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.     

Dwarfism in the Alaskan Malamute: Ultrastructural features of dwarf growth plate chondrocytes

Summary This study was performed in order to reexamine the ultrastructural morphology of the chondrocytes in the growth plates of dwarf Alaskan Malamutes and to obtain semiquantitative cytochemical data about the proteoglycans. Growth plates from age-matched dwarf and homozygous nonaffected Alaskan Malamutes were processed for routine transmission electron microscopy and also stained with ruthenium red. Chondrocytes in dwarf plates were observed to occur in clumps or cell nests. Within some of these nests, chondrocytes in the upper half of the zone of chondrocyte proliferation had bizarre shapes ranging from V-shaped to whorled or rounded. These chondrocytes contained profiles of markedly dilated rough endoplasmic reticulum (RER). Material within the RER cisternae stained positively with ruthenium red and was partially digestible with testicular hyaluronidase. The material could, therefore, represent either chondroitin sulfate or hyaluronate. The RER in these dwarf chondrocytes was not oriented parallel to the long axis of the cells; instead, it consisted of irregularly dilated cisternae. Granule counts performed on the zone of chondrocyte proliferation revealed a significant decrease in the number of ruthenium red granules in the interterritorial matrix of dwarf chondrocytes when compared to those of the homozygous nonaffected chondrocytes.     

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).     

Ultrastructure of the rat epiphyseal growth plate following chronic ethanol ingestion

Summary We have previously demonstrated that ethanol has a direct toxic effect on the rat skeleton characterized by decreased trabecular bone volume. In the present study, we examined the ultrastructure of the distal radial epiphyseal growth plates in these same animals. Eight weeks of ethanol administration to 12 male rats results in serum alcohol levels of 140 mg/dl but did not alter the width or light microscopic appearance of the radial growth plate. Quantitative electron microscopy failed to demonstrate morphologic evidence of toxicity in the skeletal cells. We conclude that although ethanol appears to have a direct effect on rat bone characterized by enhanced resorption, toxicity is not attended by ultrastructural changes in the skeletal cells.     

组织工程软骨移植修复兔生长板缺损

目的探讨一种新的修复生长板缺损的方法,应用组织工程软骨治疗生长板缺损并发肢体畸形。方法分离收集1月龄兔关节软骨,于离心管内培养2周形成组织工程软骨。36只6周龄新西兰白兔随机分为3组,于右侧胫骨上端生长板内侧造成1/3～1/2缺损,A组即刻植入培养软骨,B组及C组分别于术后4周、术后8周再次手术,切除缺损内修复组织后再移植培养软骨。左侧胫骨经相同手术造成缺损,但无植入物充填,仅作为对照组。A组于手术后16周、B组于再手术后12周、C组于再手术后8周进行双下肢X线、组织学及免疫组织化学等检查,测量双侧胫骨长度和胫骨角,观察生长板缺损修复情况。结果培养软骨呈圆盘状,直径8mm,厚1.5mm。A、B组右侧胫骨生长显著优于左侧,内翻和短缩畸形右侧较左侧轻(P<0.01)。A、B组右侧生长板缺损经组织修复后恢复正常生长板结构和性质,Ⅱ型胶原和IGF-IRa免疫组织化学染色呈阳性;左侧胫骨生长板缺损由新生骨充填。C组双侧胫骨均发生严重畸形(P>0.05),生长板闭合或接近闭合。结论离心管培养组织工程软骨移植可以有效防止4周内生长板缺损并发早期肢体畸形,但对生长板缺损8周已形成畸形者无效。     

过量氟化物摄入对实验大鼠胫骨骺板软骨细胞分化的影响

目的：观察及分析过量氟化物对大鼠骺板软骨生长发育的毒性作用。方法：SD大鼠分为对照组,低过量氟组和高过量氟组,分别于实验10周和20周观察大鼠胫骨骺板软骨光镜,电镜下组织形态学变化。结果：与对照组比较,光镜下过量氟组大鼠骺板软骨增厚,增殖层软骨细胞和肥大层软骨细胞滞留和堆积,排列紊乱,并出现软骨半岛,软骨岛和原纤维显露,且骺板软骨损伤随染氟剂量和时间延长而明显加重,电镜下骺板软骨细胞坏死增加,存活细胞功能活跃,可继续合成和分泌胶原蛋白。结论：过量氟对大鼠骺板软骨细胞分化有明显的损伤作用,可进一步干扰软骨内化骨。     

Alterations of the growth plate in chronic renal failure

Chronic renal failure modifies the morphology and dynamics of the growth plate (GP) of long bones. In young uremic rats, the height of cartilage columns of GP may vary markedly. The reasons for this variation are unknown, although the severity and duration of renal failure and the type of renal osteodystrophy have been shown to influence the height of GP cartilage. Expansion of GP cartilage is associated with that of the hypertrophic stratum. The interference of uremia with the process of chondrocyte differentiation is suggested by some morphological features. However, analysis by immunohistochemistry and/or in situ hybridization of markers of chondrocyte maturation in the GP of uremic rats has yielded conflicting results. Thus, there have been reported normal and reduced mRNA levels for collagen X, parathyroid hormone/parathyroid hormone-related peptide receptor, and matrix metalloproteinase 9, as well as normal mRNA and protein expression for vascular endothelial growth factor and chondromodulin I, peptides related to the control of angiogenesis. In addition, a decreased immunohistochemical signal for growth hormone receptor and low insulin-like growth factor I mRNA in the proliferative zone of uremic GP are supportive of reduced chondrocyte proliferation. Growth hormone treatment improves chondrocyte maturation and activates bone metabolism in the primary spongiosa.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Histomorphometric analysis of the epiphyseal growth plate in rats after prenatal alcohol exposure.

We studied the effect of prenatal alcohol exposure on growth in the proximal tibial growth plate in 0- and 15-day-old rats, using histomorphometric methods. Body weight and tibial length were reduced in all alcohol-exposed rats. In 15-day-old rats, these parameters were lower than in the 15-day-old controls, thus showing a persistence of the effects of ethanol. The proximal tibial growth plate showed alterations, principally in 15-day-old rats. The most notable of these was a decrease in growth plate height produced by a significant reduction in hypertrophic zone height. Likewise, there were fewer cells in this zone in alcohol-exposed rats than in controls. This work shows that prenatal ethanol exposure induces growth retardation which may be due to growth plate alterations that might reflect impaired cell function.     

Impact of growth plate senescence on catch-up growth and epiphyseal fusion

In mammals, longitudinal bone growth occurs rapidly in prenatal and early postnatal life, but then slows and eventually ceases. This deceleration, which reflects a decline in chondrocyte proliferation, was previously attributed to a hormonal or other systemic mechanism. However, new evidence suggests that it is due to a local mechanism within the growth plate. In particular, recent findings suggest that growth plate chondrocytes have a finite proliferative capacity that is gradually exhausted, causing growth to slow and finally stop. This concept has provided insight into clinical phenomena including catch-up growth after transient growth inhibition, catch-down growth after transient estrogen exposure, and epiphyseal fusion.J. Baron is a commissioned officer in the U.S. Public Health ServiceThis work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Structural changes in reassembled growth plate aggregates

To investigate possible structural changes in reassembled proteoglycan aggregates during cartilage mineralization, we examined the molecular architecture and dimensions of growth plate proteoglycan aggregates by electron microscopy. The ends of fetal bovine femurs and tibias were separated into three regions: the epiphysis; the cartilage growth plate, consisting of the proliferative zone and the unmineralized portion of the hypertrophic zone; and the calcified portion of the hypertrophic zone along with part of the metaphysis. Aggregates from all three regions had the same molecular architecture. They consisted of central hyaluronic filaments with multiple attached monomers. Monomers consisted of two segments: a peripheral thick segment, which represents primarily the chondroitin sulfate-rich region, and a thin segment attached directly to the hyaluronic acid filament. The length of aggregated monomers did not differ between the growth plate cartilage and the metaphysis, nor did the lengths of the thin and thick segments, indicating that the chondroitin sulfate-rich region of aggregated monomers is not degraded during cartilage mineralization. Between the growth plate cartilage and the metaphysis, aggregates became shorter and had fewer monomers and wider spacing between monomers. These structural alterations in proteoglycan aggregates may be one of the events that prepares the matrix for mineralization.     

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 × 10⁶ cells per 35-mm plate and grown for 17 days in BGJ_b medium supplemented with 10% fetal bovine serum, 50 μg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive ⁴⁰Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.     

Increased cAMP production after short-term capacitively coupled stimulation in bovine growth plate chondrocytes

Growth plate chondrocytes from newborn calf costochondral junctions grown in monolayer were subjected to a capacitive AC signal of 500 V peak to peak (P-P) at 60 kHz for 48 h and were analyzed for [3H]thymidine uptake. The stimulated chondrocytes showed a 130% greater uptake over unstimulated controls. Other newborn calf growth plate chondrocytes were stimulated at 500 V P-P at 60 kHz for 2.5, 5.0, 10.0, and 20.0 min and were analyzed for cAMP. Chondrocytes stimulated for 2.5 and 5.0 min showed a 142.8% (p less than 0.05) and 394.5% (p less than 0.01) increase over controls, respectively. The chondrocytes stimulated for 10.0 and 20.0 min showed no significant difference from the controls. It is concluded that short-term exposure of growth plate chondrocytes to an appropriate capacitively coupled field stimulates cAMP production, but longer-term application of the electrical field is ineffective.     

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.     

Demonstration of type I and type II somatomedin receptors on bovine growth plate chondrocytes

The chondrocytes of the epiphyseal growth plate are the presumed target cells for hormones regulating skeletal growth. The somatomedins, a family of low molecular weight peptides, are thought to play a stimulatory role in this regulation. The cellular actions of the somatomedins are themselves determined by binding to specific receptors on target cells. Previous studies have characterized a specific receptor for somatomedin-C (Sm-C) or insulin-like growth factor I (IGF-I) on bovine growth plate chondrocytes (GPCs). We now report the characterization of a second type of somatomedin receptor on these cells that is more specific for another class of somatomedin represented by multiplication-stimulating activity (MSA) or rat insulin-like growth factor II (rIGF-II). Binding of [125I]MSA/rIGF-II to isolated GPCs was time dependent and saturable. Unlabeled Mr 7,100 MSA/rIGF-II and Sm-C/IGF-I were approximately equipotent in competing with [125I]MSA/rIGF-II for binding, while Mr 8,600 MSA/rIGF-II was an order of magnitude less potent. Low levels of competition by insulin appeared in some studies at concentrations of 10(-7) M and higher, suggesting displacement of [125I]MSA/rIGF-II binding to the Sm-C/IGF-I receptor. In affinity-labeling studies, [125I]MSA/rIGF-I labeled a complex of Mr greater than 300,000 (unreduced) and of Mr 140,000 (reduced), consistent with a type I somatomedin receptor composed of disulfide-linked subunits. [125I]MSA/rIGF-II labeled a Mr 240,000 moiety (unreduced) and Mr 260,000 (reduced), consistent with a type II somatomedin receptor. Both affinity-labeling and kinetic data revealed cross-binding of MSA/rIGF-II and insulin with the type I receptor and of Sm-C/IGF-I with the type II receptor. In contrast, the type II receptor did not recognize insulin. These data suggest a complex pattern of graded specificity of these receptors for their ligands. These data are consistent with the hypothesis that IGF-II as well as Sm-C/IGF-I participate in the stimulation of skeletal growth.     

The effect of oxygen tension on proteoglycan synthesis and aggregation in mammalian growth plate chondrocytes

Growth plate chondrocytes isolated from the proliferative and hypertrophic zones of bovine costochondral junctions were grown in vitro in the presence of various oxygen tensions ranging from 3 to 60%. Using [35S] sulfate as an index of glycosaminoglycan synthesis, incorporation was found to be maximal at 21% O2. In contrast, proteoglycan aggregation under the same conditions was found to be maximal at 3% O2. There were no consistent differences in response between cells from the different morphologic zones even though they are exposed to different oxygen tensions in situ. These results show that proteoglycan synthesis and aggregation in growth plate chondrocytes in vitro are differentially affected by the ambient oxygen environment.     

Histological and biological changes in the epiphyseal plate during fracture healing

The alterations that the epiphyseal plate undergoes during fracture healing are well documented microscopically, yet there are no reports in the literature which discuss the cellular and molecular changes that accompany this process. We studied fracture healing in 49 Wistar rats (5 weeks old) in which we inflicted a fracture to the distal third of the femur of the right hind leg (experimental side). The rats were killed 2 weeks later, and we dissected both hind legs from the hip joint to the knee joint, detaching all the surrounding soft tissues. We manually detached the distal epiphyses and the epiphyseal plates from both femurs. A piece of the epiphyseal plate was removed from the epiphyseal side of the femurs. In 25 animals, we analyzed the DNA content. In 8 animals, the specimen was studied under an electron microscope, and in the remaining 16 animals, the control and experimental sides were studied histologically. We found that healing was accompanied by an increase in DNA content, by a change in cellular activity, and by greatly accelerated apoptosis. Received: December 7, 2000 / Accepted: August 22, 2001     

Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis

OBJECTIVE: Glucocorticoid treatment of children often leads to growth retardation, and the precise target(s) in the growth plate responsible for this effect are unknown. Angiogenesis is an important part of the endochondral ossification process, and VEGF expressed in the growth plate is essential for proper angiogenesis to occur. Since glucocorticoid treatment down-regulates VEGF expression in cultured chondrocytes, we hypothesized that in vivo glucocorticoid treatment could result in VEGF down-regulation in the growth plate and disturbed angiogenesis, thus contributing to the growth retardation. DESIGN: We treated 6-week-old prepubertal piglets (10 kg) for 5 days with prednisolone (50 mg/day). Tibial growth plate sections were studied for apoptosis and the expression of VEGF protein and mRNA and MMP-9 protein. Capillaries in the metaphysis were visualized by CD31 immunostaining. Growth plate morphology (width of various zones) was determined by interactive measurements on hematoxylin/eosin stained sections and apoptotic cells were detected by TUNEL assay. RESULTS: In the prednisolone-treated animals, the total width of the growth plate decreased to 81% of controls (P<0.02), which was explained by a decrease of the width of the proliferative zone to 73% (P<0.05). The treatment had no effect on the orderly organization of the chondrocyte columns. In the growth plates of control animals, apoptosis was shown in 5.8% of the hypertrophic chondrocytes and was limited to the terminal hypertrophic chondrocytes. In prednisolone-treated animals, 40.5% of the hypertrophic chondrocytes was apoptotic (P<0.02), with apoptotic chondrocytes also appearing higher in the hypertrophic zone. We observed fewer capillaries and loss of their parallel organization in the metaphysis in the prednisolone-treated animals. The capillaries were shorter and chaotic in appearance. In contrast to controls, in prednisolone-treated animals VEGF mRNA and protein could not be detected in the hypertrophic zone of the growth plate. Trabecular bone length in the primary spongiosa was also diminished by the treatment. No changes were observed in the expression pattern of MMP-9, a matrix metalloproteinase, which is also important for angiogenesis and bone formation. CONCLUSIONS: These results indicate that short-term glucocorticoid treatment of growing piglets severely disturbs the width of the growth plate, apoptosis of chondrocytes, VEGF expression by hypertrophic chondrocytes, the normal invasion of blood vessels from the metaphysis to the growth plate and bone formation at the chondro-osseous junction. These effects could alter the dynamics of endochondral ossification and thus contribute to glucocorticoid-induced growth retardation.