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.     

NG2蛋白多糖在糖尿病大鼠肾脏组织中表达的改变

糖尿病肾病（DN）的发病率在全世界范围内正在显著增加，已成为诱导终末期肾衰竭需要肾替代治疗的最常见的原因。早期DN的主要特征为肾小球肥大和进行性细胞外基质（ECM）积聚，其分子机制涉及到多种因素和途径，是目前研究的热点。本实验的目的为观察NG2蛋白多糖在正常大鼠和糖尿病大鼠肾脏中的表达，探讨NG2在DN中的作用机制。     

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。     

生长因子对成人关节软骨细胞的促增殖作用

目的观察不同生长因子对成人关节软骨细胞（adult human articular chondrocytes，AHAC）增殖的影响，探索AHAC体外大量扩增的方法。方法以酶消化法从成人关节软骨分离细胞，条件培养基培养；传2代细胞分别用不同浓度成纤维细胞生长因子2（fibroblast growth factor-2，FGF-2）、转化生长因子β1（transforming growth factor β1，TGF—β1）、血小板衍生因子bb（platelet derived growth factor-bb，PDGF-hb）、肝细胞生长因子（hepatocyte growth factor，HGF）或其不同组合作用。用MTT法比较细胞增殖情况，用组化和免疫组化检测观察细胞表型变化。结果FGF-2、TGF—β1、PDGF—bb、HGF均有促AHAC增殖的作用，其最大效应剂量分别是50ng/ml、1ng／ml、1ng／ml、20ng／ml。5ng／ml FGF-2＋1ng/ml TGF-β1有最强的促增殖作用，继续加用PDGF—bb和（或）HGF无进一步促进作用；用这一因子组合培养AHAC，可以传10代以上，细胞扩增2000倍以上，且传9代细胞仍弱表达Ⅱ型胶原和aggrecan。结论FGF-2、TGF-β1、PDGF—bb、HGF均对AHAC有一定的促增殖作用；5ng／ml FGF-2＋1ng/ml TGF-β1有最大的促增殖效应，细胞短期内大量扩增，且在大量扩增的同时维持了一定的软骨细胞表型，因此是合适的AHAC体外大量扩增促进剂。     

Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro

OBJECTIVE: This study investigated the in-vitro effects of a crystalline glucosamine sulfate (GS) preparation on DNA synthesis and on proteoglycan (PG) and type II collagen (coll II) production by human articular chondrocytes isolated from human osteoarthritic articular cartilage in a 3-dimensional culture system for 4, 8, and 12 days. MATERIALS AND METHODS: Human articular chondrocytes from osteoarthritic femoral heads were isolated from their matrix by collagenase digestion and then cultured in suspension. Under constant agitation, cells aggregated and formed a cluster within a few days. The effects of GS (1-100 micrograms/ml) on chondrocytes were determined by quantifying DNA synthesis (by measurement of [3H]-thymidine uptake) as well as PG and coll II production using radiommunoassays (RIAs) specific for coll II and to human human cartilage PG. Cross-reaction with GS in the RIAs was not detected. Moreover, PG size distribution was determined by exclusion chromatography under associative conditions to determine the association of PG monomers with hyaluronic acid (HA) to form large molecular weight PG aggregates. RESULTS: Under the above conditions, PG production in culture media and chondrocyte clusters was increased by GS (10-100 micrograms/ml). DNA synthesis and coll II production were not modified by GS. In addition, GS did not modify the physico-chemical form of PG produced by cells during culture. CONCLUSIONS: Glucosamine sulfate did not affect DNA synthesis nor coll II production but caused a statistically significant stimulation of PG production by chondrocytes from human osteoarthritic cartilage cultured for up to 12 days in 3-dimensional cultures.     

Induction of apoptosis of articular chondrocytes and suppression of articular cartilage proteoglycan synthesis by heat shock

We investigated cellular and matrix responses of articular cartilage to heat shock. Rat articular cartilage was pretreated at 37 degrees C for 24 h before being exposed to 48 degrees C for 10 min and subsequently incubated at 37 degrees C for 1, 2, 4, 7, 10, and 14 days. Following heat shock, a terminal deoxynucleotidyl transferase nick end labeling assay showed that articular chondrocyte apoptosis appeared at day 1, peaked at day 7, and declined by day 14. Analysis by transmission electron microscopy confirmed that the chondrocytes had characteristic morphological features of apoptosis; immunohistochemical staining revealed that caspase-3 activity in chondrocytes increased, 3-B-3-positive articular chondrocytes decreased in number, and the expression of 3-B-3 native epitope in articular chondrocytes was reduced. Safranin-O staining revealed that depletion of proteoglycans in the matrix was not found in any group. Morphological and biochemical evidence from this study suggested that heat shock at 48 degrees C induced articular chondrocyte apoptosis and suppressed proteoglycan synthesis of articular cartilage in vitro. This study thus provides evidence of the onset of osteoarthritis induced by heat shock and a basis for choosing a temperature at which malignant bone tumor cells can be killed with minimal damage to articular cartilage.     

Biological freezing of human articular chondrocytes

AIM: To preserve viable, metabolically active chondrocytes cultured in alginate beads at -196 degrees C for further use in in vitro and in vivo studies. METHODS: Human articular chondrocytes were isolated from femoral condyles within 24 h post mortem. To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimethyl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before being thawed and the cell viability was determined by Trypan Blue exclusion test. To investigate the effect of control-rate freezing on chondrocyte metabolism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks. Non-frozen chondrocytes cultured in agarose served as controls. Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degrees C. Non-frozen alginate beads containing chondrocytes and incubated with 5% DMSO served as a control. After 2 weeks in culture, chondrocytes in agarose or in alginate were sulfated with 10 microCi(35)SO(4)/ml for 48 h. The total production of aggrecans, and the aggrecan subtypes, were subsequently determined. RESULTS: Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocytes. Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells. The proportion of aggrecan in the aggregate form of control-rate frozen chondrocytes kept in agarose remained unaltered. Chondrocytes, control-rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrecan synthesis rates in culture when compared with the non-frozen chondrocytes. The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the control situation. Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death. There was no difference in the synthesis of the different aggrecan subtypes between frozen and non-frozen chondrocytes in alginate. CONCLUSION: Human articular chondrocytes can be stored at -196 degrees C for 24 h without important decreases in their aggrecan synthesis rates when control-rate frozen as a cell suspension in 5% DMSO. Proportions of the aggrecan subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged. The control-rate freezing procedure in the alginate beads pre-incubated with 5% DMSO for 5 h produced no decrease in total aggrecan synthesis rates and no change in the synthesized aggrecan subtypes. Further experiments have to confirm the suitability of this freezing method for long-term storage of chondrocytes allowing us to set up a 'chondrocyte' bank for further use in in vitro and in vivo manipulations.     

Assessment of the profiling MicroRNA expression of differentiated and dedifferentiated human adult articular chondrocytes

MicroRNA has an important role in regulating gene expression during cell differentiation. In this study we identified the expression pattern of microRNA in the differentiated and dedifferentiated chondrocytes. Adult human articular chondrocytes were cultured in monolayer. RNA was isolated from the differentiated chondrocytes (collected after isolation) and the fifth‐passage (dedifferentiated) chondrocytes, and subjected to gene expression analysis using microRNA and cDNA microarray analysis. Real‐time RT‐PCR was also performed to confirm the differentially expressed genes. Furthermore, we integrated microRNA and cDNA microarray data together with computational approaches, such as microRNA gene target prediction algorithms, to reveal the role of microRNAs involved in chondrocyte homeostasis. The results showed a dramatic change in expression of microRNA between the two cell types. Thirteen up‐regulated and 12 down‐regulated microRNAs were detected in differentiated chondroctes. We also revealed microRNA–gene target pairs potentially involved in dedifferentiation process. Our results revealed novel findings of differential expression of microRNA in dedifferentiation, and microRNA could have an important role in the maintenance of chondrocytes homeostasis. MicroRNA may be a target for cartilage tissue engineering and regenerative medicine. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1578–1584, 2011     

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro

Transplantation of chondrocytes by injection or within carrier matrices has shown promise for augmenting the repair of articular cartilage defects. In vivo, transplanted chondrocytes are exposed to mechanical forces. This in vitro study examined the effect of a step application of compressive load to chondrocytes after the cells had been seeded onto a cartilage surface. Bovine chondrocytes were transplanted onto bovine cartilage disks, allowed to attach for 1 hour or 4 days, and subjected to compression through overlying cartilage disks in a confined compression configuration. Before use, the disks were lyophilized to lyse the endogenous chondrocytes and thereby allow assessment of the metabolic activity of the transplanted cells. During a 16-hour application of compressive stress of 0-24-0.72 MPa, proteoglycan synthesis, assessed as [³⁵S]sulfate incorporation into macromolecules. was inhibited by approximately 68% after the 1-hour attachment and by approximately 45% after the 4-day attachment. Cell retention after the application of load was assessed by use of [³H]thymidine-tagged chondrocytes and quantitation of the displacement of radioactivity. After the 1-hour seeding period, loading induced a dose-dependent dislodgment of [³H]radioactivity (as much as 35%) from the tissue bilayer. In contrast, after the 4-day seeding period, there was no detectable effect of loading on chondrocyte dislodgment with an 8–12% release of radioactivity. The inhibitory effect of a 16-hour compression of 0.48 MPa applied after the 4-day seeding period was studied further. This protocol did not appear to have an irreversible effect on chondrocyte metabolism; at 2 days after the release of load, proteoglycan synthesis by the loaded cells was stimulated by 41% compared with transplanted cells that were not subjected to loading. These results suggest that the application of static compressive stress to chondrocytes at a cartilage surface may affect biosynthesis by these cells and thus subsequent integrative cartilage repair. Such an effect may have implications for optimization of the tightness of the press fit of a cell-laden cartilaginous construct into an articular defect.     

外源性人胰岛素样生长因子-1基因在关节软骨细胞中的表达

目的探讨含有绿色荧光蛋白(GFP)的外源性人胰岛素样生长因子(hIGF)-1基因真核表达载体在关节软骨细胞内稳定表达的可行性，建立IGF-1基因工程化的关节软骨细胞。方法利用基因重组技术，将GFPcDNA片段插入pcDNA3．1-hIGF-1载体中，构建重组载体pcGI。然后采用脂质体方法，转染原代关节软骨细胞，经G418筛选后，继续体外单层培养4周。原位杂交和免疫细胞化学检测hIGF-1的表达，荧光显微镜观察GFP的表达，Ⅱ型胶原免疫细胞化学检测软骨细胞表型，四甲基偶氮唑蓝(MTT)比色法检测软骨细胞增殖能力。结果根据重组载体pcGI内的酶切位点，设计采用BamHI酶切显示为线性化，XbaI酶切显示为512bp、1768bp、5915bD三个片段，HindⅢ则酶切下3023bp、5172bp二个片段，证明所构建的质粒方向及大小正确，含有GFP和hIGF-1cDNA片段。稳定转染pcGI的软骨细胞原位杂交、免疫细胞化学和荧光显微镜证实了hIGF-1和GFP的表达，同时MTT显示转染后的软骨细胞增殖能力增强，并能维持Ⅱ型胶原的表达。结论外源性hIGF-1和GFP基因在关节软骨细胞内获得稳定表达，体外单层培养的转染关节软骨细胞增殖能力增强，同时维持软骨细胞表型。     

Mitochondrial proteomic characterization of human normal articular chondrocytes

OBJECTIVE: Mitochondrial dysfunctions have been associated with apoptosis, aging and osteoarthritis (OA). Chondrocyte mitochondrial proteins are attractive targets for the study of the metabolism of cartilage degradation. The copurification of "contaminating" proteins has been the major problem in all phases of mitochondrial proteome research. Therefore, we set up a procedure for the proteomic analysis of human chondrocyte mitochondrial proteins. METHOD: Four types of protein extracts were obtained from primary cultured chondrocytes isolated from healthy donors: (1) initial total chondrocyte extract (CE), (2) cytosol-enriched supernatant fraction (CY), (3) crude mitochondria fraction (CM), and (4) pure mitochondria fraction (PM). Mitochondria were purified by density gradient ultracentrifugation. Mitochondrial proteins were separated by means of two-dimensional gel electrophoresis (2-DE) and silver stained. Protein spots were then identified by mass spectrometry using MALDI-TOF/TOF technology. RESULTS: The best 2-DE reference map of mitochondrial proteome was constructed employing PM fraction. Thirty-nine percent of the identified proteins were functionally distributed in the mitochondria, 14% in the endoplasmic reticulum and 36% in the cytoplasm. Examining their biological function, 22% are involved in protein targeting, 12% in signaling, 12% in glycolysis, 10% in RNA, DNA or protein synthesis, 10% in oxidative phosphorylation and 4% in redox. The analysis of mitochondrial Mn-superoxide dismutase (SODM) revealed an age-dependent decrease of this protein. CONCLUSION: PM fraction allowed the obtention of a high quality proteomic map for the study of mitochondrial proteins in human articular chondrocytes. This proteomic approach may be also efficient to analyze both quantitative and qualitative modulations of the mitochondrial proteome in human chondrocytes during aging and pathological conditions such as OA.     

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.