Anabolic and catabolic function of chondrocyte ex vivo is reflected by the metabolic processing of type II collagen

OBJECTIVE: The aim of the present study was to investigate collagen metabolism after anabolic and catabolic stimulation of chondrocytes ex vivo. DESIGN: Metabolic activities in ex vivo bovine cartilage explants were stimulated with insulin-like growth factor I (IGF-I) or a combination of tumor necrosis factor alpha (TNFalpha) and oncostatin M (OSM). Supernatants were assessed for changes in biochemical markers, N-terminal propeptide of type II (PIINP) collagen and fragments of C-telopeptide of type II collagen (CTX-II). Matrix metalloproteinases (MMP) were added to metabolic inactivated cartilage and evaluated by the two biochemical markers for formation or degradation, respectively. Finally, urinary CTX-II and PIINP were evaluated for assessment of type II collagen turnover in patients with rheumatoid arthritis (RA). RESULTS: In the bovine articular cartilage explants, IGF-I induced an increase in PIINP level up to 4.8+/-1.1[ng/ml]/mg cartilage whereas CTX-II remained below 0.1+/-0.1[ng/ml]/mg cartilage. In the catabolic stimulated explants both PIINP and CTX-II were released to the supernatant, reaching concentrations of 9.0+/-1.4 and 9.1+/-2.2[ng/ml]/mg cartilage, respectively. RA patients had significantly lower serum concentrations of PIINP (3.4+/-3.7 ng/ml) compared with those healthy individuals (18.7+/-12.41 ng/ml, P<0.001). In contrast, RA patients had significantly higher urinary CTX-II (0.8+/-0.8 mg/mmol) compared to the healthy controls (0.1+/-0.08 mg/mmol, P=0.004). CONCLUSIONS: This study is the first to demonstrate that precursors and degradation products of type II collagen released into the supernatant can effectively reflect the anabolic and catabolic activities of stimulated cartilage explants.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

A type XI collagen mutation leads to increased degradation of type II collagen in articular cartilage

OBJECTIVE: To determine in articular cartilage whether degraded type II collagen is more abundant in Col11a1 mutant cho/+ than in age-matched +/+ mice and whether collagen degradation occurs in a generalized or localized fashion. DESIGN: Knee joints from cho/+ and +/+ mice at 6, 9, 12 and 15 months of age were dissected, fixed, cryosectioned, and stained with antibody COL2-3/4m against denatured type II collagen using a FITC-conjugated secondary antibody. Sections were viewed and photographed under a fluorescence microscope and areas of staining were quantified. RESULTS: Before 12 months of age, little degraded collagen staining was detectable in +/+ or cho/+ mice. By 15 months, however, cho/+ mice showed significantly more degraded type II collagen than age-matched controls. Degraded collagen staining was localized at the articular surface, not distributed generally throughout the articular cartilage. CONCLUSIONS: The results suggest a model in which cumulative biomechanical stresses trigger increased collagen synthesis and degradation in both +/+ and cho/+ mice at around 12 months of age. Cho/+ mice, however, are less able to synthesize and assemble normal replacement collagen fibrils because of the Col11a1 mutation. Degradation is further activated, resulting in the accumulation of degraded type II collagen in the articular cartilage extracellular matrix. Similar mutations that do not overtly affect skeletal development may likewise predispose humans to increased collagen degradation and resultant osteoarthritis.     

Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice.

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human chondrodysplasias, including achondroplasia, the most common form of dwarfism in humans. From in vitro studies, the skeletal defects observed in these disorders have been attributed to constitutive activation of FGFR3. Here we show that FGF9 and FGFR3, a high-affinity receptor for this ligand, have similar developmental expression patterns, particularly in areas of active chondrogenesis. Targeted overexpression of FGF9 to cartilage of transgenic mice disturbs postnatal skeletal development and linear bone growth. The growth plate of these mice exhibits reduced proliferation and terminal differentiation of chondrocytes similar to that observed in the human disorders. The observations provide evidence that targeted, in vivo activation of endogenous FGFR3 inhibits bone growth and demonstrate that signals derived from FGF9-FGFR3 interactions can physiologically block endochondral ossification to produce a phenotype characteristic of the achondroplasia group of human chondrodysplasias.     

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells

The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress‐related response in meniscus cells remains unclear. We investigated mechanical stretch‐regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry‐type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real‐time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch‐related protein–DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA‐binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch‐induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:468–474, 2012     

Tissue engineering: chondrocyte cultures on type I collagen support. Cytohistological and immunohistochemical study

Cartilaginous tissue has limited capacity for regeneration after damage, since the natural repair process leads to the formation of fibrocartilaginous tissue which does not have the resistance and capability of deformation under load, typical of hyaline cartilage which covers the articular surfaces. The possibility of transplanting human chondrocytes for cartilage reconstruction has been demonstrated in orthopaedics. The scope of our study was to evaluate the possibility of cultivating and expanding human chondrocytes seeded on a pure equine type I collagen support. Human articular cartilaginous cells multiplied and grew on a type I collagen substrate with production of extracellular matrix. This chondrocyte culture showed a correct morphology and phenotype as shown by alcian-PAS staining to indicate the presence of mucopolysaccharides and by immunohistochemical methods to identify type II collagen. The use of scaffolds may lead to improvement in the surgical technique, by making it possible to hold the cells physically in the area to be repaired and by allowing optimum spatial adaptation inside injuries of all shapes.     

Identification of collagen type I and type II in chondroid tissue

Summary This paper deals with investigations concerning the matrix of chondroid tissue. Among the 73 human fetus or child mandibles and the 42 cat mandibles we have studied histologically and microradiographically, 8 human and 3 cat mandibles were used to determine the collagen composition in chondroid tissue matrix, and 10 cat mandibles were analyzed in order to have an ultrastructural approach to chondroid tissue. Both in human and cat mandibles, types I and II collagen were identified by indirect immunofluorescence and immunoperoxidase techniques. Electron microscopic analysis shows large collagen fibrils which correspond to type I collagen, and smaller collagen fibrils, principally located at the periphery of the chondroid cells. From our investigations, chondroid tissue should be considered as being different from both bone and cartilage, although it is not a transitional tissue, since no transformation of chondroid tissue into bone is observed; it should be classified as an intermediate tissue between cartilage and bone because of its morphological characteristics.     

Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.

Chondrogenic differentiation by mesenchymal progenitor cells (MPCs) is associated with cytokines such as transforming growth factor-beta 1 (TGF-beta1) and dexamethasone. Extracellular matrix (ECM) also regulates the differentiation by MPCs. To define whether ECM plays a functional role in regulation of the chondrogenic differentiation by MPCs, an in vitro model was used. That model exposed to dexamethasone, recombinant human TGF-beta1(rhTGF-beta1) and collagens. The results showed that MPCs incorporated with dexamethasone and rhTGF-beta1 increased proliferation and expression of glycosaminoglycan (GAG) after 14 days. Type II collagen enhanced the GAG synthesis, but did not increase alkaline phosphatase (ALP) activity. When adding dexamethasone and rhTGF-beta1 MPCs increased mRNA expression of Sox9. Incorporation with type II collagen, dexamethasone and rhTGF-beta1, MPCs induced mRNA expression of aggrecan and enhanced levels of type II collagen, and Sox9 mRNA. In contrast, incorporation with type I collagen, dexamethasone and rhTGF-beta1 MPCs reduced levels of aggrecan, and Sox9 mRNA, and showed no type II collagen mRNA. Altogether, these results indicate that type I and II collagen, in addition to the cytokine effect, may play a functional role in regulating of chondrogenic differentiation by MPCs.     

Osteochondrodysplasia determined genetically by a collagen type II gene mutation]

Chondrodysplasias are a heterogenous group of skeletal dysplasias, affecting the growing cartilage. The main part of chondrodysplasias is caused by mutations in various types of collagen genes. The current classification within this group of disorder relies on clinical, histological and radiographic features. Type II collagenopathies comprise part of chondrodysplasias, consisting of hereditary disorders caused by defects in the type II collagen. Collagen type II is coded by a large gene--COL2A1. The chromosomal location for the human COL2A1 gene is 12q13.11-q13.12. Defects in collagen type II are caused by point mutations in the COL2A1 gene. Type II collagenopathies form a wide spectrum of clinical severity ranging from lethal achondrogenesis type II, hypochondrogenesis, through severe forms like spondyloepiphyseal dysplasia congenita, spondyloepimetaphyseal dysplasia congenita, Marshall syndrome, to the mild forms--Stickler syndrome and early osteoarthritis. The pathological changes in the patients are observed in the growth plate, nucleus pulposus and vitreous body, where the abnormal collagen type II is distributed. This article presents the genetic background of collagenopathies type II and the results of current molecular studies of the patients. Both the molecular and the clinical studies may promise a better understanding of the relationship between the genotype and the phenotype. We present the patients, who were diagnosed at the Department of Medical Genetics and in the Orthopaedic Department in Poznań.     

Impaired angiotensin II type 1 receptor signaling contributes to sepsis-induced acute kidney injury

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Effect of different solvents and crosslinkers on cytocompatibility of Type II collagen scaffolds for chondrocyte seeding

Type II collagen extracted from porcine costal cartilage was evaluated as scaffolds for cartilage tissue engineering. Chemical crosslinkers were employed to improve the mechanical properties and the resistance toward degradation. Films and porous scaffolds were prepared from collagen solutions dissolved in 3% acetic acid (designated A) or in deionized water (designated W) and crosslinked by an epoxy (designated E) or by a carbodiimide (designated C). Immortalized rat chondrocytes and rabbit chondrocytes were used to assess cytocompatibility of crosslinked collagen matrices. Cell adhesion rate onto the films made by different preparations ranked in the order of WE > or = WC > AC > or = AE. Cell proliferation ranked in the order of AC > WC > AE > or = WE. Cells maintained round morphology only on AC and WC films. In 3-D seeding, AC scaffolds also were found to be the most cytocompatible. WC scaffolds, however, had better dimensional stability. It was concluded that Type II collagen scaffolds, when prepared by using deionized water as the solvent and carbodiimide as the crosslinker, could promote chondrocyte growth and matrix production.     

骨炎定方对一氧化氮抑制人骨关节炎软骨细胞Ⅱ型胶原合成的影响

[目的]观察骨炎定方对一氧化氮抑制离体人骨关节炎软骨细胞Ⅱ型胶原合成的影响,探讨中医药补肾益气行血法保护软骨细胞的作用途径.[方法]髋关节炎行关节置换术中获取离体股骨头关节软骨,行软骨细胞分离培养传代,通过设立空白血清对照组,IL-1组、IL-1加NAME组、补肾益气行血法中药骨炎定方含药血清组加入传代软骨细胞,采用MTT法于不同时间测定软骨细胞增殖影响,并运用逆转录聚合酶链技术检测对软骨细胞一氧化氮合酶（iNOS）和Ⅱ型胶原mRNA的影响.[结果]骨炎定方含药血清组在不同时间段均有促进软骨细胞增殖的作用.和对照组相比,3组（IL-1组、IL-1加NAME组、IL-1＋骨炎定方含药血清组）均明显了增加iNOS mRNA的表达,但加入NAME组及骨炎定方含药血清组和IL-1组相比,可以抑制iNOS mRNA的表达,和IL-1组相比,有明显的差异性（P〈0.05）.和IL-1组相比,3组（对照组、IL-1加NAME组、IL-1＋骨炎定方含药血清组）均明显增加了Ⅱ型胶原mRNA的表达,但各组间无差异（P〉0.05）.培养细胞上清液中亚硝酸盐含量测定显示：关节软骨细胞几乎不产生NO,但在IL-1刺激下可产生NO,而加入NO合酶抑制剂NAME后,可部分抑制NO产生,骨炎定方含药血清组也显示出可以部分抑制NO的产生,其作用与NO合酶抑制剂NAME统计学处理没有明显差异.[结论]骨炎定方可以促进人软骨细胞增殖、能抑制iNOS的表达,增加Ⅱ型胶原的表达,这种作用与使用一氧化氮合酶抑制剂得出的结果没有明显差异,可能是中医药补肾益气行血法保护软骨细胞作用途径之一.     

MMP9 production by human monocyte-derived macrophages is decreased on polymerized type I collagen

The production of matrix metalloproteinases (MMPs), such as MMP9, by macrophages may be a critical factor in the rupture of unstable atherosclerotic plaques and aortic aneurysms. Therefore, we studied the role of matrix and soluble cytokines in the regulation of monocyte/macrophage expression of MMP9. Although freshly isolated monocytes synthesize little MMP9, cells cultured on tissue-culture plastic differentiate into macrophages and synthesize maximal amounts of MMP9. Differentiated macrophages cultured on plastic are unresponsive to further stimulation by interleukin 1beta, tumor necrosis factor alpha, or platelet-derived growth factor BB. In contrast, monocytes cultured on polymerized collagen synthesize much less MMP9 than cells cultured on plastic and demonstrate a more than three-fold increase in MMP9 synthesis in response to interleukin 1beta, tumor necrosis factor alpha, and platelet-derived growth factor BB. To determine whether the physical state of the collagen was critical for the decrease in basal synthesis of MMP9, monocytes were cultured in suspension for 5 days to allow differentiation and then seeded onto monomer or polymerized collagen. Synthesis of MMP9 was significantly decreased in cells on polymerized collagen and modestly increased in macrophages seeded on monomer collagen. These results suggest that MMP9 synthesis by macrophages in the vessel wall may be under negative control by native, polymerized collagen and that disruption of this native conformation could increase MMP9 production. In addition, cells in contact with the collagen matrix are potentially more responsive to soluble mediators such as platelet-derived growth factor, interleukin 1beta, and tumor necrosis factor alpha.     

SOX9、L—SOX5、SOX6及Ⅱ型胶原在青少年特发性脊柱侧凸患者顶椎终板软骨的共表达及意义

目的：观测SOX9、L—SOX5、SOX6及Ⅱ型胶原在青少年特发性脊柱侧凸（adolescent idiopathic scoliosis，AIS）患者顶椎终板软骨中的表达，探讨其与AIS发生、发展的关系。方法：12例AIS患者，每例在前路手术时截取顶椎终板软骨1份，每份标本均包含凸侧与凹侧。切片行HE染色观察其组织细胞形态；行免疫组织化学染色，应用病理图像分析系统观测凸侧与凹侧的SOX9、L—SOX5、SOX6及Ⅱ型胶原表达的阳性细胞数和累积光密度（IOD），并计算这4个因子表达的相关性。结果：HE染色显示终板软骨有类似四肢长骨骺板样软骨内成骨组织形态．与凹侧比较凸侧显示了更强的软骨细胞活性。凸侧SOX9、L—SOX5、SOX6及Ⅱ型胶原表达的阳性细胞数和前三者的IOD值均明显大于凹侧，差异均有显著性（P〈0．05），凸侧Ⅱ型胶原表达的IOD值与凹侧比较无显著性差异（P〉0．05），4个因子表达的阳性细胞数之间的相关系数为0．46-0.91（P〈0．05），IOD之间为0．64～0．98（P〈0．05），均呈正性相关。结论：AIS患者顶椎凸侧与凹侧终板软骨的组织形态学及SOX9、L—SOX5、SOX6和Ⅱ型胶原的表达均存在差异，其可能是脊柱不同部位间机械应力差异下的继发性变化：4个因子的共表达异常触发AIS的可能性小。     

Urinary and serum type II collagen: markers of renal fibrosis

Background. The progression of chronic renal failure is characterized by the progressive fibrosis of the kidneys. Such fibrosis reflects the increased deposition of collagens (I, III, and IV) as well as fibronectin within scarred kidneys. In this study, we determined whether changes in renal extracellular matrix (ECM) components are reflected by parallel changes in their circulating or urinary levels. Patients and methods. We studied 40 patients with a range of subacute and chronic nephropathies who underwent a renal biopsy. At the time of the biopsy, their serum and urinary levels of collagens III (amino terminal peptide of procollagen III; PIIINP) and IV, as well as fibronectin were measured. Clinical, biochemical and histological parameters were correlated. Multiple regression analysis was applied to determine the predictive value of circulating and urinary ECM components for the severity of renal fibrosis. Results. We noted an increase in circulating and urinary levels of collagens III and IV but not fibronectin in patients with nephropathies compared to healthy volunteers. Increased immunoreactivity for these ECM components was also detected in kidney biopsies when compared to normal kidneys. A strong positive correlation was detected between circulating and urinary procollagen III (PIIINP) and the severity of renal interstitial fibrosis (serum PIINP: r=0.49, P<l0.01; urine PIINP: r=0.51, P<0.01). Conclusions. We conclude that the measurements of urinary collagen III (PIINP), and to a lesser extent serum collagen III (PIIINP), are useful indicators of the extent of renal fibrosis. This may have diagnostic implications and may prove useful for the monitoring of disease progression.