A model for intramembranous ossification during fracture healing.

We have developed a method to study the molecular basis of intramembranous fracture healing. Unlike intramedullary rods that permit rotation of the fractured bone segments, our murine model relies on an external fixation device to provide stabilization. In this study we compare stabilized fracture callus tissues with callus tissues from non-stabilized fractures during the inflammatory, soft callus, hard callus, and remodeling stages of healing. Histological analyses indicate that stabilized fractures heal with virtually no evidence of cartilage whereas non-stabilized fractures produce abundant cartilage at the fracture site. Expression patterns of collagen type IIa (colIIa) and osteocalcin (oc) reveal that mesenchymal cells at the fracture site commit to either a chondrogenic or an osteogenic lineage during the earliest stages of healing. The mechanical environment influences this cell fate decision, since mesenchymal cells in a stabilized fracture express oc and fail to express colIIa. Future studies will use this murine model of intramembranous fracture healing to explore, at a molecular level, how the mechanical environment exerts its influence on healing of a fracture.     

分别构建膜内成骨和软骨内成骨骨修复模型的研究

[目的]分别构建膜内成骨骨修复模型和软骨内成骨骨修复模型。[方法]取BALB/c小鼠64只,随机分为骨皮质钻孔模型组和骨膜划痕模型组。骨皮质钻孔模型组在小鼠右侧胫骨前内侧实施单纯性骨皮质钻孔损伤用于构建膜内成骨模型;骨膜划痕模型组在小鼠右侧胫骨前内侧实施骨膜划痕损伤用于构建软骨内成骨模型。术后第7、10、14和21 d,每组每个时间点处死8只小鼠,观察损伤部位的组织修复。[结果]骨皮质钻孔模型组小鼠损伤后第7 d在损伤部位出现新生小梁骨构成的骨痂组织。损伤后第10 d新生小梁骨充满损伤骨皮质及骨髓腔。损伤后第14 d新生骨痂组织进入改建过程,至第21 d,多数骨痂组织基本改建完成。在修复过程中无软骨细胞出现。骨膜划痕模型组小鼠损伤后第7 d在损伤部位出现新生软骨组织构成的软骨痂,并有部分软骨细胞已分化为成熟软骨细胞。损伤后第10 d软骨痂中心区域软骨基质溶解,第14 d后新生小梁骨出现,逐渐替代软骨组织。损伤后第21 d,软骨痂已基本被骨小梁替代。在修复过程中,首先出现软骨基质形成的骨痂组织,随后骨化中心形成,骨性骨痂组织逐渐替代软骨,完成骨修复。[结论]成功构建了以膜内成骨方式愈合的骨修复模型和以软骨内成骨方式愈合的骨修复模型,为今后深入研究骨愈合机制提供了基础。     

Runx1 dose-dependently regulates endochondral ossification during skeletal development and fracture healing

Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1(L148A) ) and generated Runx1(L148A/-) mice in which >50% of Runx1 activity was abrogated. Runx1(L148A/-) mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9-, Runx2-, and Runx3-positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1-Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud-derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral-expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage.     

Adhesion molecules in skeletogenesis: I. Transient expression of neural cell adhesion molecules (NCAM) in osteoblasts during endochondral and intramembranous ossification.

We report that neural cell adhesion molecules (NCAM) are expressed transiently in developing chicken osteoblasts during osteogenesis using immunostaining on cryostat sections. NCAM is strongly expressed in most osteoblasts along bone trabeculae that coincide with the presence of collagen I and alkaline phosphatase activity. In endochondral ossification, NCAM is highly expressed in osteogenic buds as seen in the epiphysis and diaphysis of tibia and vertebrae. In intramembranous ossification, NCAM is seen in osteogenic condensation of calvaria and in the periosteum of tibial diaphysis. The expression is transient because NCAM is not expressed in mesenchymal cells before osteogenic condensation and NCAM expression is lost in osteocytes in later stages. The staining pattern suggests that NCAM is present on the cell membrane of osteoblasts. Using a specific monoclonal antibody, the osteoblast NCAM is shown to contain polysialic acid, which is enriched in embryonic brain. Northern blot analysis using chicken brain NCAM cDNA as probes showed two major sizes of mRNA at 6.4 and 4.2 kb in calvarial mRNA as opposed to bands at 7.2, 6.4, and 4.2 kb in the brain. An immunoblot showed major proteins at Mr 165 and 110 kd, unlike brain NCAM, which are 180, 140, and 120 kD. That NCAM is involved in bone morphogenesis is consistent with the general hypothesis that NCAM plays pivotal roles in mesenchymal condensation, as shown in the formation of muscle, kidney, skin, and cartilage. The results establish NCAM as a cell surface molecule expressed transiently during osteoblast lineage. The implication that NCAM may mediate osteoblast interaction and regulate skeletal morphogenesis is discussed.     

Possible roles of Epstein-Barr virus in Castleman disease

Background  

Complete resection seemed to be curative in patients with Castleman disease of any location but the disease is likely to be reactive in its pathogenesis. The relation between Epstein-Barr virus and Castleman disease has not been elucidated. We tried to define the role of Epstein-Barr virus in the pathogenesis of Castleman disease.     

高泳动家族性别决定基因9(Sox9)启动子转染软骨肉瘤细胞的实验研究

[目的]研究观察Sox9启动子在软骨肉瘤细胞中对软骨相关基因的调控与影响。[方法]将合成Sox9启动子表达质粒转染SW1535软骨肉瘤细胞,观察其在人体软骨肉瘤细胞中对Sox9基因和Ⅱ型胶原蛋白基因Col2a1蛋白及mRNA表达的影响。[结果]转染Sox9启动子后人体软骨肉瘤细胞中Sox9基因和Ⅱ型胶原蛋白基因Col2a1的蛋白表达及mRNA表达均较对照细胞强。[结论]外源性Sox9启动子可以稳定转染SW1535人体软骨肉瘤细胞,转染后可以增加Sox9基因和Ⅱ型胶原蛋白基因Col2a1蛋白及mRNA的表达。     

Sox9、CTGF及BMP-2对促进腱骨结合处早期愈合的研究

[目的]观察SRY相关高迁移组box基因9蛋白(Sox9)及结缔组织生长因子(CTGF)、骨形态发生蛋白2(BMP-2)在促进腱骨结合处愈合的作用。[方法]72只成年新西兰大白兔随机分为Sox9组、CTGF组、BMP-2组及对照组。Sox9组、CTGF组、BMP-2组分别于骨与肌腱的缝合处注入浓度为200 ng/ml的Sox9、CTGF及BMP-2各2 ml,对照组只手术但不注射任何材料。建立动物模型后对四组行动物实验并分别于术后4、8及12周行组织学及生物力学检查,统计学分析结果。[结果]术后第4、8、12周,Sox9组、CTGF组的组织学愈合情况均好于对照组,生物力学分析中Sox9组、CTGF组腱骨愈合界面的横截面积均低于对照组,拉断负荷及极限拉应力均高于对照组(P0.05),而BMP-2组各项指标均与对照组差异无统计学意义。[结论]Sox9以及CTGF组能够促进腱骨结合处的早期愈合,并能够增加其生物力学强度,而BMP-2对骨肌腱结合部位愈合并无明显作用。     

Sox9基因促软骨形成作用机制研究进展

Sox9基因作为一个重要转录因子,在软骨形成中起着极其重要的调控作用,相关研究对软骨缺损修复等组织再生工程发展具有十分重要的意义。软骨形成过程中软骨细胞增殖、分化调控过程复杂,参与的细胞因子较多,目前比较明确的是Sox9结合于软骨细胞特异增强子Col2α1,直接调控其表达。L-Sox5、Sox6与Sox9共同形成蛋白复合物协同作用于Col2α1增强子序列,增强其转录,并通过结合于PTHrP基因的启动子区上调PTHrP基因启动子活性,刺激软骨细胞早期阶段增殖和发育。Sox9与Wnt/β-catenin和TGF-β/Smad信号转导通路相互作用,调控软骨细胞分化。最新研究显示,Sox9基因在软骨形成过程中还与其他物理因子和生物因子如低氧、电磁场等相互影响、相互作用。     

Possible roles of mast cell-derived chymase for skin rejuvenation

The relationships between mast cell-derived chymase, angiotensin II, and extracellular matrix production in the skin after intense pulsed light (IPL) were clarified in hamsters. Dorsal areas of the hamsters were irradiated once or twice a week by IPL. The index of extracellular matrix production in the skin was defined as the depth stained with Azan-Mallory stain from the epidermis to the dermis at the point of maximum thickness. The index had significantly increased 7 days after IPL irradiation in sections treated once or twice with IPL compared with that of untreated control sections. The numbers of mast cells, chymase-positive cells, and angiotensin II-positive cells had also significantly increased in IPL-irradiated areas. Significant increases in chymase and angiotensin II activities were observed in the extracts obtained from IPL-irradiated skin. Mast cell-derived chymase may be involved via angiotensin II formation in the dermal extracellular matrix production that occurs after IPL irradiation.     

白细胞介素1对椎间盘细胞软骨特异性基因Sox9 mRNA的调节作用

目的探讨白细胞介素1（interleukin-1,IL-1）对人椎间盘细胞软骨特异性基因Sox9和Ⅱ型胶原基因表达的调节作用。方法应用RT—PCR技术检测IL-1对培养的椎间盘细胞中软骨特异性基因so西和Ⅱ型胶原基因mRNA表达的调节作用。结果在IL-1浓度为0．1ng／ml、1ng／ml和10ng／ml培养24h时,其对椎间盘细胞Sox9和Ⅱ型胶原基因mRNA可起到显著的负向调控作用;10ng／ml的IL-1随着培养时间的延长对椎间盘细胞中Sox9和Ⅱ型胶原基因mRNA出现显著的负向调控作用。结论IL-1可以按照剂量及时间依赖方式负向调节椎间盘细胞Sox9和Ⅱ型胶原基因的表达。