大鼠胫骨骺板软骨细胞P物质的表达

目的 研究P物质（SP）在大鼠胫骨骺板软骨细胞中的表达及其在长骨纵向生长中的意义。方法 应用免疫组化技术检测8周龄大鼠胫骨骺板不同组织学层次软骨细胞的SP表达状况。结果 骺板静止及增殖期软骨细胞未见SP免疫阳性染色，肥大区及钙化区软骨细胞可见SP免疫组化阳性染色。结论 骺板肥大区及钙化区软骨细胞可以表达SP，提示SP在长骨的纵向生长中可能起调节作用。     

组织工程骺板软骨移植修复兔胫骨上骺板缺损

目的　探讨在体外以三维支架构建的组织工程骺板软骨修复胫骨上骺板缺损的效果 ,了解促进植入工程化软骨与受区骺板融合方法的效果。　方法　酶消化分离幼兔骺板软骨细胞并接种培养 ,收获第 1代软骨细胞 ,制成 2 .5× 10 7/ ml的细胞凝胶混悬液 ,接种于聚磷酸钙纤维 / L-聚乳酸 (CPPf/ PL L A)复合支架体外构建组织工程化软骨。采用幼兔右侧胫骨上骺板 4 0 %缺损模型 ,将日本大耳白兔 72只分为 4组 ,每组 18只 :A组缺损区植入同窝幼兔来源的工程化软骨 ,并以相同的细胞凝胶混悬液充填其间隙 ;B组充填复合有生物凝胶而无细胞的 CPPf/ PL L A支架材料 ;C组植入皮下脂肪 ;D组不做任何充填。分别于术后 2、4、6、8、12和 16周摄 X线片、大体及组织学观察。　结果　A组术后2周组织工程化软骨在骺板缺损区即衍生为典型的骺板组织结构 ,并与受区骺板组织很好融合 ;4周修复骺板组织 ,结构正常、胫骨无畸形 ;8周后骺板修复区出现早闭的组织学表现 ,胫骨出现短缩和内翻畸形 ;16周修复区骺板已闭合 ,胫骨畸形明显 ,生长功能恢复率为 4 3.6 %。B、C、D3组术后 2周胫骨即出现畸形 ;4周骺板缺损区均已骨性闭合 ,畸形明显 ;16周畸形严重。后三组间差异无统计学意义 ,骺板缺损区无骨生长。　结论　组织工程化的骺板软骨     

兔髂骨骺板软骨细胞体外构建骺板样软骨组织

目的　利用组织工程学技术体外构建骺板样软骨组织。方法　从 4～ 5周龄兔髂骨骺板软骨处获取软骨细胞 ,在离心管内轻微离心后 ,体外培养。行组织学观察。结果　培养至第 7天时 ,细胞呈现定向分化 ,形态与体内骺板软骨细胞相类似 :肥大软骨细胞体积较大、呈圆形或椭圆形 ;增殖、成熟软骨细胞体积小 ,呈圆形或扁圆形 ;细胞周围充满大量的细胞外基质。这些不同分化阶段的细胞形成了分化区带 ,肥大软骨细胞位于上侧 ,增殖、成熟细胞位于中间 ,其次是散在静止软骨细胞。培养第 14天 ,分化区带更加明显 ,增殖、成熟细胞和肥大软骨细胞呈现纵向定向排列。培养第 2 1天 ,组织表面出现膜样的结构。结论　体外构建的骺板样软骨组织与天然骺板的组织学形态极为相似。从髂骨处骺板处获取肥大软骨细胞进行体外构建骺板软骨材料 ,更具有临床实用性     

兔髂骨骺板软骨细胞体外构建骺板样软骨组织

目的 利用组织工程学技术体外构建骺板样软骨组织。方法 从4～5周龄兔髂骨骺板软骨处获取软骨细胞，在离心管内轻微离心后，体外培养。行组织学观察。结果 培养至第7天时，细胞呈现定向分化，形态与体内骺板软骨细胞相类似：肥大软骨细胞体积较大、呈圆形或椭圆形；增殖、成熟软骨细胞体积小，呈圆形或扁圆形；细胞周围充满大量的细胞外基质。这些不同分化阶段的细胞形成了分化区带，肥大软骨细胞位于上侧，增殖、成熟细胞位于中间，其次是散在静止软骨细胞。培养第14天，分化区带更加明显，增殖、成熟细胞和肥大软骨细胞呈现纵向定向排列。培养第21天，组织表面出现膜样的结构。结论 体外构建的骺板样软骨组织与天然骺板的组织学形态极为相似。从髂骨处骺板处获取肥大软骨细胞进行体外构建骺板软骨材料，更具有临床实用性。     

骺板损伤影像学诊断的进展

骨骺及软骨板损伤又称为骺板损伤，是儿童期常见的骨骼创伤，有时诊断比较困难。临床容易误诊和漏诊，主要原因是骨骺部的软骨在X线片上不显影，因此，通过软骨部份的骨折线不能被直接看到。但诊断上的困难并非无法克服。只要我们认真分析临床表现，细心观察影像学资料，善于总结经验就能找出规律。     

骺板软骨细胞复合三维支架体外构建组织工程软骨的研究

目的探讨将骺板软骨细胞复合三维支架经体外培养,构建组织工程软骨的效果及其生物学特点. 方法将3周龄幼兔第1代骺板软骨细胞与液态的生物凝胶混合,接种于聚磷酸钙纤维/L-聚乳酸(CPPF/PLLA)三维支架材料,构建组织工程软骨组织块,连续培养4周.行大体、倒置显微镜及组织学、Ⅰ型和Ⅱ型胶原免疫组织化学光镜观察,定量检测硫酸糖胺多糖(GAG)含量. 结果构建的组织工程软骨块在培养过程中能保持其初始外形,种子细胞呈稳定的三维均相分布,外观逐渐呈乳白色、半透明,硬度亦不断增加.培养1周有软骨细胞陷窝形成,2周后形成富含Ⅱ型胶原和蛋白聚糖、具有典型软骨组织结构的工程化软骨,且Ⅰ型胶原逐渐转为阴性.4周时构建软骨的组织结构与天然骺板软骨相类似,硫酸GAG含量平均为天然骺板软骨的34%以上. 结论骺板软骨细胞复合三维支架体外培养可生成典型软骨,且可形成类似天然骺板软骨的组织结构,能满足修复骺板缺损的基本要求.体外培养1～2周可能是植入体内修复骺板缺损的较佳时机.     

可吸收内固定材料对骺板影响的研究进展

目前，可吸收内固定材料的基础和临床研究日益增多。本文综述了可吸收内固定材料的生物降解吸收性能、机械性能及其对骨矿物质密度的影响，骺板的结构及损伤，可吸收内固定材料对骺板的影响及临床应用研究，并指出存在的问题和今后研究的重点。     

骺板损伤的诊断与治疗进展

骺板损伤可导致骨骺与干骺端之间形成骨桥，骺板提前闭合，造成肢体短缩和(或)成角畸形，影响儿童生长发育。早期诊断较困难，X线、CT、MRI等影像学检查及“骨桥地图法”可协助诊断。传统治疗有骨桥切除后以脂肪、骨水泥等填塞，但效果不一。近年来，随着显微外科及组织工程等新技术的开展，用培养的骺软骨细胞、间充质干细胞等移植代替无活力的填充物植入，它能不断增殖并抑制骨桥形成，将为骺板损伤治疗提供更好的解决方案。本文结合国内外最新研究成果对骺板损伤的现状进行回顾，并对其前景作一展望。     

胎儿及婴儿股骨近端骺板的组织学结构及发育演变

作者应用胎儿，死后婴儿股骨近端进行大体解剖和切片组织学检查，对骺板各层发育演变进行了描述，根据其组织学特点将骺板分为长短两臂，长臂由颈骺板和转子骺板组成，短臂由头骺板和钩骺板构成。２岁时颈骺板消失；观察了骺板内血管的分布形式。胎儿时间有交通血管存在，来源于骨骺侧，至新生儿期消失，血运屏障建立，骨骺和干骺端形成两个独立的血液循环系统，探讨了软骨周围骨化沟与骺板横径增殖的关系，认为骨化沟参与骺板横径增     

低营养要素对大鼠骺板和干骺端发育的影响

目的 配制低营养要素饲料,比较低营养要素、病粮以及普通饲料喂养大鼠骺板和干骺端发育的差异,探讨低营养要素对大鼠骺板和干骺端发育的影响以及对大骨节病病因研究的启示.方法 Wistar大鼠78只雌雄各半,体重60～65g,随机分为三组:A组为普通饲料组;B组为低营养要素饲料组;C组为病粮饲料组,每组26只.于0周各组处死2只大鼠,第1、2、3、4周取材,每次每组6只大鼠取左膝关节为标本.大体观察后,行HE染色和Masson染色.结果 低营养要素饲料为低蛋白、低维生索、低硒.B组在第4周可见灶状坏死,钙化带细胞排列不整齐,骨化线不整齐,干骺端骨小梁排列紊乱,有较多成纤维细胞充填.结论 低营养要素饲料可使大鼠骺板软骨细胞出现灶状坏死,干骺端发育有异常.这些异常是否和大骨节改变有关系有待进一步研究.     

大鼠胫骨生长板软骨细胞的分离与培养鉴定

目的 探讨高效生长板软骨细胞分离方法 和体外培养条件.方法 采用二步酶消化法对6只SD大鼠胫骨生长板的软骨细胞进行分离,采用含炭吸附过的胎牛血清培养基培养,按5×105个/瓶的密度接种细胞并对软骨细胞进行形态学观察和鉴定,描绘原代软骨细胞在无激素培养基中的生长曲线.结果 软骨细胞贴壁较慢,12 h后开始附壁,第8天时90%融合,互相连接成"铺路石"样结构.原代软骨细胞胞质Ⅱ型胶原免疫着色强阳性,传代后染色减弱.结论 本研究所采用的方法 能高效快速获得原代软骨细胞,原代软骨细胞最接近体内生理状态,最适合进行实验研究.     

Steindler lecture. Binding sites in fetal and growth plate cartilage

In addition to genetic and nutritional factors, linear growth during the prenatal and postnatal periods is controlled by peptide, steroid, and thyroid hormones interacting with the receptors present on the membrane or in the cytosol and nuclei of growth plate cartilage. Using standard procedures, insulin and "nonsuppressible insulin-like activity" (a somatomedin) showed significant binding in 600, 15,000, and 105,000 g membrane fractions of epiphyseal cartilage of immature animals. The binding of growth hormone and prolactin was small and probably not significant. Specific uptake of glucocorticoid was demonstrated in viable canine chondrocytes, but not of androgen, estrogen, or vitamin D3 metabolite. A triiodothyronine receptor was present in nuclei from dog epiphyseal cartilage. Hormones that lack binding may affect cartilage only indirectly. Hormone receptors were studied in those portions of fetal growth cartilage that will later evolve into an ossification center, articular cartilage, and epiphyseal cartilage. Cytosol fractions contained a receptor for glucocorticoid but not for androgen or estrogen. Zonal analysis showed a higher level in the peripheral and central sections than in the palisade section. Triiodothyronine binding was also detected in nuclei prepared from whole fetal cartilage. Heterogeneity of cell function was obvious in fetal cartilage. Cell division was high in the central and peripheral zones as well as the upper half of the palisade zone, but low in the lower palisade section. Proline and sulfate incorporation predominated in the palisade section compared with the central and peripheral sections. Disease states with changes of metabolic activities in the cartilage may perhaps be better understood with a clearer knowledge of receptor levels and interactions.     

Regeneration of growth plate cartilage induced in the neonatal rat hindlimb by reamputation

Following primary hindlimb amputations dividing the lower femur or the central tibiofibula, the neonatal rat innately regenerates the distal growth plate(s) with a frequency of about 20-30%. One or two reamputation procedures were performed in an effort to increase the frequency of physeal regeneration, noting that such procedures, and related forms of tissue stimulation, have been repeatedly shown to induce regenerative growth at limb amputation sites of some amphibians that display little innate regenerative capacity. The present reamputation sequences divided the skeletal stump through the cartilaginous mass arising at its distal end. Following first reamputation an approximate three fold increase in the frequency of growth plate cartilage regeneration was observed at transfemoral and transtibiofibular sites. Only after second reamputation, however, did tibiofibular physeal cartilage regeneration equal in frequency that observed after first reamputation through the lower femur. Ectopic growth plate cell architecture was identified in cartilaginous extensions arising from the side of the distal femoral shaft, and also within the regrown secondary cartilage body, which unites the lower tibia and fibula in the shank of the rat. Moreover, among 3 of 11 femoral amputees that had sustained reamputations, regrowth of the distal femoral condylar mass and profile were achieved to varying degrees. It is concluded that a regimen of reamputation, known to induce regenerative growth in the amphibian limb, also induces skeletal regneration in the mammalian limb, and lead to the appearance of ectopic growth plate cell architecture at adjacent sites.     

Molybdenum-induced changes in the epiphyseal growth plate

Summary Molybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.     

Reimplantation of growth plate chondrocytes into growth plate defects in sheep

Defects in growth plates due to trauma, infection, or genetic causes can result in bone formation across the defect, bridging the epiphysis and metaphysis, resulting in growth arrest and limb deformation. We have investigated the capacity of implanted chondrocyte cultures to prevent this process. Sheep growth plate chondrocytes were isolated, and after culture at high density produced easily manipulated cartilaginous discs. The tissue was implanted into growth plate defects produced in lambs and the response was assessed histologically. Following implantation, cultures continued to proliferate and maintain a cartilage-like matrix. After 8 to 12 weeks, hypertrophic maturation chondrocyte columnation, and associated endochondral calcification were observed. Culture implantation was always associated with local immune inflammatory reaction, which continued throughout the course of investigation. Cellular survival was variable and resulted in the presence of viable implants as well as residual cartilage matrix devoid of chondrocytes; however, implanted chondrocyte discs always prevented bone bridge formation. These findings encourage the expectation that cultured chondrocytes may provide a useful replacement for the inert interpositional materials currently used in the treatment of growth arrest. The potential of this technique for growth plate replacement, however, requires a more predictable rate of implant survival. The likely reasons for implant loss are discussed.     

Growth differentiation factor-5 stimulates the growth and anabolic metabolism of articular chondrocytes

Objective： To observe the effect of growth differentiation factor-5 （GDF-5） on the growth and anabolic metabolism of articular chondrocytes. Methods： The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱ collagen by RT-PCR, the collagen phenotypic expression of chondrocytes detected by immunofluorescence. Results： After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱcollagen because of the col2al mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/ml, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Chondrocytes were cultured with GDF-5 for 21 days, immunofluorescent staining of type Ⅱ collagen was clear, the type Ⅰ and Ⅹ collagen were negative. Conclusion： GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation, but did not change the collagen phenotypic expression of chondrocytes in mono-layer culture.     

Cytosolic calcium concentration in bovine growth plate chondrocytes

The cytosolic free calcium ion concentration for mammalian cell systems is believed to be maintained within a narrow range compatible with cellular homeostasis. Growth plate chondrocytes have been shown to accumulate large quantities of calcium within their mitochondria, but the cytosolic free calcium concentration has not been determined. This study measures the cytosolic free ionic calcium concentration in growth plate chondrocytes using two variations of the Quin II fluorescence technique. The results indicate that in isolated growth plate chondrocytes, the cytosolic free ionic calcium concentration is similar to other nonmineralizing mammalian cell types (106–137 nM).     

Effect of growth hormone on fluoride balance

Fluoride balances were determined in nine children, aged 4 to 18 years, undergoing treatment with human growth hormone. Urinary F was increased in 6 of the 9 subjects during the period of initial treatment. The hyperfluoruria occurred in the face of a preexisting negative F balance. Fecal F did not change, and since the magnitude of the hyperfluoruria could not be correlated with changes in renal function it is likely that it, together with the increases in Ca and hydroxyproline excretion, represents a direct effect of the hormone (or possibly “sulfation factor”) on bone resorption. During the control periods, the F balance data reveal that 10–90% of dietary F (all of which came from food) appeared in the feces; these values are generally higher than those reported for subjects whose intake was primarily from water.     

Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography

The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.     

The role of hepatocyte growth factor in growth plate cartilage

To investigate the physiological role of hepatocyte growth factor (HGF) in endochondral bone formation, we examined the expression of HGF and its receptor c-met and the effects of HGF on growth plate chondrocytes. HGF was highly expressed in the prehypertrophic zone and hypertrophic zone in rat costal growth plate cartilage. The expression of HGF increased in rabbit chondrocytes as they matured in culture. Conversely, c-met expression was down regulated along maturation of growth plate chondrocytes. HGF had weak stimulatory effects on DNA and proteoglycan synthesis of growth plate chondrocytes. However, HGF strongly inhibited expression of terminal differentiation-related phenotypes, such as type X collagen and alkaline phosphatase (APase) synthesis and cartilage matrix mineralization. When HGF was removed from the cultures, cells quickly expressed type X collagen and APase. Once chondrocytes differentiated to mature chondrocytes, HGF did not inhibit further differentiation of these cells. These results suggested that HGF is a negative regulator of terminal differentiation of growth plate chondrocytes.. Received: Feb. 12, 1998 / Accepted: March 12, 1998