Metabolic effects of avocado/soy unsaponifiables on articular chondrocytes

Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1-10 mug/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23-37%), labeled sulfate release (15-23%) and PGE2 synthesis (45-58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes.     

Effects of Misoprostol and Salicylate on Canine Osteoarthritis

The ability of misoprostol to reverse the deleterious changes induced in cartilage by sodium salicylate was tested using osteoarthritic canine and chondrocytes. Adult mongrel dogs were subjected to anterior cruciate ligament transection and dosed with either misoprostol, salicylate, or misoprostol plus salicylate. No significant differences were noted among the three groups in either gross and histological changes or general biochemical changes. This approach was abandoned since the levels of misoprostol attained by oral dosing were much lower than those required for domonstration of misoprostol effects in vitro. Next, chondrocytes were isolated from the osteoarthritic and contralateral knee joint cartilage of dogs 12 weeks after anterior cruciate ligament transection and cultured in alginate beads. The cultures were incubated with (3)H-proline and both the genetic types of collagen synthesized and the net synthesis of (3)H-hydroxyproline were determined. No drug or combination of drugs affected the genetic types of collagen synthesized. Total protein and collagen synthesis by chondrocytes was reduced in the presence of salicylate and increased in the presence of misoprostol. When osteoarthritic chondrocytes were incubated with both agents, the salicylate effect was reversed by misoprostol. Collagen synthesis by the chondrocytes from the contralateral knee was also suppressed by salicylate, but addition of misoprostol failed to restore synthesis. In summary, misoprostol, even at very high doses, has limited chondroprotective activity in canine cartilage, as judged from collagen synthetic activity.     

低氧对脂肪干细胞和关节软骨细胞三维共培养成软骨能力的影响

背景：多项体内外研究表明低氧和共培养均促进干细胞向软骨细胞方向分化。目的：观察低氧对脂肪干细胞和关节软骨细胞三维共培养成软骨能力的影响。方法：脂肪干细胞和关节软骨细胞二者按3∶1比例混合,以5×1010 L-1接种于聚乳酸-羟基乙酸共聚物/明胶支架上,分别在常氧（体积分数为20%O2）、低氧（体积分数为5%O2）环境下培养6周。苏木精-伊红染色进行组织学分析,阿尔新蓝染色鉴定糖胺多糖的合成,免疫组化鉴定Ⅱ型胶原的表达,并测定各组支架-细胞复合物的DNA、糖胺聚糖、羟脯氨酸含量。结果与结论：低氧组苏木精-伊红染色显示大量细胞及细胞外基质生成,阿尔新蓝染色显示有大量糖胺多糖生成,免疫组化显示Ⅱ型胶原表达强阳性,且DNA、糖胺聚糖、羟脯氨酸等各项指标均高于常氧组。表明低氧促进脂肪干细胞和关节软骨细胞共培养成软骨分化。     

京尼平苷对大鼠软骨细胞增殖的影响

背景:京尼平苷能够促进韧带成纤维细胞的增殖和胶原的合成,促进组织的修复和韧带损伤的愈合.目的:观察京尼平苷对大鼠软骨细胞增殖的影响.方法:采用二次酶消化法获得大鼠关节软骨细胞进行传代培养,并取第4 代细胞给予0,25,50,100mmol/L 京尼平苷干预,利用MTT 比色、流式细胞仪检测细胞周期等方法检测药物干预后第4 代软骨细胞的增殖情况.结果与结论:体外培养的软骨细胞随着传代次数的增加,细胞形态由原代的多角形逐渐变为长梭形;且随着培养时间的延长及京尼平苷浓度的升高,软骨细胞增殖更为显著(P < 0.01).     

Development of a refined tenocyte expansion culture technique for tendon tissue engineering

The aim of this study was to efficiently expand less differentiated tenocytes with minimum use of fetal bovine serum (FBS) for tenocyte‐based tendon tissue engineering. To achieve this goal, human tenocytes were cultured in different concentrations of FBS and combinations of growth factors PDGF_BB, IGF‐1 and bFGF. A number of growth factors were selected that could support tenocyte expansion at reduced differentiated state with minimum FBS usage. Results showed that the expansion of the tenocytes cultured for 14 days with 1% FBS, 50 ng/ml PDGF_BB and 50 ng/ml bFGF was similar to that cultured in the 10% FBS control group. The tenocytes cultured in the treatment group showed significantly lower collagen synthesis and down‐regulation of mRNA expression of tendon differentiation markers. Cell morphology confirmed that tenocytes cultured in the growth factors had reduced collagen fibril formation compared to tenocytes cultured in 10% FBS. Our findings confirm the feasibility of inducing human tenocyte expansion in vitro with the least amount of FBS usage, while controlling their differentiation until required. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of Collagen Gene Expression by Prostaglandins and Interleukin-1beta in Cultured Chondrocytes and Fibroblasts

To compare the modulatory effects of different prostaglandins on collagen gene expression in human chondrocytes, PGE(2), PGE(1), misoprostol (PGE(1) analog), and PGF(2alpha) (10, 50 and 100 ng ml(minus sign1)) were added to human chondrocytes with or without interleukin-1beta (IL-1beta) in the presence of indomethacin to inhibit endogenous prostaglandin synthesis and the effects evaluated on chondrocyte morphology, collagen synthesis, and procollagen mRNA levels. The effects of prostaglandins on the expression of collagen gene regulatory sequences were examined using transient transfection assays of reporter gene constructs in human chondrocytes and BALB/c3T3 fibroblasts, PGE(1), misoprostol, and PGF(2alpha), similar to PGE(2), inhibited type I collagen gene expression in fibroblasts and promoted type II collagen gene expression in chondrocytes. PGE(2), the major inflammatory prostaglandin produced by IL-1-activated chondrocytes and fibroblasts, and PGF(2alpha) were somewhat more potent than the anti-inflammatory prostaglandins PGE(1) and misoprostol in counteracting the IL-1-induced suppression of type II collagen gene expression by chondrocytes and stimulation of type I collagen gene expression by fibroblasts. Rather than promoting degradation of the cartilage matrix in joint diseases, prostaglandins may be somewhat protective, suppressing fibrosis, and maintaining or promoting appropriate cartilage repair.     

组织块法培养成年兔肌腱细胞

背景:体外分离培养肌腱细胞,熟悉其生物学特性是研究肌腱愈合机制、改善肌腱愈合内环境的前提和基础。目的:采用组织块法体外培养成年兔肌腱细胞,观察其细胞形态、生长增殖情况以及Ⅰ型、Ⅲ型胶原蛋白的表达。方法:无菌条件下取成年新西兰白兔双侧后肢趾屈肌腱,手术显微镜下剥离、去除肌腱外膜组织,剪成组织小块,0.25%胰蛋白酶和0.1%胶原酶Ⅰ消化10～15min,离心去上清,将组织小块转移到培养瓶中,贴壁后加入1mL培养液,待细胞游出贴壁生长时,再加培养液继续培养,每3d换液1次,当细胞长到80%～90%融合时按1:3传代。结果与结论:一般10d左右细胞会从组织块游出贴壁生长,此时细胞多呈星形或不规则形,随着时间延长细胞逐渐增多,呈梭形的成纤维细胞样。传代细胞刚接种时圆形,4～6h开始贴壁并伸展为梭形,排列逐渐规则成群。从传代细胞的生长曲线可看出,前4d细胞生长较慢,为潜伏期,第5天后进入快速增殖期,7d以后进入平台期。免疫荧光法证实Ⅰ型胶原染色阳性,而Ⅲ型胶原染色呈阴性。结果表明采用组织块法可在体外成功培养成年兔肌腱细胞。     

京尼平苷对大鼠软骨细胞增殖的影响

背景：京尼平苷能够促进韧带成纤维细胞的增殖和胶原的合成,促进组织的修复和韧带损伤的愈合。目的：观察京尼平苷对大鼠软骨细胞增殖的影响。方法：采用二次酶消化法获得大鼠关节软骨细胞进行传代培养,并取第4代细胞给予0,25,50,100mmol/L京尼平苷干预,利用MTT比色、流式细胞仪检测细胞周期等方法检测药物干预后第4代软骨细胞的增殖情况。结果与结论：体外培养的软骨细胞随着传代次数的增加,细胞形态由原代的多角形逐渐变为长梭形;且随着培养时间的延长及京尼平苷浓度的升高,软骨细胞增殖更为显著（P〈0.01）。     

In vitro two‐dimensional and three‐dimensional tenocyte culture for tendon tissue engineering

In order to examine the differentiation potential of the tenocytes expanded in our defined culture medium (reported previously) and the effect of sequential combination of the two culture conditions on human tenocytes, a two‐dimensional and three‐dimensional experimental approach was used. Human tenocytes were sequentially exposed to 1% fetal bovine serum (FBS) + 50 ng/ml platelet‐derived growth factor‐BB (PDGF_BB) + 50 ng/ml basic fibroblast growth factor (bFGF) for the first 14 days (expansion phase) followed by a further 14‐day culture in the presence of 10 ng/ml transforming growth factor β‐3 plus 50 ng/ml insulin‐like growth factor 1, but in the absence of serum (differentiation phase). The results showed that by sequential treatment of human tenocytes maintaining a long‐term two‐dimensional tenocyte culture in vitro for up to 28 days was possible. These findings were further verified using a three‐dimensional scaffold (Bombyx silk) whereby the tendon‐like constructs formed resembled macroscopically and microscopically the constructs formed in 10% FBS supplemented culture media and the human hamstring tendon. These findings were further substantiated using haematoxylin and eosin staining, scanning electron microscopy and by immunohistochemical detection of type I collagen. In addition, the mechanical properties of the three‐dimensional constructs were determined to be significantly superior to that of the natural human hamstring tendon. This is the first report to demonstrate a possible approach in expanding and differentiating human tenocytes for tendon tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

类胰岛素样生长因子I在人鼻中隔软骨细胞体外培养中的作用

目的:为加速体外培养的人鼻中隔软骨细胞的增殖,观察类胰岛素样生长因子I(IGF-I)对体外培养软骨细胞的影响。方法:体外培养人鼻中隔软骨细胞,MTT法观察IGF-I对软骨细胞增殖的影响;流式细胞仪检测IGF-I对软骨细胞周期的影响;斑点杂交法了解IGF-I对软骨细胞II型胶原mRAN的影响。结果:IGF-I浓度大于5ng/ml即可促进体外培养软骨细胞的增殖,缩短软骨细胞增殖周期,有助于II型胶原mRNA的合成。结论:IGF-I促进人鼻中隔软骨细胞增殖与缩短细胞周期有关,并可能通过促进软骨细胞II型胶原mRNA的合成使软骨细胞保持表型稳定。     

Enhanced repair of the anterior cruciate ligament by in situ gene transfer: evaluation in an in vitro model.

The inability of the ruptured anterior cruciate ligament (ACL) of the knee joint to heal spontaneously presents numerous clinical problems. Here we describe a novel, gene-based approach to augment ACL healing. It is based upon the migration of cells from the ruptured ends of the ligament into a collagen hydrogel laden with recombinant adenovirus. Cells entering the gel become transduced by the vector, which provides a basis for the local synthesis of gene products that aid repair. Monolayers of bovine ACL cells were readily transduced by first-generation, recombinant adenovirus, and transgene expression remained high after the cells were incorporated into collagen hydrogels. Using an in vitro model of ligament repair, cells migrated from the cut ends of the ACL into the hydrogel and were readily transduced by recombinant adenovirus contained within it. The results of experiments in which GFP was used as the transgene suggest highly efficient transduction of ACL cells in this manner. Moreover, during a 21-day period GFP+ cells were observed more than 6 mm from the severed ligament. This distance is ample for the projected clinical application of this technology. In response to TGF-beta1 as the transgene, greater numbers of ACL cells accumulated in the hydrogels, where they deposited larger amounts of type III collagen. These data confirm that it is possible to transduce ACL cells efficiently in situ as they migrate from the ruptured ACL, that transduction does not interfere with the cells' ability to migrate distances necessary for successful repair, and that ACL cells will respond in a suitable manner to the products of the transgenes they express. This permits optimism over a possible clinical use for this technology.     

Phosphonoacetic Acid-Resistant Mutants of Herpes Simplex Virus: Effect of Phosphonoacetic Acid on Virus Replication and In Vitro Deoxyribonucleic Acid Synthesis in Isolated Nuclei

Phosphonoacetic acid (PAA) inhibits the replication of herpes simplex virus in BSC-1 cells and the in vitro synthesis of deoxyribonucleic acid (DNA) in isolated nuclei. Phosphonopropionic acid at a concentration of 100 mug/ml had no effect on herpes simplex virus replication. PAA-resistant mutants were obtained at a rate of 1 in 10(4) plaque-forming units after 5-bromodeoxyuridine mutagenization of the virus. These mutants replicate in BSC-1 cells in the presence of 100 mug of PAA per ml and induce a PAA-resistant DNA polymerase that synthesizes DNA in vitro in the presence of PAA.     

Hepatocyte collagen production in vivo in normal rats.

Although hepatocytes produce collagen in vitro, their contribution to hepatic collagen synthesis in vivo is unknown. To answer this question, we injected rats intraperitoneally with [3H]proline and [14C]ornithine. [3H]Proline labeled prolyl-t-RNA in both hepatocytes and nonparenchymal cells. In contrast, [14C]ornithine was rapidly converted to [14C]arginine via the urea cycle only in hepatocytes, labeling arginyl-t-RNA. Approximately 60% of the 14C in albumin and transferrin was present as arginine while the remainder was found in proline and related amino acids. As expected for proteins that have the same proline/arginine ratio and that are produced solely by the hepatocyte, the [3H]proline/[14C]arginine ratio was very similar in albumin and transferrin. Conversely, in nonparenchymal cells a negligible percentage of 14C was present as arginine. A sizeable percentage of the 14C in hepatic collagen was present as arginine; given the greater proline(+hydroxyproline)/arginine ratio in hepatic collagen, our data indicate that in normal rats, hepatocytes contribute most of newly synthesized hepatic collagen.     

Collagen in the human lung. Quantitation of rates of synthesis and partial characterization of composition.

The presence of collagen in lung is fundamental in normal lung structure and function. Methods have been developed to examine human fetal and adult lung collagen with respect to its composition and synthesis. The second trimester fetal lung has a large number of cells per unit lung mass (36.6 plus or minus 2.7 mug DNA/mg dry wt) and relatively small amounts of collagen (17.0 plus or minus 5.3 mug collagen/mg dry wt). The number of cells per unit lung mass in the adult lung (11.1 plus or minus 3.4 mug DNA/mg dry wt) is 30% of the number of cells in the fetal lung, but the adult has 11 times more collagen (196 plus or minus 25 mug collagen/mg dry wt). The composition of fetal lung collagen can be partially characterized by extraction with salt at neutral pH, acetic acid, or guanidine. The extracted chains, representing 10% of the total lung collagen, chromatograph as alpha1 and alpha2 chains, each with a mol wt of 100,000 and an animo acid composition characteristic for collagen but not specific for lung. Short-term explant cultures of fetal and adult lung synthesize alpha chains which can be isolated by ion-exchange chromatography. These chains, representing 30-40% of the total collagen synthesized by the explants, coelectrophorese with extracted collagen chains on acrylamide gels: they are destroyed by clostridial collagenase and they have a mol wt of 100,000. Although the composition of the collagen synthesized by these explants can be only partially characterized, the rate of synthesis of both collagen and noncollagen protein can be quantitated. In fetal lung, 4.0 plus or minus 1.2% of the amino acids incorporated into protein per hour are incorporated into collagen. In normal adult lung, this percentage (4.2 plus or minus 0.9%) is remarkably similar. These values are almost identical to the relative rate of collagen synthesis in rabbit lung in the same age range. This technology should be applicable to answer specific questions regarding collagen synthesis and degradation in human lung disease.     

生长激素对大鼠胫骨生长板软骨细胞胶原Ⅱ表达的影响

目的探讨生长激素（growth hormone,GH）对离体培养的大鼠胫骨生长板的软骨细胞Ⅱ型胶原（collagenⅡ,colⅡ）表达的影响。方法采用两步酶消化法分离培养5～8只三周龄大鼠生长板的软骨细胞,用RT—PCR和免疫组化的方法分别检测不同浓度GH对软骨细胞Ⅱ型胶原mRNA和蛋白的表达。结果GH能促进软骨细胞ColⅡ的表达并呈浓度依赖性,GH在10～500ng/ml之间能促进ColⅡ的表达,与对照组比较差异均有统计学意义（P〈0．05）,并且以100ng／ml时表达最强,随着GH浓度的加大,其促ColⅡ表达的效应逐渐减弱,1000ng／ml与对照组比较无差异（P〉0．05）。结论GH通过促进软骨细胞ColⅡ的表达能维持软骨细胞的表型,GH在使用过程中不会导致骨龄的增加。     

Effect of amphotericin B and clotrimazole on lymphocyte stimulation

Human lymphocytes were stimulated in vitro by phytohemagglutinin, concanavalin A, pokeweed mitogen, purified protein derivative-tuberculin, and allogenic cells. The deoxyribonucleic acid synthesis of the lymphocytes was inhibited in increasing degree by 4 to 8 mug of amphotericin B per ml of culture irrespective of lymphocyte stimulant used. The effect of clotrimazole on the deoxyribonucleic acid synthesis varied between experiments with different and also between experiments with the same stimulant. Ten micrograms of clotrimazole per ml was generally inhibiting, whereas in one experiment 2 mug or more per ml inhibited the purified protein derivative-induced deoxyribonucleic acid synthesis. The effects of amphotericin B and clotrimazole were neutralized by serum.     

Antiviral activity of arabinosyladenine and arabinosylhypoxanthine in herpes simplex virus-infected KB cells: selective inhibition of viral deoxyribonucleic acid synthesis in synchronized suspension cultures.

The drug 9-beta-d-arabinofuranosyladenine (ara-A) significantly suppressed the formation of herpes simplex virus type 1-induced syncytia in BHK-21/4 cells at concentrations as low as 0.1 mug/ml. Optimal activity was noted when the drug was added before initiation of viral deoxyribonucleic acid (DNA) synthesis (3.5 h postinfection). The deaminated derivative of ara-A, 9-beta-d-arabinofuranosylhypoxanthine (ara-H), was at least 10 times less effective in suppressing the development of herpes simplex virus-induced syncytia. The replication of herpes simplex virus was measured by assaying fluids and cells from infected drug-treated cultures by using a plaque production technique. Ara-A at drug levels of >10 < 32 mug/ml completely blocked the replication of infectious virus particles. Ara-H was less effective than ara-A in reducing the replication of virions. Rates of host and viral DNA synthesis were monitored by pulse labeling herpes simplex virus-infected synchronized KB cells with [(3)H]thymidine and subsequently separating viral from cellular DNA in CsCl density gradients. During synthetic (S) phase, ara-A or ara-H at concentrations ranging from 3.2 to 32 mug/ml selectively inhibited viral DNA synthesis. At 3.2 mug of ara-A per ml, viral DNA synthesis was reduced 74% although total cellular DNA synthesis was unaffected. Increasing concentrations of ara-A produced increasing temporal delays in the maximal rate of host DNA synthesis. This time shift was not observed in cells treated with ara-H.     

Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae

The sulfur-containing antibiotic thiolutin has been shown to be a potent, reversible inhibitor of the growth of Saccharomyces cerevisiae. Viability was unaffected over the concentration range of 4 to 100 mug/ml. At concentrations as low as 2 mug/ml, the drug inhibited ribonucleic acid (RNA) and protein synthesis in whole cells and spheroplasts. At these low concentrations, protein synthesis continued for a short period of time after RNA synthesis was completely stopped. With higher drug concentrations (greater than 20 mug/ml) protein synthesis was inhibited; concentrations of thiolutin up to 100 mug/ml did not affect translocation or peptide bond formation in cell-free protein-synthesizing systems from yeast. The effect of thiolutin on the activity of partially purified deoxyribonucleic acid-dependent RNA polymerases was examined, and the drug was found to be a potent inhibitor of RNA synthesis in vitro. Inhibition was greatest when the polymerase was preincubated with thiolutin. Several mechanisms are discussed to explain the multiple effects of thiolutin on S. cerevisiae. Since the action of the drug is easily reversed, thiolutin may prove to be of use in studies of various stages of yeast growth.     

Doxorubicin-induced inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures. Relevance to imparied wound healing.

Previous clinical and experimental observations have indicated that wound healing is impaired as a result of treatment with doxorubicin, a chemotherapeutic agent. In this study, the effects of doxorubicin were examined in human skin fibroblast cultures with respect to collagen production and fibroblast proliferation. The results indicated that the synthesis of hydroxyproline as a marker of collagen production was markedly reduced, with an approximate concentration of inhibitor yielding 50% inhibition of 1 microM. This inhibition could be explained, in part, by generalized inhibition of total protein synthesis, but in addition, there was a significant inhibition of prolyl hydroxylation during collagen biosynthesis, as indicated by a reduction in the ratio of [3H]hydroxyproline/([3H]hydroxyproline + [3H]proline). The latter effect was shown to result from inhibition of prolyl hydroxylase by doxorubicin. As a consequence of reduced prolyl hydroxylation, the stability of newly synthesized procollagen triple helix was shown to be compromised. At the same time, doxorubicin significantly reduced fibroblast proliferation in vitro, as determined by [3H]thymidine incorporation. Thus, reduced collagen production and inhibition of fibroblast proliferation may explain the reduced wound healing in patients undergoing treatment with doxorubicin.     

软骨细胞形态、表型与胞间通讯的研究

背景在体外培养时软骨细胞会像成纤维细胞样成片生长并失去表型,软骨细胞胞间通讯与细胞表型的关系尚无报告.目的了解体外培养的不同形态的软骨细胞的增殖、基质合成、荧光染料摄入以及缝隙连接的关系.设计以诊断为依据的实验研究.地点和对象实验在解放军总医院骨科研究所和基础研究所完成,实验对象为兔关节软骨细胞,人股骨头软骨.干预体外培养的兔软骨细胞,分别进行苏木精-伊红(HE)、亮绿-藩红花O染色,显微镜下测量细胞大小、激光共聚焦显微镜下测量细胞内的荧光强度.激光淬灭软骨细胞内的荧光染料.主要观察指标软骨细胞的外观、细胞大小、细胞内的荧光强度.结果细胞投影面积测量软骨细胞变大,失去球形外观后增殖加快,平均面积1755.1 μm2,投影面积差别可达50倍.基质合成消失,缝隙连接消失.显微镜下软骨细胞的荧光强度测量球形软骨细胞CFDA-AM摄入多,(平均2057/30个细胞),扁平细胞摄入少(平均83/30个细胞).体外培养的球形兔软骨细胞间,人关节软骨生发层陷窝内的细胞间有胞间通讯.结论软骨细胞的密度、形态、表型和缝隙连接之间有一定的关系.而细胞外形可能决定细胞的表型.