Proteoglycans synthesized by chondrocytes of human nonarthritic and osteoarthritic cartilage

Chondrocyte cultures were developed from the cell outgrowths of explanted human nonarthritic and osteoarthritic human cartilage. Two significant differences in sulfated proteoglycan synthesis were demonstrated between the chondrocytes obtained in this manner. With 35SO4 to measure newly synthesized proteoglycan, we found that chondrocytes derived from osteoarthritic cartilage secreted significantly less (P less than 0.05) high density proteoglycan into the culture medium than did chondrocytes from nonarthritic cartilage after 20 hr of radiolabeling. This reduced amount of high density proteoglycan was sustained when chondrocytes were maintained in unlabeled culture medium ("chase" medium). In addition, the osteoarthritic chondrocytes secreted an increased amount of low density proteoglycan when compared with their nonarthritic counterparts. The elution profile of secreted high density proteoglycan isolated from the osteoarthritic chondrocyte culture medium was assessed by gel filtration on Sepharose CL-2B and revealed the presence of two proteoglycan subpopulations (Kav, 0.25, 0.58), whereas only one proteoglycan series (Kav, 0.37) was seen in the high density fraction of nonarthritic chondrocyte culture medium. Similar gel filtration profiles were also obtained when chondrocytes were maintained in chase medium. The results of this study demonstrated that stable differences in proteoglycan synthesis, but not in intracellular processing, exist between nonarthritic and osteoarthritic chondrocytes. The findings are noteworthy in that these differences were not previously apparent when organ-cultured cartilage was used to assess putative alterations in proteoglycans between the two groups.     

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.     

The fate of chondrocytes during ageing of human thyroid cartilage

Human laryngeal cartilages, especially thyroid cartilage, exhibit gender-specific ageing. In contrast to male thyroid cartilages, the ventral half of the female thyroid cartilage plate remains unmineralized until advanced age. In cartilage specimens from laryngectomies and autopsies, apoptosis was studied immunohistochemically and the oxidative mitochondrial enzyme nicotinamide adenine dinucleotide hydride tetrazolium reductase (NADH-TR) was localized histochemically. In addition, very fresh specimens from laryngectomies were fixed under addition of ruthenium hexamine trichloride or tannin to fixation solution to study cell organelles of chondrocytes by electron microscopic methods. In general, apoptotic chondrocytes decreased in thyroid cartilages of both genders, especially after the second decade. In the age group 41–60 years, thyroid cartilage from male specimens revealed a significantly higher percentage of apoptotic cells than did thyroid cartilage from women (P = 0.004), whereas in the age groups 0–20 years and 61–79 years no statistically significant gender difference was determined. In general, thyroid cartilage from women contained more living chondrocytes into advanced age than men. Chondrocytes adjacent to mineralized cartilage were partly positive for apoptosis and NADH-TR and partly negative. Apoptotic chondrocytes often were localized in areas of asbestoid fibres where vascularization and mineralization took place first. Electron microscopy revealed remnants of chondrocytes in asbestoid fibres. Taken together, it can be assumed that some chondrocytes in thyroid cartilage die by apoptosis and that these chondrocytes are characterized by absent reactivity for the mitochondrial enzyme NADH-TR. A possible influence of sexual hormones on apoptotic death of thyroid cartilage cells requires further elucidation.     

Glycosyltransferase activities in chondrocytes from osteoarthritic and normal human articular cartilage

Six glycosyltransferases (mannosyl-, glucosyl-, N-acetyl-glucosaminyl-, galactosyl-, sialyl- and fucosyltransferases) are studied and characterized for their optimal conditions and their relations with interfering reactions (glycosyl-nucleotide pyrophosphatases, glycosidases and proteinases) in chondrocytes from osteoarthritic and normal human articular cartilage. Osteoarthritis induces increased activities for five glycosyl-transferases. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, glycosidases or proteolytic enzymes.     

Proteomics of osteoarthritic chondrocytes and cartilage

Osteoarthritis (OA) is characterized by irreversible destruction of the articular cartilage. OA affects more than 100 million individuals worldwide and has a major impact on patients' quality of life. The lack of effective therapy that prevents, inhibits or reverses the progress of OA often leaves only the option of surgical interventions. Thus, identification of the factors that contribute to OA pathogenesis is necessary for better understanding of OA pathobiology and discovery of effective therapies. Recent proteomic studies have been conducted to identify pathological mediators and biomarkers of OA, which have pinpointed novel pathways involved in cartilage degeneration. This article summarizes the recent findings, compares major techniques used in OA proteomics and discusses key proteins in OA and their potential use as therapeutic targets.     

Proteomics of osteoarthritic chondrocytes and cartilage

Osteoarthritis (OA) is characterized by irreversible destruction of the articular cartilage. OA affects more than 100 million individuals worldwide and has a major impact on patients’ quality of life. The lack of effective therapy that prevents, inhibits or reverses the progress of OA often leaves only the option of surgical interventions. Thus, identification of the factors that contribute to OA pathogenesis is necessary for better understanding of OA pathobiology and discovery of effective therapies. Recent proteomic studies have been conducted to identify pathological mediators and biomarkers of OA, which have pinpointed novel pathways involved in cartilage degeneration. This article summarizes the recent findings, compares major techniques used in OA proteomics and discusses key proteins in OA and their potential use as therapeutic targets.     

Effects of introducing cultured human chondrocytes into a human articular cartilage explant model

Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue.     

Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.     

Tissue engineering: chondrocytes and cartilage

Tissue engineering offers new strategies for developing treatments for the repair and regeneration of damaged and diseased tissues. These treatments, using living cells, will exploit new developments in understanding the principles in cell biology that control and direct cell function. Arthritic diseases that affect so many people and have a major impact on the quality of life provide an important target for tissue engineering. Initial approaches are in cartilage repair; in our own programme we are elucidating the signals required by chondrocytes to promote new matrix assembly. These principles will extend to other tissues of the musculoskeletal system, including the repair of bone, ligament and tendon.     

Calmodulin-dependent collagenase and proteoglycanase activities in chondrocytes from human osteoarthritic cartilage.

Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation.     

In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction

To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.     

Dielectric characterization of costal cartilage chondrocytes

Background

Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

Methods

Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes.

Results

The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance.

Conclusions

We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues.

General significance

The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.     

Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage

Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.     

Caspase-9抑制剂对SD大鼠椎间盘软骨终板细胞凋亡的影响

目的探讨caspase-9抑制剂对低胎牛血清培养诱导的大鼠椎间盘软骨终板细胞凋亡影响的研究。方法取3月龄SD大鼠椎间盘软骨终板,序贯消化法获取细胞原代培养,以1%FBS培养48 h为诱导凋亡条件。实验分为1%FBS凋亡组、caspase-9抑制剂组（Z-LEHD-FMK）及DMSO对照组,分别处理细胞48 h,后经流式细胞仪检测细胞凋亡率、Western blot检测procaspase-9,active caspase-9及active caspase-3的表达。结果流式细胞仪检测显示,caspases-9抑制剂组细胞凋亡率（26.3±2.56）%与1%FBS组（40.8±0.84）%及DMSO组（40.2±1.56）%相比凋亡率较低,有显著统计学差异（P〈0.05）;Western blot检测caspases-9抑制剂组active caspase-9及active caspase-3较1%FBS凋亡组及DMSO对照组表达均明显减少,有显著统计学意义（P〈0.05）。结论 Caspase-9抑制剂能明显抑制低胎牛血清培养诱导的大鼠椎间盘软骨终板细胞凋亡,有望成为治疗椎间盘退变的新型药物。     

Expression of ICAM-1 on intact cartilage and isolated chondrocytes

Summary A major factor in cellular cytotoxicity is the interaction between LFA-1 on leukocytes and ICAM-1 on targets. Because several inflammatory cartilage diseases are characterized by the presence of leukocyte infiltrates, the expression of ICAM-1 on human cartilage, cultured chondrocytes, and transplanted cartilage was investigated using monoclonal antibodies. Frozen tissue sections, chondrocytes in suspension, as well as total cellular mRNA were prepared from human cartilage samples. ICAM-1 expression was studied with two different monoclonal antibodies directed against ICAM-1 by immunohistochemical APAAP-staining and additional flow cytometric analyses. The expression of ICAM-1-mRNA in cartilage tissue was analyzed using the northern blot hybridization technique. Furthermore, chondrocytes were treated in culture with interleukin-1 (IL-1) and gamma-interferon (gamma-IFN). ICAM-1 expression after culture was quantified using flow cytometric analysis. We could detect ICAM-1 mRNA in cartilage tissue, however, the immunostaining of tissue sections using monoclonal antibodies did not give clear positive reactions. Isolated chondrocytes showed strongly positive staining patterns in comparison with adequate negative controls as assessed by flow cytometry. A dose-dependent increase of the expression of ICAM-1 on chondrocytes was observed when stimulated with IL-1 and gamma-IFN. Finally, two of the three studied transplanted autologous cartilage samples with advanced resorption showed the presence of ICAM-1 molecules as assessed by immunohistochemistry. This expression of ICAM-1 suggests that the molecule plays a role in severe cartilage inflammatory processes, where tissue damage leads to the exposure of chondrocyte surfaces.     

The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture

Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen produced is cartilage specific collagen (type II). This work was supported in part by grant HD-05505 from NIH.     

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two-photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non-degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non-degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over approximately 0-480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4 degrees C) was identical over a range of culture medium osmolarities ( approximately 0-515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non-degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (>/=2,500 microm(3)) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non-degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180-515 mOsm).     

Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.