二胺四乙酸脱钙，然后石蜡包埋以制备7微米切片。采用抗人PDGF - A和 - B链的多克隆抗体，通过抗生物素蛋白 - 生物素 - 过氧化物酶法进行免疫组织化学。对于原位杂交，我们使用通过单向聚合酶链反应产生的、针对小鼠PDGF受体α和β的地高辛标记单链DNA探针。在骨折后第2 - 4天的炎症期，聚集在骨折部位的间充质细胞表达PDGF - B链和β受体mRNA。在第7天软骨痂形成阶段，增殖和肥大软骨细胞上PDGF - A和 - B链的免疫反应性以及增殖软骨细胞上α和β受体mRNA的信号变得明显。在第14 - 21天骨痂和骨重塑阶段，在破骨细胞和成骨细胞上均观察到PDGF - B链和β受体的主要表达。在第28天，PDGF配体蛋白和受体mRNA的信号减弱。PDGF配体及其受体的巧合定位意味着旁分泌和自分泌机制。我们的数据表明，PDGF在一定程度上有助于促进骨折愈合早期至中期间充质细胞的软骨形成和成骨变化；由β受体介导的功能，包括细胞迁移，可能是初始步骤中间充质细胞募集以及骨重塑阶段破骨细胞和成骨细胞之间相互作用的先决条件。
