Enlargement of growth plate chondrocytes modulated by sustained mechanical loading

BACKGROUND: Mechanical compression and distraction forces are known to modulate growth in vertebral growth plates, and they have been implicated in the progression of scoliosis. This study was performed to test the hypothesis that growth differences produced by sustained compression or distraction loading of vertebrae are associated with alterations in the amount of increase in the height of growth plate chondrocytes in the growth direction. METHODS: Compression or distraction force of nominally 60% of body weight was maintained for four weeks on a caudad vertebra of growing rats by an external apparatus attached, by means of transcutaneous pins, to the two vertebrae cephalad and caudad to it. Growth of the loaded and control vertebrae was measured radiographically. After four weeks, the animals were killed and histological sections of the loaded and control vertebrae were prepared to measure the height of the hypertrophic zone (average separation between zonal boundaries), the mean height of hypertrophic chondrocytes, and the amount of increase in cell height in the growth direction. RESULTS: Over the four weeks of the experiment, the growth rates of the compressed and distracted vertebrae averaged 52% and 113% of the control rates, respectively. The reduction in the growth rate of the compressed vertebrae was significant (p = 0.002). In the compressed vertebrae, the height of the hypertrophic zone, the mean chondrocyte height, and the amount of increase in cell height averaged 87%, 85%, and 78% of the control values, respectively, and all were significantly less than the corresponding control values. In the distracted vertebrae, these measurements did not differ significantly from the control values. The height of the hypertrophic zone and the mean chondrocyte height correlated with the growth rate (r (2) = 0.29 [p = 0.03] and r (2) = 0.23 [p = 0.06], respectively), when each variable was expressed as a proportion of the control value. The percentage changes in the measurements of the chondrocytic dimensions relative to the control values were smaller than the percentage changes in the growth rates, a finding that suggested that the rate of chondrocytic proliferation was also modulated by the mechanical loading. Conclusions: Mechanical loading of tail vertebrae in rats modulated their growth rate, which correlated with changes in the height of hypertrophic chondrocytes. The effects of compression were greater than those of distraction. Clinical Relevance: Information about the growth rate and chondrocytic response to mechanical loads in rat vertebrae undergoing mechanically modulated growth will be helpful in determining how human vertebral growth might respond to altered loading states during progression or treatment of scoliosis and other growth-related angular skeletal deformities.     

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.     

Compressive mechanical modulation alters the viability of growth plate chondrocytes in vitro

The aim of this study was to investigate the effect of compressive modulation parameters (mode, magnitude, duration, as well as frequency and amplitude for cyclic modulation) on the viability of growth plate chondrocytes. Swine ulnar growth plate explants (n = 60) were randomly distributed among 10 groups: baseline (n = 1 × 6); culture control (n = 1 × 6); static (n = 3 × 6); and dynamic (n = 5 × 6). Static and dynamic samples were modulated in vitro using a bioreactor. Different compression magnitudes (0.1 MPa or 0.2 MPa), durations (12 h or 24 h), frequencies (0.1 Hz or 1.0 Hz), and amplitudes (30% or 100%) were investigated. Viability was assessed by automatic quantification of number of live/dead cells from confocal images of Live/Dead labeled tissues. Chondrocyte viability was found to be dependent on compression magnitude, duration, frequency, and amplitude in a way that increasing each parameter decreased viability in certain zones of growth plate. More specifically, proliferative and hypertrophic chondrocytes were found to be more sensitive to the applied compression. This study provides an in vitro protocol for studying the effects of compressive modulation on biomechanical and biological responses of growth plate explants, which will be useful in finding efficient and non‐detrimental parameters for mechanical modulation of bone growth exploited in scoliosis fusionless treatments. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1587–1593, 2015.     

Regulation of proliferation and platelet-derived growth factor expression in palmar fibromatosis (Dupuytren contracture) by mechanical strain

Palmar fibromatosis (Dupuytren contracture) causes fibrosis of specific palmar fascial bands. These bands are subjected to repetitive mechanical strain in situ. Primary cell cultures were derived from (a) palmar fibromatosis from eight patients, (b) uninvolved palmar fascia (Skoog's fibers) from four of these patients and (c) normal palmar fascia from four additional patients. The cells were plated onto collagen-coated membranes either subjected to cyclic strain (25% maximal strain at 1 Hz) or without strain. Bromodeoxy uridine incorporation showed an increase in proliferation in all cultures subjected to strain. This increase was highest for palmar fibromatosis (10 to 40% nuclear incorporation, p = 0.02). Skoog's fibers and fascia from the normal individuals showed a trend (not significant) toward increase with strain (8 to 25%, p = 0.15 for Skoog's fibers, and 8 to 15%, p = 0.45 for normal fascia). Cyclic strain increased the expression of platelet derived growth factor-A relative to glyceraldehyde-3-phosphate dehydrogenase in palmar fibromatosis (2.2 to 3.5, p = 0.05) and Skoog's fibers (0.8 to 2.0, p = 0.04). The expression of platelet-derived growth factor-B relative to glyceraldehyde-3-phosphate dehydrogenase was enhanced by cyclic strain only in the fibromatosis tissue (0.7 to 2.1, p = 0.04). The normal fascia did not express platelet-derived growth factor. Platelet-derived growth factor neutralizing antibody decreased bromodeoxyuridine incorporation in fibromatosis cultures subjected to cyclic strain to near levels for those grown in the absence of strain (38 to 16%, p = 0.05). Conditioned medium from fibromatosis cells grown under stain showed a trend toward increased proliferation in additional fibromatosis cultures compared with conditioned medium from fibromatosis cells grown without strain (9 to 15% nuclear incorporation, p = 0.20). The observed palmar fibromatosis contracture can be partially explained on the basis of the cell's response to cyclic strain, which may be mediated by plateletderived growth factor.     

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.     

Regulation of growth plate chondrocytes by 1,25-dihydroxyvitamin D3 requires caveolae and caveolin-1.

We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice. INTRODUCTION: 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology. MATERIALS AND METHODS: Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons. RESULTS: Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis. CONCLUSIONS: The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology.     

周期性张应变力对不同年龄兔关节软骨细胞产生糖胺多糖的影响

目的 观察周期性张应变力(CTS)对体外培养不同年龄兔软骨细胞产生糖胺多糖(GAG)的影响。方法 选取雄性新西兰大白兔9只,按照年龄分为幼年组（2月龄）、成年组（8月龄）及老年组（31月龄）（每组3只）,无菌条件下取双膝关节,将各年龄组兔膝软骨细胞进行消化分离和体外培养。将每个年龄组兔原代软骨细胞分别培养于2个BioFlex 6孔培养板上,随机分为CTS组和对照组（每组6个样本）,同置于培养箱内,CTS组每天CTS (sin1 0％,0.5 Hz,6h/次)作用6h,对照组不予特殊处理。CTS组与对照组在首次作用后第12、24、36、48、60、72小时分别吸取培养细胞上清液,Alcian blue染色沉淀法测定上清液GAG含量,比较两组GAG分泌的变化。结果 CTS组与对照组相比,各年龄组兔软骨细胞GAG增加量在不同时间]点差异均有统计学意义(P＜0.05)。老年组在起始时,两组GAG的增加量与年轻组的增加量无明显差异,但从第48小时起老年组增加量开始低于年轻组,差异有统计学意义(P＜0.05)。结论 CTS可以促进兔软骨细胞产生GAG,且CTS刺激后年轻细胞比老年细胞产生更多的GAG。     

A型激酶锚定蛋白2基因表达对生长板软骨细胞功能的影响

目的:探讨A型激酶锚定蛋白2(A-kinase anchoring protein 2,AKAP2)基因表达对生长板软骨细胞(growth plate chondrocytes,GPCs)增殖、分化及细胞外基质代谢的影响及作用机制。方法:设计AKAP2过表达和基因敲除质粒转染GPCs对AKAP2进行过表达或干扰,构建AKAP2过表达阴性对照组(Vector组)、AKAP2过表达组(AKAP2 OE组)、AKAP2干扰阴性对照组(si-NC组)、AKAP2干扰组(si-AKAP2组)和AKAP2 OE+U0126组[U0126:细胞外信号调节蛋白激酶1/2 (extracellular signal regulated kinase 1/2,ERK1/2)通路阻滞剂],记录各组GPCs细胞增殖活力及钙盐沉积情况,检测细胞外基质中Ⅱ型胶原α1(collagen typeⅡalpha 1,COL2A1)、碱性磷酸酶(alkaline phosphatase,ALP)、Ⅱ型胶原(collagen typeⅡ,COLⅡ)、增殖细胞核抗原(proliferating cell nuclear an...     

周期性应力下胰岛素样生长因子1受体对大鼠软骨细胞功能的影响

目的探讨周期性机械应力条件下胰岛素样生长因子1型受体(IGF1R)对大鼠软骨细胞增殖及细胞外基质合成的影响。 方法体外分离培养大鼠软骨细胞,随机分为4组：加压0 h组、加压0.5 h组、加压1 h组、加压2 h组,采用Western Blot法检测各组细胞IGF1R的表达及磷酸化水平,并在此基础上将同代无处理的软骨细胞在加压各组检测的同时给予同样检测即为静态组,应用Gel-Pro Analyzer软件进行半定量灰度分析比较各时间段静态和加压两两各组磷酸化IGF1R/总IGF1R水平差异。另取大鼠软骨细胞随机分为静态组、加压对照组、IGF1R阻断组、加压后IGF1R阻断组,IGF1R阻断采用顺式－3－[8－胺基－1－(2－苯基－喹啉－7－基)－咪唑并[1,5－a]吡嗪－3－基]－1－甲基－环丁醇(OSI－906)或者以IGF1R shRNA两种方式。阻断IGF1R 1 h后Western Blot法检测各组细胞磷酸化细胞外信号调节激酶1/2(ERK 1/2)表达及磷酸化水平;8 h后实时荧光定量PCR检测各组2型胶原(collagen Ⅱ)、蛋白聚糖(aggrecan)表达;3 d后采用直接细胞计数方式及细胞计数试剂盒(CCK-8)对软骨细胞增殖状况进行测定。应用SPSS 18.0软件进行相关统计学分析,两组间差异采用t检验比较。 结果磷酸化IGF1R/总IGF1R蛋白水平在加压0 h组与静态组之间差异无统计学意义(t＝0.255, P＝0.811),而在0.5 h组,1 h组,2 h组较相对应的静态组增高,差异具有统计学意义(t＝－5.881、－6.172、－10.518,P均小于0.05)。IGF1R被OSI-906或shRNA抑制后,加压处理的ERK 1/2磷酸化水平显著降低(OSI-906阻断后t＝3.074,shRNA阻断后t＝3.990,P均小于0.05),细胞外基质collagen Ⅱ(OSI-906阻断后t＝3.243, shRNA阻断后t＝3.621,均为P<0.05)、aggrecan(OSI-906阻断后t＝3.128,shRNA阻断后t＝3.608,P均小于0.05)基因表达水平显著降低,大鼠软骨细胞增殖能力减弱,差异均具有统计学意义(OSI-906阻断后t＝2.835、shRNA阻断后t＝3.467,均为P<0.05)。 结论周期性机械应力通过压力感受器IGF1R将机械信号转变为生物化学信号,激活ERK 1/2信号通路促进软骨细胞增殖和细胞外基质合成。     

Regulation of bone mass by mechanical strain magnitude

Summary Thein vivo remodeling behavior within a bone protected from natural loading was modified over an 8-week period by daily application of 100 consecutive 1 Hz load cycles engendering strains within the bone tissue of physiological rate and magnitude. This load regime resulted in a graded dose:response relationship between the peak strain magnitude and change in the mass of bone tissue present. Peak longitudinal strains below 0.001 were associated with bone loss which was achieved by increased remodeling activity, endosteal resorption, and increased intra-cortical porosis. Peak strains above 0.001 were associated with little change in intra-cortical remodeling activity but substantial periosteal and endosteal new bone formation.     

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).     

生长板软骨细胞促进骨髓基质干细胞血管内皮生长因子的表达

目的观察骨髓基质干细胞(BMSCs)在生长板软骨细胞旁分泌作用下血管内皮生长因子(VEGF)的表达规律及其与成骨分化的相关性。方法大鼠BMSCs与生长板软骨细胞进行间接共培养,培养终末期做细胞化学染色,定量测定碱性磷酸酶(ALP)活性,用RT-PCR方法半定量检测VEGFmRNA的表达。结果生长板软骨细胞持续高表达VEGF。BMSCs随共培养时间的延长,ALP活性升高,BMSCs的VEGF的表达也逐渐增强。培养液加入两种分泌型VEGF中和抗体后,VEGF表达趋势不变,ALP活性仍为升高趋势,也不影响培养终末期钙化结节的形成。培养终末期BMSCs的CD31和CD34均阴性。结论BMSCs成骨分化过程中VEGF的表达符合成骨细胞分化基因的表达规律,与成骨细胞特征性基因的表达趋势一致,体外条件共培养条件下,中和VEGF后并不能阻碍BMSCs的成骨分化。     

Selective enrichment of microRNAs in extracellular matrix vesicles produced by growth plate chondrocytes

Matrix vesicles (MVs) are membrane organelles found in the extracellular matrix of calcifying cells, which contain matrix processing enzymes and regulate the extracellular environment via action of these enzymes. It is unknown whether MVs are also exosomic mediators of cell–cell communication via transfer of RNA material, and specifically, microRNA (miRNA). We investigated the presence of RNA in MVs isolated from cultures of costochondral growth zone chondrocytes. Our results showed that the average yield of MV RNA was 1.93 ± 0.78 ng RNA/10⁴ cells, which was approximately 0.1% of the parent cell's total RNA. MV RNA was well-protected from RNase by the lipid membrane and was highly enriched in small RNA molecules compared to cells. Moreover, coding and non-coding small RNAs in MVs were in proportions that differed from parent cells. Enrichment of specific miRNAs was consistently observed in all three miRNA detection platforms that we used, suggesting that miRNAs are selectively packaged into MVs. MV-enriched miRNAs were related to different signaling pathways associated with bone formation. This study suggests a significant role for MVs as “matrisomes” in cell–cell communication in cartilage and bone development via transfer of specific miRNAs.     

Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine

Summary Postnatal bone growth occurs by the process of endochondral ossification in cartilaginous growth plates at the ends of long bones. The rate and extent of long bone growth is determined by a combination of chondrocytic proliferation, matrix production, and increase in chondrocytic height in the direction of growth during cellular enlargement. In this study, single pulse and/or repeated pulse labeling with the thymidine analog bromodeoxyuridine (BrdU) was used to study the role of cellular proliferation in controlling long bone growth. Variables studied included progression of the label over time following a pulse, and patterns and progression of the label over time following repeated pulse labeling for 24 and 48 hours. Examination was made of the proliferative characteristics of chondrocytes, the spatial pattern of cellular proliferation, and cell cycle kinetics. With respect to the spatial pattern of proliferative chondrocytes, results suggest that chondrocytes within a column are more synchronized with each other than are chondrocytes in different columns. This is consistent with the concept that each column represents a clonal expansion of a stem cell, which may proceed independently from adjacent columns. Despite this apparent heterogeneity, all chondrocytes in the proliferative zone complete at least one cell cycle in 24–28 hours. This estimate of the cell cycle time is significantly shorter than previous estimates of cell cycle times in similar growth plates. Our results also suggest that chondrocytes entering the cell cycle in the proximal part of the growth plate spend an average of 4 days in the proliferative cell zone, representing approximately four cellular divisions. After leaving the cell cycle, an additional 48 hours is required for the label to reach the terminal chondrocyte, which represents the time required to complete hypertrophy. These data are important when considering hypotheses concerning both the role of controls on proliferation in the determination of overall rate of long bone growth, as well as the interplay between proliferation and hypertrophy in regulating the overall amount of growth achieved by a given growth plate.     

A poptosis of hypertrophic chondrocytes in rat cartilaginous growth plate

Calcifying cartilages undergo endochondral ossification, a process in which cartilage is replaced by bone. These tissues contain chondrocytes that proliferate, leading, to differentiation and hypertrophy. Recent histological and biochemical studies suggest that hypertrophic chondrocytes undergo apoptosis. We investigated the process of this cell death to determine when fragmentation of DNA, a hallmark of apoptosis, occurs during cellular commitment to hypertrophy, and to test the hypothesis that the chondrocytes are intrinsically programmed to undergo apoptosis. End-labeling of fragmented DNA of rat proximal tibiae revealed that a majority of hypertrophic cells bore fragmented DNA, indicating that apoptosis was in progress in this zone. In pelleted chondrocyte cultures isolated from, rat rib growth plates and employed in an in vitro model of a growth plate, hypertrophic cells were also positive for end-labeling. Gel electrophoresis of DNA isolated from the chondrocyte cultures at 1–3 weeks yielded the ladder formation characteristic of apoptosis. We conclude that the chondrocytes in the growth plate are programmed to self-annihilate by apoptosis and that the apoptotic process is closely associated with the commitment to hypertrophy.