骨折愈合的细胞基础

骨折愈合大致可分为炎症期、修复期和重建期三个阶段,每个阶段都有一定的特性。炎症期主要有各类炎性细胞和破骨细胞的出现,为骨折修复铺平道路。修复期主要出现成纤维细胞、成软骨细胞和成骨细胞,在骨折部形成纤维骨痂、软骨痂和骨性骨痂。最终骨性骨痂通过骨改建来恢复正常旨形态。     

软骨生长板基因调控机制的研究进展

脊椎动物骨骼的形态与大小因功能各异而相差悬殊,但多数骨骼却是以相同的生长方式-软骨内成骨-长成的.位于骨骺与骨干交界处的软骨生长板(growth plate)-即骺板(epiphyseal plate)-不断增生、增厚,间质发生钙化,软骨细胞凋亡,继而新骨沉积,形成骨干的纵向生长.在幼年时期,软骨的增生、退化与新骨生成的速率保持平衡,从而保证了在骨干长度增加的同时,生长板能够保持一定的厚度.当软骨生长板发育到成熟阶段,软骨增殖与成骨活性中止,生长板完全被骨化,骨的纵向生长也随之停止.因此,软骨生长板调节控制骨的纵向生长速度与骨的最终长度.骨的生长与分化受局部因素(如骨形态发生蛋白、成纤维细胞生长因子、胰岛素样生长因子等)与全身因素(如生长激素、性激素、甲状腺素等)的调控.本文就近年来局部因素对软骨生长板基因调控机制的研究进展进行综述.     

自噬在软骨内成骨中的调控作用研究进展

哺乳动物的骨发生有两种不同的形式,一种是膜内成骨,另一种是软骨内成骨,后者是骨骼形成的主要方式。软骨内成骨是由间充质细胞先分化为软骨,随着血管的产生,软骨逐渐被骨组织取代,此过程受到一系列激素及生长因子相互作用、相互调节的严密调控。骨骺生长板中的软骨细胞分裂、成熟、肥大是软骨内成骨的关键过程。自噬是一种广泛存在于细胞中的高度保守的细胞降解代谢途径,利用溶酶体降解胞内物质,重复利用大分子的过程。缺氧和营养缺乏的内环境能使自噬活性相关控制基因激活,大量吞噬溶酶体形成,提高自噬水平。生长板无血管的结构使得位于生长板中间的软骨细胞处于氧气和营养物质相对缺乏的内环境中,对氧气和营养物质较敏感,自噬活性改变,产生相应的调控作用。近年来,不仅自噬在癌症、衰老、中枢神经系统损伤等领域的研究取得了较大进展,学者们对自噬在软骨细胞中的作用及其机制给予广泛关注,发现自噬有利于软骨细胞的生存与细胞外基质的降解。本文主要从自噬调控软骨内成骨的作用及相关调控因子,对自噬在软骨内成骨中的调控进行相关综述,以期能对相关骨代谢性疾病的治疗提供新的思路。     

骨骺生长板PTHrp-Ihh信号轴及相关因子调节机制研究进展

甲状旁腺素相关肽(PTHrp)-印第安刺猬蛋白(Ihh)信号轴是骨骺生长板中调节软骨和成骨细胞增殖、分化、肥大和矿化的重要负反馈轴.围绕该信号轴有多种生长因子如Sox9、BMP、TGF-β等,参与调控软骨及成骨细胞生长.新近研究表明,PTHrp通过Sox9阻止软骨前体细胞向肥大软骨细胞转化,通过抑制BMP-6阻止软骨细胞成熟,通过抑制BMP-4阻止软骨细胞凋亡;通过下调Ihh、BMP-6表达,TGF-β抑制软骨细胞分化;通过激活FGFR-3,可下调PTHrp、Ihh表达,抑制软骨细胞增殖;Ihh除具有促进软骨细胞增殖的作用外,还促进软骨膜上成骨细胞的分化,从而加速成骨.彻底弄清骨骺生长板中PTHrp-Ihh信号轴及相关因子的调节机制,对于治疗一些遗传性疾病、修复软骨及骨缺损有着重要的临床意义.该文围绕PTHrp-Ihh信号轴及相关因子对软骨细胞调控机制研究进展作一综述.     

bFGF对关节软骨缺损的修复作用

目的观察碱性成纤维细胞生长因了对关节软骨缺损修复的影响。方法在兔双侧股骨髁间窝造成骨软骨缺损．一侧应用碱性成纤维细胞生长因子．另一侧作对照。术后3个月．利用组织切片及扫描电镜等方法，观察两侧骨软骨缺损修复情况。结果修复3个月后．对照侧软骨缺损难以完全修复．修复细胞形态多样，似成纤维细胞。蛋白多糖颗粒较少。应用碱性成纤维细胞生长因子删软骨缺损基本修复．修复细胞似软骨细胞．有较多的蛋白多糖颗粒与胶原纤维结合。缺损修复组织评分。碱性成纤维细胞生长因子组优于对照组。结论碱性成纤维细胞生长因子可促进骨软骨缺损的修复。     

软骨内化骨过程中软骨细胞的新命运

软骨内化骨是骨发生、生长和损伤修复的一种成骨方式。在成骨过程中软骨组织中的肥大软骨细胞分泌基质、钙化、死亡，软骨组织退变、崩解、被吸收，血管侵入，成骨细胞产生骨组织。目前对体外（ｉｎｖｉｔｒｏ）、在体（ｉｎｖｉｖｏ）软骨、骨组织和细胞活动的研究手段主...     

转化生长因子—β与骨,软骨代谢及修复

转化生长因子－β作为一族具有多种功能的多肽生长因子,调节着骨、软骨细胞的增殖、分化和细胞外基质合成。本文概述了转化生长因子－β在骨、软骨代谢和损伤后修复中的重要作用,以及利用转基因技术修复骨和软骨损伤的应用前景。     

细胞因子与软骨损伤及修复研究进展

骨性关节炎(OA)发病中存在软骨细胞数量减少和软骨基质破坏,软骨损伤和修复贯穿于OA全过程,各种细胞因子起着重要的作用.白介素、肿瘤坏死因子-α、白血病抑制因子、趋化因子等细胞因子对软骨细胞的作用多呈损伤性,骨形态发生蛋白、成纤维细胞生长因子、胰岛素样生长因子和生长分化因子-5等生长因子对软骨细胞的作用多呈修复性.细胞因子的作用并非单一作用,而是存在于一个复杂的调节网络之中.目前对这些细胞因子在软骨损伤与修复中的作用研究已取得一定的进展.     

骨折愈合过程中转化生长因子—β1基因表达

目的：研究转化生长因子－β１基因在骨折后不同修复细胞中的表达及其变化，了解其在骨折愈合过程中的来源及产生机理。材料与方法：大鼠肋骨骨折标本，制作脱钙石蜡切片，用原位杂交技术检测骨折修复细胞中的转化生长因子－β１信使核糖核酸。结果：骨折后２、５、１０、１５天分别在相应修复细胞中检测到转化生长因子－β１ｍＲＮＡ的阳性信号，即骨膜生发层细胞、膜内成骨区的成骨细胞、软骨形成区的软骨细胞及软骨内成骨区的成骨细胞。其中成熟软骨细胞内信号最强，而肥大软骨细胞内显示阴性信号。结论：转化生长因子－β１可以在骨折局部由修复细胞自身产生，其产生量与细胞的分化状态、功能水平有关，它可能以自分泌或旁分泌方式参与骨折愈合的调节。     

β-转化生长因子诱导骨生成局部肿瘤坏死因子-αmRNA和蛋白质的表达

β－转化生长因子（transforminggrowthfactorbeta,TGF－β）作为一种多功能的细胞因子,可调节多种正常细胞或瘤细胞的生长、分化犤１犦。TGF－β在体内无骨形态发生蛋白（bonemorphogeneticprotein,BMP）的诱导异位成骨作用,但是在骨组织中可启动新生骨与软骨的生成犤２犦。近来的研究表明,肿瘤坏死因子－α（tumornecrosisfactoralpha,TNF－α）可由T细胞、自然杀伤（naturalkiller,NK）细胞、中性粒细胞、内皮细胞及平滑肌细胞等多种细胞分泌,参与细胞生长分化、免疫和炎症等过程。此外,TNF－α对骨代谢也有重要的调节…     

TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats.

TNF-alpha is known to inhibit osteoblast differentiation in vitro and yet it is essential for bone fracture repair. Roles of TNF-alpha in the bony repair of injured growth plate were examined in young rats treated with a TNF-alpha antagonist. The results show that TNF-alpha mediates p38 activation, which influences the recruitment, proliferation, and osteoblast differentiation of mesenchymal cells and negatively regulates bone formation at the injured growth plate. INTRODUCTION: TNF-alpha inhibits expression of osteoblast differentiation factor cbfa1 and osteoblast differentiation in vitro and yet TNF-alpha signaling is essential for bone fracture healing. Roles of TNF-alpha in the bony repair of injured growth plate cartilage are unknown. MATERIALS AND METHODS: Roles of TNF-alpha in the activation of p38 mitogen activated protein (MAP) kinase and the subsequent bony repair of the injured growth plate were examined in young rats receiving the TNF-alpha inhibitor ENBREL or saline control. Activation of p38 was determined by Western blot analysis and immunohistochemistry. Inflammatory cell counts on day 1, measurements of repair tissue proportions, and counting of proliferative mesenchymal cells on day 8 at growth plate injury site were carried out (n = 6). Expression of inflammatory cytokines TNF-alpha and IL-1beta, fibrogenic growth factor (FGF)-2, cbfa1, and bone protein osteocalcin at the injured growth plate was assessed by quantitative RT-PCR. Effects of TNF-alpha signaling on proliferation, migration, and apoptosis of rat bone marrow mesenchymal cells (rBMMCs) and the regulatory roles of p38 in these processes were examined using recombinant rat TNF-alpha, ENBREL, and the p38 inhibitor SB239063 in cultured primary rBMMCs. RESULTS: p38 activation was induced in the injured growth plate during the initial inflammatory response, and activated p38 was immunolocalized in inflammatory cells at the injury site and in the adjacent growth plate. In addition, activation of p38 was blocked in rats treated with TNF-alpha antagonist, suggesting a role of TNF-alpha in p38 activation. Whereas TNF-alpha inhibition did not alter inflammatory infiltrate and expression of TNF-alpha and IL-1beta at the injured growth plate on day 1, it reduced mesenchymal infiltrate and cell proliferation and FGF-2 expression on day 8. Consistently, TNF-alpha increased proliferation and migration of rBMMCs in vitro, whereas p38 inhibition reduced rBMMC proliferation and migration. At the injured growth plate on day 8, TNF-alpha inhibition increased expression of cbfa1 and osteocalcin and increased trabecular bone formation at the injury site. There was a significant inverse correlation between TNF-alpha and cbfa1 expression levels, suggesting a negative relationship between TNF-alpha and cbfa1 in this in vivo model. CONCLUSIONS: These observations suggest that TNF-alpha activates p38 MAP kinase during the inflammatory response at the injured growth plate, and TNF-alpha-p38 signaling seems to be required for marrow mesenchymal cell proliferation and migration at the growth plate injury site and in cell culture. Furthermore, TNF signaling has an inhibitory effect on bone formation at the injured growth plate by suppressing bone cell differentiation and bone matrix synthesis at the injury site.     

Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area

Injury to the growth plate is common and yet the injured cartilage is often repaired with undesirable bony tissue, leading to bone growth defects in children. Using a rat tibial growth plate injury model, our previous studies have shown sequential inflammatory, fibrogenic, osteogenic and bone maturation responses involved in the bony repair. However, it remains unclear whether there is progressive accumulation of bone within the injury site and any potential degenerative changes at the adjacent non-injured area of the growth plate. This study examined effects of growth plate injury on the structure, composition and some cellular and molecular changes at the injury site and adjacent uninjured area. Micro-CT analysis revealed that while the bone volume within the injury site at day 14 was small, the bone bridge was considerably larger at the injury site by 60 days post-injury. Interestingly, formation of bone bridges in the adjacent uninjured area was detected in 60% of injured animals at day 60. Immunohistochemical analyses revealed reduced chondrocyte proliferation (PCNA labelling) but increased apoptosis (nick translation labelling) in the adjacent uninjured area. RT-PCR analysis on adjacent uninjured growth plate tissue found increased expression of osteocalcin at day 60, differential expression of apoptosis-regulatory genes and alterations in genes associated with chondrocyte proliferation/differentiation, including Sox9 and IGF-I. Therefore, this study has demonstrated progressive changes in the structure/composition of the injury site and adjacent uninjured area and identified cellular and molecular alterations or degeneration in adjacent uninjured growth plate in response to injury.     

Transforming growth factor-β1 and fibroblast growth factors in rat growth plate

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-β1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-β1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-β1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope. Interestingly, extracellular staining for transforming growth factor-β1 was observed only around chondrocytes in the hypertrophic zone. These results suggest a role for these growth factors in the regulation of proliferation and maturation of chondrocytes and in endochondral ossification.     

Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats

With the intensified use of chemotherapy and improved survival rates for childhood malignancies, it has become increasingly apparent that some children or adult survivors show poor bone growth and develop osteoporosis. As a step to investigate underlying mechanisms, this project examined short-term effects in rats of chemotherapy agent 5-fluorouracil (5-FU) on cell proliferation, apoptosis, and bone formation at tibial growth plate cartilage and its adjacent bone-forming region metaphysis. In addition, since insulin-like growth factor (IGF-I) is important for bone growth, we examined whether IGF-I pretreatment would potentially protect growth plate cartilage and bone cells from chemotherapy damage. Two days after a single high dose of 5-FU injection, proliferation of growth plate chondrocytes and metaphyseal osteoblasts/preosteoblasts was dramatically suppressed, and apoptosis was induced among osteoblasts and preosteoblasts. As a result, there was a reduction in the chondrocyte number and zonal height at the proliferative zone and a decline in the number of osteoblasts and preosteoblasts on the metaphyseal trabecular bone surface. At day 2, no obvious deleterious effects were observed on the height of the growth plate hypertrophic zone and the bone volume fraction of the metaphyseal primary spongiosa trabeculae. At day 10, while cell proliferation and growth plate structure returned to normal, there were slight decreases in trabecular bone volume, body length increase, and tibial length. While pretreatment with 1-week IGF-I systemic infusion did not attenuate the suppressive effect of 5-FU on proliferation in both growth plate and metaphysis, it significantly diminished apoptotic induction in metaphysis. These results indicate that growth plate cartilage chondrocytes and metaphyseal bone cells are sensitive to chemotherapy drug 5-FU and that IGF-I pretreatment has some anti-apoptotic protective effects on metaphyseal bone osteoblasts and preosteoblasts.     

Neurotrophin‐3 Induces BMP‐2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill‐hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT‐3, and NT‐4 and their Trk receptors. NT‐3 and its receptor TrkC showed the highest induction. NT‐3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT‐3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT‐3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP‐2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT‐3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP‐2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT‐3 osteogenic effect in vitro because it can be almost completely abrogated by co‐addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT‐3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co‐treatment with anti‐VEGF. This study suggests that NT‐3 may be an osteogenic and angiogenic factor upstream of BMP‐2 and VEGF in bony repair, and further studies are required to investigate whether NT‐3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.     

骨骺损伤修复过程中骺板形态及VEGF表达的变化

 目的 探讨大鼠骨骺损伤修复过程中骺板形态结构及骺板内血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)表达的变化。方法 取4~5 周龄SD大鼠30 只, 制作胫骨近端骨骺损伤动物模型。随机分为五组, 每组6 只, 分别于术后2、4、6、10、21 天处死, 取双侧胫骨。测量双侧胫骨长度, 计算实验侧长度和对照侧长度比值。行双侧胫骨X 线摄片、Micro CT扫描, 观察骺板大体形态及骺板内骨桥形成情况。通过组织切片HE 染色及免疫组织化学染色, 观察骺板内软骨细胞变化及VEGF表达。结果 双侧肢体长度于术后第4 天出现差异, 第10 天差异最大, 第21 天肢体长度差异缩小。术后第6 天开始骺板内逐渐出现纤细骨质, 终至骨桥形成。HE 染色提示伤后早期出现静止区软骨细胞聚集成团, 细胞规律性排列丧失、分化加速。免疫组织化学染色显示术后生长板内出现VEGF 高反应区, 阳性表达区逐渐扩大, 跨越骺板全层, 直后血管长入、骨化。结论 骨骺损伤修复过程中, 骺板形态早中期表现为干骺端肥大, 肢体短缩, 骺板内纤细骨质形成;随修复进展, 骺板内VEGF 表达反应性增强, 出现贯通骺板的表达带, 与骨桥形成相关。     

Fibroblast growth factor expression in the postnatal growth plate

Fibroblast growth factor (FGF) signaling is essential for endochondral bone formation. Mutations cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. Most previous work in this area has focused on embryonic chondrogenesis. To explore the role of FGF signaling in the postnatal growth plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Toward this aim, rat proximal tibial growth plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. Growth plate expressed FGFs 2, 7, 18, and 22. Perichondrial expression was generally greater than growth plate expression, supporting the concept that perichondrial FGFs regulate growth plate chondrogenesis. Nevertheless, FGFs synthesized by growth plate chondrocytes may be physiologically important because of their proximity to target receptors. In growth plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL1, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing growth velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF1, FGF7, FGF18, and FGF22 were upregulated. In summary, we have analyzed the expression of all known FGFs and FGFRs in the postnatal growth plate using a method that is quantitative and highly sensitive. This approach identified ligands and receptors not previously known to be expressed in growth plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the growth plate. We also found temporal changes in FGF and FGFR expression which may contribute to growth plate senescence and thus help determine the size of the adult skeleton.     

Impaired growth plate function in bmp-6 null mice

Bone morphogenetic protein 6 (BMP-6) is expressed by different skeletal cells including osteoblasts and growth plate chondrocytes, suggesting roles in bone formation and growth regulation. To address these possibilities, we examined whether cancellous and cortical bone parameters, or indices of growth plate function, are altered in bmp-6 null mice as assessed under basal conditions, and following stimulation of bone formation and suppression of growth by estrogen treatment. Ten-week-old female littermate bmp-6 null and wild-type (WT) mice were administered vehicle or E(2) 4, 40, 400 or 4,000 microg/kg/day by daily sc injection for 28 days (6-8 per group). Tibias were removed, and detailed histomorphometric analysis of the proximal metaphysis and growth plates, and tibial diaphysis were performed on longitudinal and transverse sections respectively. Long bone area as measured by DXA was reduced in vehicle-treated bmp-6 null mice compared with WT littermate controls. In addition, vehicle-treated bmp-6 null mice had a reduced cross-sectional area at the tibial mid-diaphysis as assessed by histomorphometry, whereas cancellous bone indices were unaffected. Histomorphometry of the proximal tibial metaphysis demonstrated a defect in bone formation immediately adjacent to the growth plate in bmp-6 null mice compared to WT mice following E(2) treatment. E(2) administration was also associated with a dose-responsive decrease in longitudinal growth rate, and proliferative and hypertrophic zone parameters of the growth plate (p<0.0001). Significantly greater reductions following E(2) treatment were observed in longitudinal growth rate (p<0.01), proliferating and hypertorphic zone widths (p<0.001), and proliferating (p<0.0002) and hypertrophic (p<0.002) cells per column of bmp-6 null mice compared to WT mice. Our observation that long bones are reduced in size compared to wild-type mice primarily through a decrease in cortical cross-sectional area, whilst cancellous bone mass is unaltered, suggests a non-redundant role for BMP-6 in periosteal but not trabecular bone formation. Moreover, growth plate function was reduced in bmp-6 null mice receiving estrogen, leading to an impaired cancellous bone response to estrogen at the highest dose, suggesting that BMP-6 also plays a physiological role in maintaining growth plate function.