The Use of Growth Factors in the Proliferation of Avian Articular Chondrocytes in a Serum-Free Culture System

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor β (TGF-β) significantly stimulated ³H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-β, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.     

Stimulation of Chondroitin Sulfate Proteoglycan Production by Chondrocytes in Monolayer

Chondrocytes in monolayer undergo morphological and biochemical changes which culminate in the establishment of cartilage nodules in vitro. Chondroitin sulfate or heparin, added to the culture media of these cells, stimulates the production of chondroitin sulfate proteoglycan over the entire period of culture with a maximum effect during the log phase of growth. In addition, a lag of 2–3 hours is required before an increase in sulfate incorporation into polysaccharide is observed. The responsiveness of chondrocytes is influenced by several factors, such as cell density, conditioned media and enzyme treatment. Furthermore, puromycin abolishes the endogenous as well as the stimulated synthesis, demonstrating the necessity for core protein synthesis in both synthetic processes. Addition of j3-D-xylosides (which presumably act as initiators of chondroitin sulfate polysaccharide synthesis) and chondroitin sulfate, concurrently, stimulate sulfate incorporation to levels higher than either agent alone, indicating that these compounds act by different mechanisms.     

Culture and Growth Characteristics of Chondrocytes Encapsulated in Alginate Beads

Methodology is described for the culture of avian and mammalian chondrocytes in ionotrophically gelled “semisolid” and “hollow” alginate beads. Chondrocytes grown in “semi-solid” gels exhibited a spherical shape as opposed to a fibroblastic morphology observed in monolayer culture. In the “semi-solid” beads, the cells grew as small clumps and as large aggregates. The aggregates were round or elliptical in appearance and surrounded by a dense Alcian Blue positive halo. Preliminary studies with collagen and chitosan matrixes encapsulated in “hollow” beads suggest that cell growth and morphology are profoundly influenced by the composition of the cellular environment. Chondrocyte structure and function in the “semi-solid” and “hollow” beads were partially characterized by light microscopy, histochemical and biochemical means. The encapsulation methodology is readily applicable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture.     

Proteoglycans Synthesized by Fetal Guinea Pig Chondrocytes in Culture

Short term cultures were carried out with chondrocytes and tissue fragments from fetal guinea pig epiphyseal cartilage. Proteoglycans were isolated from these cultures and their properties were compared with those of pro-teoglycans from adult hyaline cartilage. It was concluded that the proteoglycans synthesized in culture were essentially similar to those present in cartilage matrix in vivo. The authors therefore suggest that fetal guinea pig chondrocytes cultured in monolayer or as aggregates in suspension constitute a useful system for the study of synthesis and secretion of proteoglycans.     

Orientation of Primary Cilia of Articular Chondrocytes in Three‐Dimensional Space

Primary cilia have functions as sensory organelles integral to signal transduction and establishment of cell polarity. In articular cartilage the primary cilium has been hypothesized to function as an antenna to sense the biomechanical environment, regulate the secretion of extracellular matrix components, and maintain cellular positional information, leading to high tissue anisotropy. We used analysis of electron microscopy serial sections to demonstrate positional attributes of the primary cilium of adult equine articular chondrocytes in situ. Data for ～500 axonemes, comparing superficial to radiate chondrocytes from both load‐bearing and non‐load‐bearing regions, were graphed using spherical co‐ordinates θ, φ. The data demonstrate the axoneme has a definable orientation in 3D space differing in superficial and radiate zone chondrocytes, cells that differ by 90° in the orientation of their major axes to the articular surface. Axonemal orientation is more definable in load‐bearing than in non‐load‐bearing areas. The position of emergence of the axoneme from the cell also is variable. In load‐bearing regions of the superficial zone, extension of the axoneme is from the cellular side facing the subchondral bone. In radiate zone cells, axonemes extend from either face of the chondrocyte, that is, both toward the articular surface or toward the subchondral bone. In non‐load‐bearing regions this consistency is lost. These observations relate to current hypotheses concerning establishment of tissue anisotropy in articular cartilage during development, involving both migration of cells from the joint periphery and a restricted zone of division within the tissue resulting in the columnar arrangement of radiate zone cells. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

The Effects of Long Term Monolayer Culture on the Proteoglycan Phenotype of a Clonal Population of Mature Human Malignant Chondrocytes

Articular cartilage chondrocytes maintain biosynthetic heterogeneity in cell culture, but undergo irreversible dedifferentiation of their proteoglycan phenotype, as defined by keratan sulfate content. A recently described cell line of malignant human chondrocytes, 105KC, was the first to maintain a differentiated keratan sulfate-proteoglycan phenotype in long-term culture. A clone of 105KC, labeled KC2H3, is currently described and represents a distinct and metabolically more homogeneous population of mature chondrocytes than 105KC. KC2H3 cells universally express keratan sulfate biosynthesis, as defined by indirect immunofluorescence. In addition, KC2H3 expresses a more mature proteoglycan phenotype than 105KC, as demonstrated by the keratan sulfate content: 24% of glycosaminoglycan content of the aggregating proteoglycans of KC2H3 versus 13% for 105KC. Further reported are the effects of long term monolayer culture on the proteoglycan phenotype expressed by KC2H3. After more than 16 months in continuous monolayer, KC2H3 cells remained morphologically indistinguishable from those maintained in suspension alternating with monolayer. In addition, the proteoglycan phenotype remained mature, without a tendency towards dedifferentiation. The flattened morphology adopted by chondrocytes while in monolayer has been considered a stimulus of dedifferentiation; the present study is the first to examine the direct effects of physical state on a homogeneous and stable population of chondrocytes.     

Increase of Decorin Content in Articular Cartilage Following Running

The effect of long distance running exercise (40 km/day for 15 weeks, five days a week) on the decorin content of articular cartilage from the knee joint was studied in female beagle dogs. Samples from load bearing sites on the lateral plateau of the tibia (TL), and pooled material from two minimum load bearing sites on the posterior section of lateral (FLP) and medial (FMP) femoral condyles were analyzed. The running exercise protocol did not lead to significant changes in the overall glycosaminoglycan content of the cartilage. However, the amount of decorin significantly increased in the TL samples, and also in the FMP pool. These results support earlier in vitro observations that decorin synthesis is stimulated by loading, independent of the synthesis of aggrecan.     

Metal lothionein Induction in Calcifying Growth Plate Cartilage Chondrocytes

Metallothionein (MT) induction was studied in mineralizing cultures of chicken growth plate chondrocytes and quantitated using a Cd-saturation assay. In serum free media, MT induction was observed for Cd concentrations of 0.1 μM and greater and at Zn concentrations of 100 μM and greater. Supplementation of culture media with cysteine and/or methionine resulted in higher levels of MT induction and reduced toxicity during Cd exposure. Maximum MT induction appeared to coincide with the earliest culture stages during which important enzymes and matrix components are being synthesized. Of non-metal MT inducers tested, sodium butyrate caused a low level induction of MT while interleukin-1 had no effect on basal MT levels. 1, 25-dihydroxyvitamin D increased MT induction. The steroid hormone dexamethasone caused a reduction in basal and induced MT levels. These findings suggest that MT regulation in growth plate chondrocytes differs significantly from what is known in other cell types and that this difference may be related to the mineralization of this tissue.     

碱性成纤维细胞生长因子对体外培养的乳兔软骨细胞中p53 mRNA表达的影响

目的 探讨在乳兔关节软骨细胞体外培养过程中,成纤维细胞生长因子（bFGF）对p53mRNA表达水平的影响及意义。方法 原代体外培养乳兔关节软骨细胞并传代,实验组加入10ng／ml的bFGF,光镜下观测各代细胞形态学变化,RT—PCR检测各代细胞中p53基因在mRNA水平上的表达。结果光镜观察体外培养的软骨细胞,对照组第4代培养细胞始出现凋亡细胞,bFGF组第7代出现少量凋亡细胞;RT—PCR检测bFGF组p53的目的基因指数（p53吸光光度值,β—actin吸光光度值）明显低于对照组（P〈0．05）。结论 在体外培养的兔关节软骨细胞中bFGF下调p53基因mRNA水平表达。     

Chondrocytes from Different Zones Exhibit Characteristic Differences in High Density Culture

Superficial and middle/deep zone chondrocytes were isolated from goat femoral cartilage by a zonal abrasion method. The cells were expanded 100-fold through two passages, then seeded into agarose wells to form high-density constructs through a self-assembling process. After 4 weeks in culture, the superficial zone constructs contracted into a dense cell mass, while middle/deep zone chondrocytes formed constructs with four distinct regions. Middle/deep zone chondrocytes produced 250% more glycosaminoglycans per dry weight and more collagen per dry weight than superficial zone chondrocytes. The superficial and middle/deep zone chondrocytes were found to retain characteristic differences even after 100-fold expansion, as evidenced by construct morphology and extracellular matrix content. This study uniquely demonstrated the ability of expanded superficial and middle/deep zone chondrocytes to form constructs of distinct characteristics without a scaffold. The goal of tissue engineering different zones of cartilage is to eventually replicate the specific function of each zone.     

Static Compressive Loading Reduces the mRNA Expression of Type II and X Collagen in Rat Growth-Plate Chondrocytes During Postnatal Growth

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.     

Effects of Cell Shape Type X Collagen Gene Expression in Hypertrophic Chondrocytes

During endochondral ossification, small flat resting and proliferating chondrocytes mature into large round hypertrophic chondrocytes that synthesize a unique collagen, type X. We have asked whether this change in cell shape during chondrocyte maturation regulates type X collagen gene expression, using immature chick vertebral chondrocytes grown in monolayer or in suspension. The freshly isolated chondrocytes contained no type X collagen RNA, but after 30 days of culture, both attached and suspended cells contained a similar large amount. However, in cells that were grown in monolayer and then resuspended three days before harvest, type X collagen gene expression increased a further 6 fold. These results suggest that the change from a flat to a round shape that occurs during chondrocyte maturation in vivo may be important for maximal expression of the type X collagen gene.     

Thioredoxin Gene Expression in Rat Knee Articular Cartilage After Full-Thickness Injury

To study the expression of the antioxidative protein thioredoxin (TRX) in intact and injured articular cartilage, we examined the presence of trxmRNA in rat knee joints by in situ hybridization. Our results showed that in the intact knee, most cells, including articular cartilage chondrocytes, expressed trx mRNA. We examined joints at 1, 7, 14, and 28 days after the infliction of full-thickness cartilage injuries on distal femoral condyles. At 1 day after injury, no significant changes were observed in the wound or in trx expression pattern. However, at 7 to 28 days after injury, the wound became filled with repair tissue. Also, trx expression was detected in differentiating mesenchymal cells in the deeper zones of the wound but not in fibroblast-like cells in the upper part of the repair tissue, toward the joint cavity. This lack of TRX expression in the fibroblast-like cells may underlie the susceptibility of the repair tissue fibrocartilage to oxidative stress.     

Expression of Growth Hormone by Lymphocytes

In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone (GH) in vitro. Studies using antibody affinity chromatography, high pressure liquid chromatography, and polyacrylamide gel electrophoresis indicate that leukocytes secrete a ～22,000 dalton molecular weight immunoreactive GH (irGH). The irGH appeared to be de novo synthesized since it could be radiolabeled with tritiated amino acids and its production blocked by prior incubation of leukocytes with cycloheximide. The levels of secreted irGH were enhanced by concanavalin A or lipopolysaccharide. By using RNA slot blot analysis, we detected specific GH mRNA present in the cytoplasm of rat leukocytes. Leukocytes from a variety of tissues in rats, including spleen, thymus, bone marrow, and peripheral blood as well as separated spleen T and B cells, were all observed to produce GH RNA and secrete GH from the cells. We conclude that lymphocytes produce a mRNA for irGH and are then able to translate that message and secrete the molecule from the cell. The data suggest a potential regulatory loop between the immune and neuroendocrine systems.     

Osteogenic Differentiation is Selectively Promoted by Morphogenetic Signals from Chondrocytes and Synergized by a Nutrient Rich Growth Environment

Cartilage formation always precedes that of bone during endochondral skeletal development. To determine if chondrocytes provide inductive signals for osteogenesis, C3H10T½ mesenchymal stem cells were co-cultured in membrane separated transwell culture chambers with chondrocytes, osteoblasts, or fibroblasts. Osteogenesis, as assessed by the expression of osteocalcin mRNAs, was strongly induced in the C3H10T½ cells co-cultured with chondrocytes but not induced by co-culture with either osteoblasts or fibroblasts. Interestingly, while only osteogenic differentiation was observed in the C3H10T½ cells co-cultured with chondrocytes, bone morphogenetic protein (BMP)-7 treatment induced an ordered endochondral progression of skeletal cell differentiation in which chondrogenic differentiation preceded osteogenesis by 2 to 4 days. A nutrient enriched growth environment enhanced osteogenic differentiation induced by either co-culture or BMP-7 treatment 2- to 5-fold. Nutrient enhanced osteogenic differentiation was associated with an activation of the retinoblastoma-mediated signal transduction pathways. In summary, these results show that osteogenesis is selectively induced by morphogenetic signals produced by chondrocytes and that a nutrient rich environment enhances both BMP-7- and co-culture-induced osteogenic differentiation.     

Ia Expression in Keratinocytes Following Ultraviolet Radiation

Injections of murine gamma interferon (IFN-gamma) into BALB/c nude mice induced Ia expression by keratinocytes. The aim of the present study was to use this murine model to determine the effect of ultraviolet radiation (UV) or cyclosporine A (CyA) on Ia expression by keratinocytes. Two sets of experiments were performed. In the first, mice were injected intraperitoneally with IFN-gamma for 6 days. The mice were divided into three groups. One group, with one ear protected by electrical tape, was exposed to UVB radiation for 15 days starting 4 days before the injection. The second group received subcutaneous injections of CyA simultaneously with the IFN-gamma and during the 10 days following the IFN-gamma injection. The third group received only IFN-gamma injections. Fifteen days after the IFN-gamma injection all mice were killed and evaluations of Ia positive cells were performed. In the second set of experiments the nude mice were treated with CyA or UVB only 10 days after the last IFN-gamma injections. In both experiments UVB inhibited and down-regulated Ia expression by keratinocytes. This effect on keratinocytes was not observed in the protected ears. Thus it appears that the effect of UVB on keratinocytes is local and not systemic. CyA failed to inhibit or down-regulate Ia expression. This study may shed some light on understanding the mechanism effect of UV radiation in a variety of skin diseases.     

股骨颈骨折后髋关节软骨退变的生物力学研究

在动物模型上研究股骨颈骨折后髋关节软骨退变在发生、发展、转归过程中软骨厚度和生物力学特性改变及其与伤后时间的关系。用新西兰白兔60只作股骨颈基底截骨制作骨折动物模型，于术后2、4、6、8、12、16周采用压凹法测定关节软骨厚度和生物力学特性。免股骨颈骨折后关节软骨持续增厚，6周达高峰，随后软骨迅速磨损、变薄；骨折后4周起软骨退变失代偿，弹性和机械强度进行性大幅度下降，退变至骨关节炎。股骨颈骨折后保守治疗形成局部低应力环境，关节软骨缺乏动态载荷，基质胶原网络迅速降解所引起的生物力学特性降低是导致并维持软骨退变不断前进的动力。