Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells

The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Qiagen), linear polyethylenimine (PEI; ExGen 500; Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl β-d-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2 μg of pSES.β was combined with either 21 μg of dendrimer, 1.7 μg of linear PEI, or 2.0 μg of branched PEI. At the same DNA/polymer ratios, the proportions of X-gal-stained cells were also the highest (31.3 ± 7.5%, 30.3 ± 9.0%, and 8.3 ± 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes. Received: May 31, 2000 / Accepted: September 22, 2000     

Implication of prostaglandin E(2) in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs

OBJECTIVE: Calpains are known as Ca(2+)-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined. METHODS: Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined. RESULTS: TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E(2) (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha. CONCLUSIONS: These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process.     

Novel mode of processing and secretion of connective tissue growth factor/ecogenin (CTGF/Hcs24) in chondrocytic HCS-2/8 cells

The synthesis, processing, and secretion of human connective tissue growth factor (CTGF/Hcs24) in a human chondrocytic cell line, HCS-2/8, were analyzed immunochemically. By metabolic pulse-labeling, chasing, and subsequent immunoprecipitation analyses, active synthesis of CTGF was observed not only in growing HCS-2/8 cells, but also in confluent cells. However, secretion and processing of CTGF were found to be regulated differentially, depending upon the growth status. During phases of growth, HCS-2/8 cells released CTGF molecules immediately without sequestering them within the cell layer. In contrast, after the cells reached confluence, the secretion slowed, resulting in an accumulation of CTGF in the cells or extracellular matrices (ECMs). Also, in confluent cell layers, a 10 kDa protein that was reactive to an anti-CTGF serum was observed. This CTGF-related small protein was not detected immediately after labeling, but gradually appeared within 6 h after chase, which suggests its entity as a processed subfragment of CTGF. Surprisingly, the 10 kDa protein was stable even 48 h after synthesis, and was not released by ECM digestion, suggesting an intracellular maintenance and function. Taken together, the behavior of CTGF in HCS-2/8 cells is remarkably different from that reported in fibroblasts, which may represent unique roles for CTGF in the growth and differentiation of chondrocytes.     

成人骨髓基质干细胞体外定向诱导分化为软骨细胞的实验研究

目的 建立临床成人骨髓基质干细胞(MSCs)体外培养、定向诱导分化为软骨细胞的途径。方法抽取成人骨髓，Percol密度梯度离心法进行体外培养，贴壁细胞传代，取第3代细胞在培养基中添加软骨分化诱导剂地塞米松、维生素C和不同剂量转化生长因子-β(TGF-β)，培养16d后,在倒置显微镜观察细胞形态，甲苯胺蓝染色蛋白多糖，逆转录一聚合酶链反应(RT-PCR)、免疫细胞化学(SABC法)检测Ⅱ型胶原表达，诱导后MSCs与新型材料聚乳酸和羟基乙醇共聚物(PL-GA)复合。结果 Percoll密度梯度离心法培养可获得均一的。MSCs；5、10ng TGF-β诱导分化的MSCs生长迅速。呈典型的软骨细胞形态，甲苯胺蓝染色阳性，Ⅱ型胶原表达阳性，MSCs对材料PL-GA黏附力强。结论 可以从成人骨髓中培养出MSCs，并可定向诱导分化为软骨细胞，5～10ng TGF-β为最佳诱导剂量，成人MSCs可用作临床自体软骨组织工程种子细胞。     

Selective Agonists of Nuclear Retinoic Acid Receptor Gamma Inhibit Growth of HCS-2/8 Chondrosarcoma Cells

Chondrosarcoma is the second most common primary bone sarcoma. Treatment of chondrosarcoma is limited to surgery due to radiation and chemotherapy resistance of this cancer. An ideal treatment for chondrosarcoma would be a well-tolerated, minimally invasive local or systemic treatment modality to halt or slow tumor growth prior to resection of local, unresectable local, or metastatic disease. Palovarotene, an agonist of nuclear retinoic acid receptor γ (RARγ) has shown therapeutic action for treatment of heterotopic ossification and osteochondroma without serious adverse effects in animal models. We hypothesized that selective agonists of RARγ would have an inhibitory effect on chondrosarcoma. All human chondrosarcoma specimens expressed RARγ as determined by immunohistochemical staining. The ΗCS-2/8 chondrosarcoma cell line, established from low-grade human chondrosarcoma, was used to examine the actions of RARγ agonists. In ΗCS2/8 pellet cultures, RARγ agonist treatment reduced the mass size and significantly decreased total glycosaminoglycan, protein amounts, and gene expression levels of cartilage matrix molecules when compared with control groups. Systemic treatment with RARγ agonists significantly inhibited the growth of ΗCS-2/8 cell transplants in vivo. Furthermore, local injection of RARγ agonist-loaded poly-lactic acid nanoparticles induced regression of the mass size of the transplants. Histologic analysis demonstrated that RARγ agonist treatment inhibited cell proliferation activity and stimulated encapsulation of the tumor. These findings indicate that RARγ agonists, including palovarotene, may have an anti-tumor effect on low-grade chondrosarcomas. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1045-1051, 2020     

Expression of NG2/human melanoma proteoglycan in human adult articular chondrocytes

OBJECTIVE: NG2 is a transmembrane chondroitin sulfate (CS) rich proteoglycan originally identified in rats. It has recently been shown to be identical to human melanoma proteoglycan (HMPG). In rats NG2 has a limited distribution in adult tissues, being expressed predominantly by neuronal and glial cells whereas during development it is also expressed in developing mesenchyme including cartilage. NG2/HMPG has putative roles in interactions between glial and melanoma cells with extracellular matrix (ECM) molecules. This study was undertaken to assess whether NG2/HMPG was expressed by normal and osteoarthritic human articular chondrocytes. DESIGN: Cryostat sections of human fetal knee joints and normal and osteoarthritic articular cartilage were immunostained with antibodies against rat NG2 (N143.8) and HMPG (M28B5, 9.2.27). Immunoprecipitation and Western blotting was carried out on protein extracts of chondrocytes from normal and osteoarthritic cartilage. Immunofluorescence of NG2 and potential ligands was carried out in vitro on cells from normal and osteoarthritic cartilage. RESULTS: Fetal and both normal and osteoarthritic adult cartilage showed strong immunoreactivity for NG2/HMPG. Western blotting showed a smeared component of molecular weight greater than 400 kDa and a faint band at 250 kDa which became predominant upon digestion with chondroitinase ABC. Immunofluorescence of chondrocytes in vitro showed NG2 to be distributed in a punctate pattern without co-localization of actin or several ECM proteins including fibronectin and type VI collagen. CONCLUSION: NG2/HMPG is expressed by human fetal and adult chondrocytes and in adult articular chondrocytes the core protein is chondroitin sulfated. The function of this molecular in human articular cartilage remains to be defined.     

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34(+)lin(-) hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well receives a single hematopoietic stem cell candidate. During an experiment lasting several days to weeks, each cell-containing well is moved sequentially and serially to a microscopic imaging system. This movement is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Biobox built on the microscopic stage. New image analysis software facilitates tracking of cell movement, recording the time of cell division, and immunophenotyping of multiple, individual, or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and may be applicable to a wide range of other systems of interest in tissue engineering.     

Preconditioning and adenosine protect human proximal tubule cells in an in vitro model of ischemic injury

Renal ischemic reperfusion injury results in unacceptably high mortality and morbidity during the perioperative period. It has been recently demonstrated that ischemic preconditioning or adenosine receptor modulations attenuate renal ischemic reperfusion injury in vivo. An in vitro model of ischemic renal injury was used in cultured human proximal tubule (HK-2) cells to further elucidate the protective signaling cascades against renal ischemic reperfusion injury. ATP depletion preconditioning (1 h of antimycin A and 2-deoxyglucose treatment followed by 1 h of recovery), adenosine, an A(1) adenosine receptor selective agonist, or an A(2a) adenosine receptor selective agonist significantly attenuated subsequent severe ATP depletion injury of HK-2 cells. In contrast, an adenosine receptor antagonist failed to prevent protection induced by ATP depletion preconditioning. Cytoprotection by ATP depletion preconditioning or A(1) adenosine receptor activation was prevented by inhibitors of extracellular signal-regulated mitogen-activated kinases, protein kinase C, and tyrosine kinases. The A(1) and A(2a) adenosine receptor-mediated cytoprotection were also dependent on G(i/o) proteins and PKA activation, respectively. It is concluded that ATP depletion preconditioning and A(1) and A(2a) adenosine receptor activation protect HK-2 cells against severe ATP depletion injury via distinct signaling pathways.     

Cathepsin K在人软骨细胞体外培养去分化过程中的表达研究

目的通过基因芯片技术研究在体外培养的去分化人软骨细胞的基因改变，从中筛选出目标基因，并对其在各代软骨细胞中的表达进行研究。方法取体外培养的P1代和去分化的P4代软骨细胞进行8300点基因芯片的DNA微阵矩杂交，筛选出差异最明显的基因，并通过RT-PCR和Western-blot方法检测各代软骨细胞中该基因的表达水平。结果体外培养的P1、P4代软骨细胞间共140个基因差异表达，其中Cathepsin K基因表达差异水平最明显，P4与P1的比值平均为29．72，RT-PCR显示在596bp的条带处，随传代次数增加逐渐增亮，半定量分析P4／P1为3．21，差异有统计学意义（P〈0．05）。Western-blot检测显示在相对分子质量为40000的蛋白条带随体外传代蛋白浓度增加，灰度分析P4／P1为3．39，差异有统计学意义（P〈0．05）。结论基质降解半胱氨酸蛋白酶Cathepsin K在去分化软骨细胞中表达强烈，提示其与软骨细胞去分化过程中的基质降解有关。     

大鼠椎间盘软骨终板细胞凋亡体外模型的建立

[目的]建立大鼠椎间盘软骨终板细胞凋亡体外模型.[方法]为了模拟椎间盘内部低营养低代谢环境,采用低胎牛血清培养法培养椎间盘软骨终板细胞分别含0％,1％,3％,5％,8％,10％胎牛血清,设置不同浓度梯度筛选最佳浓度,检测凋亡率、凋亡蛋白表达及caspase酶活性.[结果]低胎牛血清培养组细胞发生形态改变,DAPI染色阳性细胞增多;流式细胞仪检测凋亡率随着FBS浓度降低而升高,1％为最有效诱导凋亡浓度;Western blot示FAS、caspase-3、PARP、细胞色素C表达在1％ FBS组明显高于10％时,同时caspase-3/8/9酶活性增高.[结论]低胎牛血清培养法能诱导体外培养的软骨终板细胞发生凋亡,最终会引起细胞功能丧失和椎间盘退变,caspase家族可能参与了这一过程.     

不同类型人软骨细胞体外生物学特性比较

目的　通过研究人耳软骨和肋软骨两种不同类型软骨细胞体外分离、增殖、老化规律 ,为选择合适的组织工程种子细胞提供依据。方法　取小耳畸形残耳软骨和肋软骨 ,体外分别用0 .0 5 %和 0 .15 %Ⅱ型胶原酶消化 16h分离 ,台盼蓝染色计活细胞数 ,得原代细胞获得率。体外单层培养 6代 ,观察形态学改变 ,群体倍增时间 (PDT ) ,免疫细胞化学染色及逆转录 聚合酶链反应(RT PCR)检测Ⅱ型胶原和Aggrecan评定软骨细胞老化规律。 结果　人残耳软骨组织平均细胞获得率为 ( 1.5 4± 0 .14 )× 10 6/ g ,肋软骨平均获得率为 ( 0 .46± 0 .0 9)× 10 6/ g ,两类软骨细胞P1的PDT最短 ,前 3代增殖力较强 ,P3 以后PDT明显延长 ,P6代细胞不再增殖。免疫细胞化学及RT PCR均证实耳软骨细胞 3代内、肋软骨细胞 4代内软骨细胞表型稳定。结论　第 2代的耳软骨细胞与第 3代肋软骨细胞可作为人体内组织工程化软骨构建的种子细胞。     

TGF-β2转染关节软骨细胞的实验研究

目的 观察人关节软骨细胞在体外单层培养过程中的去分化，以及转染TGF-β2在关节软骨细胞内的表达和对去分化的抑制作用。方法 从手术中切除的软骨组织中分离培养成人关节软骨细胞，通过脂质体介导的方法将已构建的pcDNA3.1( )/TGF-β2转染到体外单层培养的软骨细胞中，采用RT-PCR，ELISA、组织学染色、免疫组化和原位杂交的方法．分别对转染组和未转染组的第1、6、9代细胞进行检测，比较目的基因表达、软骨细胞形态以及胶原和多糖生物合成的差异．结果 经多次传代后，未转染软骨细胞在体外单层培养过程中逐渐走向去分化．TGF-β2、Ⅱ型胶原和蛋白多糖聚糖体的表达逐渐减低．而Ⅰ型胶原的表达增高，目的基因在转染组各代软骨细胞内均得到表达，转染后细胞保持软骨细胞的形态，Ⅱ型胶原和蛋白多糖聚糖体表达虽有降低．但均高于未转染的同代细胞，而Ⅰ型胶原表达增高的程度低于未转染细胞。结论 人关节软骨细胞在体外单层培养中有去分化趋势；pcDNA3.1( )/TGF-β2真核表达载体转染人关节软骨细胞获得成功．在转染后关节软骨细胞内稳定表达．并对软骨细胞的去分化有抑制作用。     

犬骨髓基质干细胞体外定向分化为软骨细胞

目的 体外诱导犬骨髓基质干细胞(BMSCs)定向分化为软骨细胞，探讨体外诱导成软骨的方法和条件。方法 自犬肋骨取骨髓2～3ml，体外行原代和传代培养扩增，顺序加入碱性成纤维细胞生长因子(bFGF)和转化生长因子β1(TGF-β1)，以培养瓶内较高细胞浓度培养，诱导BMSCs分化为软骨细胞。甲苯胺蓝、阿新蓝染色检测软骨基质的分泌，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。结果 诱导的软骨样细胞甲苯氨蓝异染性、阿新蓝染色阳性；Ⅱ型胶原免疫组织化学检测阳性。结论 应用bFGF和TGF-β1体外可以诱导犬BMSCs分化为软骨细胞，诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

Effects of extracorporeal shock waves on human articular chondrocytes and ovine bone marrow stromal cells in vitro

Introduction We investigated the effects of extracorporeal shock waves on cytotoxicity and on the proliferation of human chondrocytes and ovine bone marrow stromal cells.Materials and methods Isolated cells were cultured to confluence, and 500 shock waves were applied at energy flux densities of 0, 0.02, 0.06, and 0.17 mJ/mm² for the cytotoxicity assay. The same energies at 100, 500, and 1000 impulses were used for the proliferation assay.Results Although bone marrow stromal cells revealed a dose- and impulse-dependent increase in the proliferation rate, no significant differences were found. Chondrocytes had less proliferative potential than untreated control groups. In the experimental set-up using 1000 impulses, proliferation was even higher in the control group. Both types of cells revealed a dose-dependent increase in cytotoxicity in the lactate dehydrogenase (LDH) assay.Conclusion As femoral head necrosis, osteochondritis dissecans, and similar disorders are increasingly treated with shock waves, their effect on human cartilage and chondrocytes deserves attention. We recommend further in vitro experiments with bone marrow stromal cells, as the latter might play an important role in the presumed multifactorial osteogenetic mechanism of shock waves due to their pluripotent character.The authors declare that the experiments comply with the current laws.     

Glucosamine sulfate modulates dysregulated activities of human osteoarthritic chondrocytes in vitro

OBJECTIVE: The efficacy of glucosamine sulfate (GS) in the symptomatic treatment of patients with osteoarthritis (OA) is suggested to be mediated by still unknown effects on the altered OA cartilage. DESIGN: Using human OA chondrocytes in culture, the effects of GS on protein synthesis, caseinase, collagenase, phospholipase A2 (PLA2) and protein kinase C (PKC) activities as well as production of nitric oxide and cyclic AMP were studied in both cells and culture medium. RESULTS: GS significantly reduced PLA2 activity, and more modestly collagenase activity, in the OA chondrocytes in a dose-dependent manner. By contrast, PLA2 and collagenase activity of the culture medium was not modified. No effects on caseinase activity was seen. GS significantly and dose-dependently increased protein synthesis. GS did not modify nitric oxide and cAMP production but significantly increased PKC production. CONCLUSION: GS modified cultured OA chondrocyte metabolism by acting on PKC, cellular PLA2, protein synthesis and possibly collagenase activation. Extrapolation of the effect to the in-vivo situation remains hypothetical but they might represent some possible mechanisms of action of the drug in human.     

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。