Distribution of chondrocytes containing alpha-smooth muscle actin in human articular cartilage.

Relatively little is known about the contractile behavior of the human articular chondrocyte. Other connective tissue cells are known to express a contractile actin isoform, alpha-smooth muscle actin, in response to injury, at selected stages of wound healing, and in certain pathological conditions. This and recent work demonstrating contractile behavior in adult canine articular chondrocytes in vitro prompted the present study of the distribution of alpha-smooth muscle actin-containing chondrocytes in human articular cartilage. Approximately 75% of the chondrocytes in the superficial region of cartilage expressed alpha-smooth muscle actin as demonstrated by immunohistochemistry. In contrast, only approximately 10% of the cells in the deep region stained for this contractile actin isoform. There was no correlation of the percentage of alpha-smooth muscle actin-positive cells in either region with Mankin grade or with age. This is the first report of a contractile potential for human articular chondrocytes. The roles of alpha-smooth muscle actin in these cells warrant further investigation. The question of whether it is necessary to refer to these cells as myochondrocytes is considered.     

Outgrowth of chondrocytes from human articular cartilage explants and expression of α-smooth muscle actin

The objectives of this study were to investigate the effect of various enzymatic treatments on the outgrowth of chondrocytes from explants of adult human articular cartilage and the expression of a specific contractile protein isoform, alpha-smooth muscle actin, known to facilitate wound closure in other connective tissues. Explants of articular cartilage were prepared from specimens obtained from patients undergoing total joint arthroplasty. The time to cell outgrowth in vitro was determined and the expression of alpha-smooth muscle actin shown by immunohistochemistry. Treatment of the explants with collagenase for 15 minutes reduced the time to outgrowth from more than 30 days to 3 days. Hyaluronidase, chondroitinase ABC, and trypsin applied for the 15-minute period had no effect on the time to cell outgrowth when compared with untreated controls. Pretreatment with hyaluronidase prior to collagenase reduced the time to outgrowth. A notable finding of this study was that the majority of chondrocytes in the adult human articular cartilage specimens and virtually all of the outgrowing cells contained alpha-smooth muscle actin. We conclude that human articular chondrocytes have the capability to migrate through enzymatically degraded matrix and express a contractile actin isoform. Collagenase treatment reduces the time required for cell outgrowth.     

Homeostasis of the extracellular matrix of normal and osteoarthritic human articular cartilage chondrocytes in vitro

OBJECTIVE: In normal articular cartilage cells, the IGFRI/insulin-like growth factor 1 (IGF-1) autocrine pathway was shown to overrule the catabolic effects of the IL-1/IL-1RI pathway by up-regulation of the IL-1RII decoy receptor. The activity of the IGF-1/IGFR1 and IL-1/IL-1R pathways, and of the IL-1RII control mechanism in the synthesis and turnover of the extracellular matrix (ECM) by chondrocytes from normal and osteoarthritic (OA) articular cartilage was compared in order to identify possible therapeutic targets of this disease. METHODS: Phenotypically stable human articular cartilage cells were obtained from normal and OA cartilage of the same knee showing focal OA. The cells were cultured in alginate beads over 1 week to re-establish the intracellular cytokine and growth factors, to reexpress the respective plasma membrane receptors and to reach equilibrium in accumulated cell-associated matrix (CAM) compounds. Following liberation of the cells from the alginate beads, the levels of cell-associated matrix (CAM) aggrecan, type II collagen and fibronectin, of intracellular IGF-1, IL-1alpha and beta and of their respective plasma membrane-bound receptors, IGFR1, IL-1RI and the decoy receptor IL-1RII, were assayed using flow cytometry. RESULTS: Coordinated production and accumulation of CAM aggrecan and type II collagen under the effect of the IGFR1/IGF-1 autocrine pathway-as documented for chondrocytes from healthy controls-was absent when the chondrocytes had been obtained from OA joints. When compared with cells obtained from normal tissues, chondrocytes from fibrillated OA cartilage expressed significantly higher intracellular IGF-1 levels and plasma membrane-bound IGFR1. At the same time, significantly higher intracellular IL-1alpha and beta levels and upregulated plasma membrane-bound IL-1RI were observed. Plasma membrane-bound IL-1RII decoy receptor was downregulated in OA chondrocytes. The levels of CAM aggrecan, type II collagen and fibronectin were significantly reduced in the chondrocytes obtained from pathological tissue. CONCLUSION: Paired analysis of normal and OA chondrocytes from the same knee joint has shown an enhanced capacity of chondrocytes from OA cartilage to produce ECM macromolecules. However, the same cells have increased catabolic signalling pathways. As a consequence of this increased IL-1 activity and the reduced amounts of IL-1RII decoy receptor, less of the produced ECM macromolecules may persist in the CAM of the OA chondrocytes.     

Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes.

Destruction of collagen within osteoarthritic cartilage depends in part on collagen-degrading matrix metalloproteases (MMP). Degradative fragments of type II collagen (Col II) occur in normal and in osteoarthritic cartilage, and may contribute to regulation of matrix turnover by interfering with normal cell-matrix communication pathways. Therefore, the effects of different types of collagen fragments on mRNA and protein levels of MMP-2, MMP-3, MMP-9, and MMP-13 in cultured bovine articular knee chondrocytes and explants were examined. Primary chondrocytes and explants were incubated with fragments from whole cartilage collagen matrix (Colf) and from purified type II collagen (Col2f), or with a synthetic 29-mer peptide representing the amino-terminal domain of type II collagen (Ntelo). Gelatin zymography revealed increases of proMMP-2, a shift towards active MMP-2 and increases in proMMP-9, depending on the type of fragment. In situ hybridization of cartilage sections displayed MMP-3 mRNA in virtually all cells. Moderate to strong increases in MMP-2, MMP-3, MMP-9, and MMP-13 mRNA levels were detected by quantitative PCR. The results demonstrate stimulating effects of collagen fragments on both mRNA and/or protein from MMP -2, -3, -9, and -13, and suggest a novel mechanism of MMP induction and activation that includes a particular role for N-telo in controlling catabolic pathways of matrix turnover.     

Effects of FGF-2 and IGF-1 on adult canine articular chondrocytes in type II collagen-glycosaminoglycan scaffolds in vitro

OBJECTIVE: Chondrocyte-seeded tissue engineering scaffolds hold the promise of enhancing certain cartilage repair procedures. The objective of this study was to evaluate the effects of selected growth factors [fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1] individually and in combination on adult canine articular chondrocyte-seeded type II collagen-glycosaminoglycan (GAG) scaffolds grown in serum-free (SF) medium. DESIGN: Approximately 500,000 second passage chondrocytes were seeded into discs of the scaffold, 4mm diameterx2 mm thick. The constructs were grown in the following media: serum-containing medium; a basal SF medium; SF with 5 ng/ml FGF-2; SF with 25 ng/ml FGF-2; SF with 100 ng/ml IGF-1; and SF with 5 ng/ml FGF-2 plus 100 ng/ml IGF-1. The DNA and GAG contents of the scaffolds were determined after 1 day and 2 weeks and the protein and GAG synthesis rates determined at 2 weeks using radiolabels. Histology and type II collagen immunohistochemistry were also performed. RESULTS: FGF-2 at 5 ng/ml was found to substantially increase the biosynthetic activity of the cells and the accumulation of GAG. The histology demonstrated chondrocytes uniformly distributed through a matrix that stained intensely for GAG and type II collagen after only 2 weeks. Of interest were the rapid degradation of the collagen scaffold, despite the fact that the scaffold was carbodiimide cross-linked, and the contraction of the constructs. There were less pronounced effects using the higher dose of FGF-2 and the combination with IGF-1. CONCLUSIONS: Chondrocyte-seeded type II collagen scaffolds cultured in SF medium supplemented with 5 ng/ml FGF-2 undergo contraction, demonstrate an increase in construct incorporation of radiolabeled sulfate, and display qualitative signs of chondrogenesis.     

Expression of alpha-smooth muscle actin in canine intervertebral disc cells in situ and in collagen-glycosaminoglycan matrices in vitro.

The objective of this study was to investigate the presence of a contractile actin isoform, alpha-smooth muscle actin, in annulus fibrosus cells in situ and in two and three-dimensional cultures. Annulus fibrosus cells were isolated from healthy adult dogs, serial passaged, and then injected into porous collagen-glycosaminoglycan copolymers consisting of either type-I or type-II collagen. Alpha-smooth muscle actin was detected in the cells in tissue samples and in culture by immunohistochemistry. The number of cells and glycosaminoglycan content of the matrices were determined after 1, 7, and 14 days, and the diameters of the specimens were measured every 2 days. Although few annulus fibrosus cells in vivo displayed the presence of the alpha-smooth muscle actin isoform, most cells in two-dimensional culture demonstrated this phenotype. The contractile behavior of these cells was shown by the cell-mediated contraction of type-I collagen-glycosaminoglycan scaffolds after 8 days in culture. Glycosaminoglycan production was not significantly different in the seeded type-I matrices than in the unseeded matrices, whereas the seeded type-II matrices had a significant increase in glycosaminoglycan production between days 1 and 14 compared with the unseeded controls. This is the first report of both the expression of the contractile alpha-smooth muscle actin isoform in intervertebral disc cells and the ability of the cells to contract a collagen matrix. This finding could aid in better understanding the nature of cells in the annulus.     

Expression of vitamin D receptors and matrix metalloproteinases in osteoarthritic cartilage and human articular chondrocytes in vitro

OBJECTIVES: To examine the in situ distributions of vitamin D receptors (VDR) and matrix metalloproteinases (MMPs) in osteoarthritic cartilage for comparison with non-arthritic, normal cartilage; and to assess the in vitro effects of 1alpha,25 dihydroxyvitaminD(3)(1alpha,25(OH)(2)D(3)) on MMPs-1, -3 and -9 and prostaglandin E(2)(PGE(2)) production by cultures of human articular chondrocytes (HAC) shown to be VDR-positive. METHODS: Using immunohistochemistry VDR expression in different specimens of osteoarthritic cartilage (N=11) was compared to that in normal cartilage (N=6), along with the immunodetection of MMPs-1, -3 and -9. The effects of 1alpha25(OH)(2)D(3)on MMP and PGE(2)production by HAC in vitro, with and without stimulation by TNFalpha or phorbol myristate acetate (PMA), was evaluated using ELISA methodology. RESULTS: VDR was demonstrated in HAC of all specimens of osteoarthritic cartilage, especially the superficial zone, whereas only two of five normal cartilage specimens were VDR(+)for a minor proportion of HAC. Immunolocalization of MMPs-1, -3 and -9 was often seen in areas where chondrocytes were VDR(+), and dual immunolocalization has demonstrated individual chondrocytes positive for both VDR and MMP-3 in situ. In vitro, 1alpha25(OH)(2)D(3)alone had no effect on MMP-1, -9 and PGE(2)production by HAC, but MMP-3 production was up-regulated by 1alpha25(OH)(2)D(3)either with or without stimulation with TNFalpha or PMA. By contrast the increased production of MMP-9 and PGE(2)induced by PMA was significantly suppressed by concomitant treatment with 1alpha25(OH)(2)D(3). CONCLUSIONS: The demonstration of VDR expression by HAC in osteoarthritic cartilage was often associated with sites where MMP expression was prevalent, observations in contrast to their virtual absence in normal age-matched cartilage. Together with HAC in vitro studies, the data suggests that 1alpha25(OH)(2)D(3)contributes to the regulation of MMP and PGE(2)production by HAC in osteoarthritic cartilage.     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

Gene expression during redifferentiation of human articular chondrocytes

OBJECTIVE: The aim of the present study was to investigate gene expression during the in vitro redifferentiation process of human articular chondrocytes isolated from clinical samples from patient undergoing an autologous chondrocyte transplantation therapy (ACT). METHOD: Monolayer (ML) expanded human articular chondrocytes from four donors were cultured in a 3D pellet model and the redifferentiation was investigated by biochemistry, histology, immunohistochemistry and microarray analysis. RESULTS: The culture expanded chondrocytes redifferentiated in the pellet model as seen by an increase in collagen type II immunoreactivity between day 7 and 14. The gene expression from ML to pellet at day 7 included an increase in cartilage matrix proteins like collagen type XI, tenascin C, dermatopontin, COMP and fibronectin. The late phase consisted of a strong downregulation of extracellular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and SOX-9, suggesting that the late phase mimicked parts of the signaling processes involved in the early chondrogenesis in limb bud cells. Other genes, which indicated a transition from proliferation to tissue formation, were the downregulated cell cycle genes GSPT1 and the upregulated growth-arrest-specific protein (gas). The maturation of the pellets included no signs of hypertrophy or apoptosis as seen by downregulation of collagen type X, Matrix Gla protein and increased expression of caspase 3. CONCLUSION: Our data show that human articular chondrocytes taken from surplus cells of patient undergoing ACT treatment and expanded in ML, redifferentiate and form cartilage like matrix in vitro and that this dynamic process involves genes known to be expressed in early chondrogenesis.     

血清对体外培养成年人关节软骨细胞生长的影响

目的观察不同血清及血清浓度对成人关节软骨的细胞（AHAC）生长的影响，为软骨细胞移植的临床应用提供血清选择。方法用无血清、胎牛血清（FBS）和人AB血清及不同浓度血清在加或不加生长因子条件下培养成年人关节软骨细胞，比较细胞增殖和生长情况。结果对于AHAC，人AB血清培养优于FBS；合适的人AB血为10％；合适的FBS浓度为20％；无血清培养细胞增殖非常缓慢；不加因子用人AB血清培养细胞梭形化明显，加用生长因子后与使用FBS在形态上一致。结论10％浓度的人血清是AHAC体外培养的合适血清和浓度选择。     

人胎关节软骨细胞体外培养的生物学特性

目的 研究软骨组织工程中传代软骨细胞与原代的差异。方法 用５个月人胎关节软骨分离培养细胞,观察细胞存活率、贴壁率、生长曲线和组织形态学的改变。结果 ⑴软骨块在４℃下,３ｄ内细胞存活率可达９３．４％～９７．６％。⑵原代细胞为圆形或三角形;第４代有一半转变成梭形,到第６代全部变为长梭形。⑶传代细胞贴壁时间（２～３ｈ）短于原代（４～７ｈ）。玻璃瓶内贴壁率传代细胞为７８．７％～８５．５％,原代８．８％。⑷     

重组腺病毒介导外源性SOX9在正常关节软骨细胞中诱导软骨基质合成的体外表达

背景：关节软骨损害是临床一种常见的损伤,软骨形成转录因子SOX9是一种调控Ⅱ型胶原合成的关键因子。 目的：观察以表达外源性SOX9的腺病毒体外成功感染关节软骨细胞后对Ⅱ型胶原和蛋白聚糖（Aggrecan）合成的影响,探讨软骨损伤后修复软骨缺损的基因治疗方法。 方法：体外构建腺病毒载体AdSOX9和AdGFP,成功感染培养的人关节软骨细胞,分别以逆转录聚合酶链式反应（RT-PCR）技术检测了病毒感染前后SOX9、II型胶原和蛋白聚糖基因mRNA的表达,同时以免疫组化技术检测了病毒感染前后关节软骨细胞中Ⅱ型胶原的表达。 结果：应用AdEasy腺病毒构建专利技术体外成功构建了能高效表达外源性SOX9的腺病毒AdSOX9和只表达绿色荧光蛋白GFP的腺病毒AdGFP;以腺病毒AdSOX9和AdGFP体外成功感染人关节软骨细胞后48h,未感染对照组和AdGFP感染组,均检测到了Ⅱ型胶原和蛋白聚糖的表达;而AdSOX9感染组的细胞中,检测到了SOX9基因mRNA的表达明显增高,与未感染对照组和AdGFP感染组相比有显著性差异（P〈0．05）,同时,Ⅱ型胶原和蛋白聚糖的表达也较未感染对照组和AdGFP感染组明显增高,差异显著（P〈0．05）。 结论：以外源性SOX9为目的基因的腺病毒介导基因治疗方法,在促进关节软骨细胞Ⅱ型胶原和蛋白聚糖合成方面得出了初步满意的结果,SOX9可能是关节软骨缺损基因治疗研究领域一个新的理想靶点,值得继续深入研究。     

Effects of shear stress on nitric oxide and matrix protein gene expression in human osteoarthritic chondrocytes in vitro.

Mechanical loading alters articular cartilage metabolism. However, mechanisms underlying intracellular signaling and communication between cells in response to mechanical stresses remain enigmatic. This study tested the hypothesis that shear stress-induced nitric oxide (NO) production participates in the regulation of matrix protein gene expression. The data presented here demonstrate that exposure of human osteoarthritic chondrocytes to a continuously applied shear stress (1.64 Pa) upregulated NO synthase gene expression and increased NO release by 1.8-, 2.4-, and 3.5-fold at 2, 6, and 24 h, respectively. Exposure of chondrocytes to a short duration of shear stress for 2 h resulted in the release of accumulation of NO in the culture medium. Exposure of chondrocytes to shear stress for 2, 6, and 24 h inhibited type II collagen mRNA signal levels by 27%, 18% and 20% after a constant post-shear incubation period of 24 h. Aggrecan mRNA signal levels were inhibited by 30%, 32% and 41% under identical conditions. Addition of an NO antagonist increased type II collagen mRNA signal levels by an average of 1.8-fold (137% of the un-sheared control) and reestablished the aggrecan mRNA signal levels by an average of 1.4-fold after shear stress (92% of the un-sheared control) (ANOVA p < 0.05). These data support the hypothesis that shear stress-induced NO release may influence the development of degenerative joint diseases by inhibiting matrix macromolecule synthesis.     

Differentiation plasticity of human fetal articular chondrocytes.

OBJECTIVE: To test chondrogenic differentiation potential, we examined the differentiation plasticity of isolated human fetal articular chondrocytes (HFACs). STUDY AND DESIGN SETTING: Culture-expanded human fetal articular chondrocytes (HFACs) were analyzed for chondrogenic, adipogenic, osteogenic capacity and neural differentiation ability in defined in vitro culture systems. RESULTS: The different assays demonstrated that culture-expanded HFACs have potential to form cartilage in pellet mass culture, to form adipose cells, osteogenic cells, and neural cells in monolayer culture. CONCLUSIONS: These results suggest that within human fetal articular cartilages there are MSC-like cells that exhibit differentiation plasticity that is comparable with that of BM-MSCs and they may be a new kind of seeding cells for head and neck cartilage reconstruction.     

Musculoskeletal connective tissue cells with muscle: Expression of muscle actin in and contraction of fibroblasts, chondrocytes, and osteoblasts

The expression of the gene for a muscle actin in certain nonmuscle cells and the contraction of these connective tissue cells has been associated with several important physiological and pathological processes; the contraction of healing skin wounds and the contracture in Dupuytren's disease being two notable examples. Studies in recent years have shown that a much wider variety of connective tissue cells than previously considered, including cells in many of the musculoskeletal tissues, e.g., chondrocytes and osteoblasts, can express the gene for alpha-smooth muscle actin and can display contractile behavior. These findings suggest that muscle actin-enabled cell contraction may also be playing important roles in the other connective tissues comprising the musculoskeletal system, namely, tendon, ligament, meniscus, intervertebral disc, articular cartilage, and bone.     

Interaction of periosteal explants with articular chondrocytes alters expression profile of matrix metalloproteinases

Periosteal tissue is a source of growth factors and of osteochondral progenitor cells which makes it suitable for implantation in chondral defects as known in autologous chondrocyte implantation. The aim of this study was to determine the interaction between periosteal tissue and articular chondrocytes with respect to catabolic effectors such as matrix metalloproteinases (MMPs) and IL‐6. Human articular chondrocytes were cultured for up to 28 days as micromass pellets in coculture either with physical contact to periosteal explants or allowing paracrine interactions only. Expression, secretion, and activation of MMPs and IL‐6 were analyzed in chondrocytes, periosteum, and culture supernatants. Both coculture conditions influence gene expression levels of MMPs and IL‐6 in a time‐, culture‐, and tissue‐dependent manner. Coculturing of periosteum with chondrocytes promotes gene expression and secretion of IL‐6. In periosteum, physical contact inhibits MMP‐2 and MMP‐13 gene expression while paracrine coculture induces expression of IL‐6, MMP‐2, ‐7, and ‐13. Pro‐MMP‐2, ‐7, and ‐13 were detected in supernatants of all culture regimens whereas pro‐MMP‐9 was secreted from periosteum only. As a balanced amount of MMP activity is likely required to achieve sufficient integration of the regenerate tissue with the surrounding healthy cartilage, an exceeding expression of proteinases might result in degradation, hypertrophy or rejection of the graft. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1576–1585, 2010