Inorganic phosphate (Pi) modulates the expression of key regulatory proteins of the inorganic pyrophosphate (PPi) metabolism in TGF-β1-stimulated chondrocytes

The balance between extracellular inorganic phosphate (ePi) and extracellular inorganic pyrophosphate (ePPi) is controlled by four membrane proteins: the transporters ANK (exporting PPi outside the cells) and PiT-1 (importing ePi into the cells), and the enzymes PC-1 (generating ePPi from nucleotides) and Tissue Non-specific Alkaline Phosphatase (TNAP, hydrolyzing ePPi into ePi). TGF-β1 was shown to stimulate ANK and PC-1 expression in articular chondrocytes, and subsequent ePPi level, as well as to increase ePi uptake by inducing PiT-1 expression in a chondrogenic cell line. Thus, we investigated the ability of ePi to modulate the effect of TGF-β1 on the regulatory proteins of the ePi/ePPi balance in chondrocytes. In the pathophysiological range of 0.01-1 mM, ePi was inactive by itself but potentiated the stimulatory effects of TGF-β1 on ANK, PC-1 or PiT-1 mRNA (RT-qPCR) and protein (Western blot) levels. PC-1 activity was also increased by TGF-β1 and further potentiated by ePi supplementation. TNAP mRNA and activity became undetectable in response to TGF-β1. These data suggest that ePi could increase ePPi level by changing the control of ANK and PC-1 expression by TGF-β1, further highlighting an adaptative regulation of the Pi/PPi balance to prevent basic calcium phosphate deposition into the joints.     

Transglutaminase Participates in the Incorporation of Latent TGFβ into the Extracellular Matrix of Aging Articular Chondrocytes

TGFβ1 is a multifunctional peptide growth factor that promotes processes associated with age-related degenerative diseases in articular cartilage. Large quantities of TGFβ1 are stored in cartilage extracellular matrix (ECM) in a latent form (LTGFβ1), and yet little is known about the factors that participate in the incorporation of LTGFβ1 into the highly specialized cartilage ECM. We previously demonstrated high levels of the protein cross-linking enzyme transglutaminase (TGase) in aging articular chondrocytes and showed that this enzyme participated in LTGFf31 activation. This work explores the hypothesis that extracellular TGase participates in LTGFβ1 incorporation into ECM in aging chondrocytes. We studied the effects of Tease inhibitors on TGFβ1 levels in ECM of old and young porcine articular chondrocytes. TGase inhibitors decreased the quantity of LTGFβ1 in the ECM in old but not in young chondrocytes to 60-70% of control values (p<.05). Fibronectin, an extracellular TGase competitive substrate, also decreased LTGFβ1 levels in ECM (p<.01). Levels of activated TGF131 also decreased in the presence of TGase inhibitors, as did levels of latent TGF13 binding protein l in the cell layer. Extracellular TGase activity was present in old but not young chondrocyte cultures. These findings support a role for extracellular TGase in the incorporation of LTGFβ1 in the ECM of aging chondrocytes.     

The potential of IGF-1 and TGFbeta1 for promoting "adult" articular cartilage repair: an in vitro study

Research into articular cartilage repair, a tissue unable to spontaneously regenerate once injured, has focused on the generation of a biomechanically functional repair tissue with the characteristics of hyaline cartilage. This study was undertaken to provide insight into how to improve ex vivo chondrocyte amplification, without cellular dedifferentiation for cell-based methods of cartilage repair. We investigated the effects of insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 (TGFbeta1) on cell proliferation and the de novo synthesis of sulfated glycosaminoglycans and collagen in chondrocytes isolated from skeletally mature bovine articular cartilage, whilst maintaining their chondrocytic phenotype. Here we demonstrate that mature differentiated chondrocytes respond to growth factor stimulation to promote de novo synthesis of matrix macromolecules. Additionally, chondrocytes stimulated with IGF-1 or TGFbeta1 induced receptor expression. We conclude that IGF-1 and TGFbeta1 in addition to autoregulatory effects have differential effects on each other when used in combination. This may be mediated by regulation of receptor expression or endogenous factors; these findings offer further options for improving strategies for repair of cartilage defects.     

Mechanical Regulation of Terminal Chondrocyte Differentiation via RGD-CAP/β ig-h3 Induced by TGF-β

RGD-CAP (βig-h3), initially cloned as a transforming growth factor (TGF)-β inducible gene in human lung adenocarcinoma cells, was demonstrated to have a negative regulatory function in mineralization in hypertrophic chondrocytes, and the expression was shown to be associated with mechanical stimulation. We hypothesized that mechanical stimulation may regulate the terminal chondrocyte differentiation through the TGF-β pathway by enhancing the RGD-CAP expression. To test this hypothesis, we investigated the effects of mechanical strain on the terminal differentiation and mineralization of growth-plate chondrocytes and assessed the mechanical regulation of TGF-β and RGD-CAP expression. A cyclic mechanical strain of 12% elongation was applied to the cultured prehypertrophic chondrocytes isolated from the rib cartilage of 4-week-old male rats at 30 cycles/min (loading and relaxation on every alternate second). The terminal differentiation and mineralization of chondrocytes were assessed by alkaline phosphatase (ALP) activity assay and alizarin red staining. The gene expressions of TGF-β and RGD-CAP, as well as chondrocytic terminal differentiation markers such as type X collagen and ALP, were examined with real-time RT-PCR. Cyclic mechanical strain decreased the ALP activity and intensity of alizarin red staining in prehypertrophic chondrocytes, as well as the gene expressions of type X collagen and ALP. TGF-β and RGD-CAP were upregulated in the prehypertrophic chondrocytes subjected to mechanical strain, whereas the level of PTHrP receptor mRNA was not affected by the mechanical strain. The neutralizing antibody for TGF-β suppressed the reduction of the mineralization of chondrocyte cultures with the downregulation of RGD-CAP. These results suggest that mechanical strain negatively regulates the terminal differentiation of chondrocytes through the signal pathway of TGF-β with the induction of RGD-CAP.     

Modulation of intracellular reactive oxygen species level in chondrocytes by IGF-1, FGF, and TGF-beta1

Growth factors are important in the development, maintenance and repair of cartilage. The principal aim of this study was to test the capacity of three growth factors with established roles in cartilage, namely insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF) and transforming growth factor (TGF)-beta 1, to alter intracellular reactive oxygen species (ROS) levels. Explants of articular cartilage from young, mature, and aged rats were pretreated with IGF-1, FGF, or TGF-beta 1 and intracellular ROS levels were quantified using the free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal microscopy, and densitometric image analysis. Viability of chondrocytes following ROS stress and growth factor treatment was assessed using the live/dead cytotoxicity assay, and the activities of the antioxidant enzymes--catalase (CAT), total superoxide dismutase (SOD), and glutathione peroxidase (GPX)--were measured spectrophotometrically by decay of the substrate from the reaction mixture. The effect of IGF-1 on ROS levels in cultured human chondrocytes also was examined. In rat cartilage, FGF did not significantly affect ROS levels or antioxidant enzyme activity in any age group. TGF-beta1 significantly increased cellular ROS levels in mature and old cartilage whereas in marked contrast, IGF-1 significantly and age-dependently reduced ROS levels. IGF-1 also had a potent antioxidant effect on cultured human chondrocytes. Pretreatment of rat cartilage with IGF-1 significantly enhanced the activity of GPX, without altering the activity of SOD or CAT, and protected chondrocytes against ROS-induced cell death. TGF-beta 1 had no significant effect on the activity of the antioxidant enzymes. Despite promoting ROS production, TGF-beta 1 was not cytotoxic. We concluded that TGF-beta 1 exhibits an acute pro-oxidant effect in cartilage that is not cytotoxic, suggesting a role in physiological cell signalling. In marked contrast, IGF-1 is a potent antioxidant in mature and aged rat and human chondrocytes, protecting cells against ROS-induced cell death probably through the enhancement of the activity of the antioxidant enzyme GPX.     

TGF-β1 and IGF-1 influence the re-differentiation capacity of human chondrocytes in 3D pellet cultures in relation to different oxygen concentrations

To prevent de-differentiation of chondrocytes in vitro, the 3D environment, growth factors and different oxygen concentrations were considered. In this in vitro study, we quantified the influence of insulin-like growth factor (IGF)-1 and/or transforming growth factor (TGF)-β1 under differing oxygen (5/21% O(2)) levels on the proliferation and synthesis rates of hyaline extracellular matrix (ECM) components in chondrogenic pellet cultures. Human chondrocytes isolated from articular cartilage were transferred into conical tubes to form pellets. Pellets were stimulated with TGF-β1 and/or IGF-1. After 2 and 5 weeks of cultivation the DNA concentration and expression of pro-collagen type 1, type 2 and aggrecan were analysed. Under hypoxia the DNA content remained stable. In contrast, under normoxia, cells showed an increase of DNA concentration after stimulation with TGF-β1/IGF-1 and TGF-β1. Nevertheless, DNA contents under normoxia did not reach the values of hypoxic-cultivated cells. Under both culture conditions a reduced synthesis of pro-collagen type 1 could be determined. Although the expression of pro-collagen type 2 was significantly higher under normoxia, a decrease in the case of TGF-β1/IGF-1- and IGF-1-stimulated cells was observed. Under hypoxia pro-collagen type 2 contents remained stable or increased for TGF-β1/IGF-1-stimulated cells. Furthermore, incubation with growth factors resulted in aggrecan accumulation under hypoxia, while a reduced expression under normoxia could be determined for TGF-β1/IGF-1- and IGF-1-stimulated cells. Our results demonstrate that the treatment with growth factors causes differences in the expression of ECM compounds within pellet cultures. While under normoxia TGF-β1 alone leads to a positive effect of the expression of hyaline cartilage-specific ECM components, an additive effect of both growth factors was only determined under hypoxia.     

Regulation of IGF-1 receptors on rabbit articular chondrocytes by inflammatory mediators

Insulin-like growth factor 1(IGF-1) is a known stimulator of proteoglycan synthesis in articular cartilage. However, arthritic cartilage shows a reduced responsiveness to IGF-1, resulting in depletion of proteoglycan moieties.A receptor binding assay for IGF-1 was set up in normal articular chondrocytes to determine whether or not growth factors and cytokines found in inflammatory tissues could influence the response of these cells to IGF-1 through modulation of target receptors.The results of the binding assays show that IGF-1 receptors on rabbit articular chondrocytes are not down-regulated by inflammatory cytokines, and are only modulated by high concentrations of IGF-1, insulin and Nu-serum (of which IGF-1 and insulin are constituents). This suggests that if monolayer cultures of chondrocytes behave similarly to chondrocytes in cartilage, reduced responsiveness to IGF-1 is unlikely to be caused by a depletion of IGF-1 receptors.     

转化生长因子-β及胰岛素样生长因子-Ⅰ修复关节软骨缺损的实验观察

目的观察转化生长因子-β（transforming growth factor-β,TGF-β）、胰岛素样生长因子-Ⅰ（insulin—like growth factor-Ⅰ,IGF-Ⅰ）对关节软骨缺损修复的作用。方法采用组织工程方法制备骨基质明胶（BMG）软骨细胞移植物。将40只4月龄的新西兰兔随机分为TGF-β组、IGF-Ⅰ组、TGF-β联合IGF-Ⅰ组、空白对照组（前三组为实验组）。各组制备关节软骨缺损模型,实验组兔膝关节腔注射对应等量人重组蛋白,对照组注射等量盐水。术后行组织学观察及免疫组化检测。结果TGF-β联合IGF-Ⅰ组软骨细胞生长较快,术后24周修复的软骨组织HE染色与正常关节软骨一致,软骨细胞呈柱状排列,免疫组化见Ⅱ型胶原染色较深;TGF-β组、IGF-Ⅰ组术后24周部分软骨细胞呈柱状排列,免疫组化见Ⅱ型胶原染色较浅;空白对照组未修复。结论联合应用TGF-β及IGF-Ⅰ可较好促进关节软骨缺损修复,其作用优于两者单独应用。     

The chrondoprotective actions of a natural product are associated with the activation of IGF-1 production by human chondrocytes despite the presence of IL-1β

Background

Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1). We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria^®), is a well-described inhibitor of NF-κB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249), possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality.

Methods

Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA), cartilage matrix degradation and nitric oxide (NO) production, under basal conditions and in the presence of IL-1β.

Results

RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P <0.05). As expected, IL-1β exposure completely silenced IGF-1 production by chondrocytes. However, in the presence of IL-1β both RNI 249 and vincaria protected IGF-1 production in an additive manner (P <0.01) with the combination restoring chondrocyte IGF-1 production to normal levels. Cartilage NO production was dramatically enhanced by IL-1β. Both vincaria and RNI 249 partially attenuated NO production in an additive manner (p < 0.05). IL-1β – induced degradation of cartilage matrix was quantified as glycosaminoglycan release. Individually RNI 249 or vincaria, prevented this catabolic action of IL-1β.

Conclusion

The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1β, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases.     

Comparison of Matrix Vesicles Derived from Normal and Osteoarthritic Human Articular Cartilage

Articular cartilage matrix vesicles (MVs) from normal human adult articular cartilage were examined for protein and enzyme content and biomineralizing capacity for comparison to MVs derived from human osteoarthritic (OA) cartilage. Femoral condylar and tibial plateau cartilage from each of 9 healthy donors ages 17–37y was enzymatically digested and serially ultracentrifuged to pellet MVs at 3 × 10⁶ g-min. MV protein content, nucleoside triphosphate pyrophospho hydrolase (NTPPPH) specific activity (SA) and capacity for ⁴⁵Ca precipitation were determined. MV precipitated mineral was examined using Fourier transform infrared spectroscopy (FTIR). Normal human cartilage yields 50% less MV pro-tein/g cartilage than OA cartilage (p <. 01). Normal human articular MVs possess 30–70x higher NTPPPH S A than cell-free digest. Mean NTPPPH S As of MVs derived from normal human cartilage are 3x higher than that of OA MVs (p <. 05) and normal MV NTPPPH SA appears to decrease with age (p <. 01). Normal human MVs support significantly higher calcium precipitation/mg MV protein in both ATP-dependent (p <. 01) and -independent (p =. 05) systems. The FTIR spectrum of MV mineral generated in the presence of ATP strongly resembles the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectrum of MV mineral generated without ATP resembles that of carbonate-substituted apatite (AP). The fact that isolated MVs from normal cartilage generate pathologically relevant crystal phases in vitro implies that matrix integrity and substrate availability may be crucial factors in the control of pathologic biomineralization.     

Effect of construct properties on encapsulated chondrocyte expression of insulin-like growth factor-1

Hydrogels are a promising type of biomaterial for articular cartilage constructs since they have been shown to enable encapsulated chondrocytes to express their predominant phenotypic marker, type II collagen. Endogenously expressed signaling molecules, such as insulin-like growth factor-1 (IGF-1), are also known to facilitate the retention of this chondrocytic phenotype. Recent investigations have attempted to enhance the ability of encapsulated chondrocytes to regenerate cartilage through delivery of exogenous signaling molecules. However, we hypothesize that by altering construct properties, such as cell density and polymer concentration, we can augment the expression of endogenous IGF-1 in chondrocytes. To this end, bovine articular chondrocytes were encapsulated within alginate hydrogels at two different cell densities (25,000 and 100,000 cells/bead) and various alginate concentrations (0.8%, 1.2%, and 2.0% w/v). These parameters were chosen to simultaneously investigate cell-to-cell distance on paracrine signaling and water content on IGF-1 diffusion by chondrocytes. At 1, 4, and 8d, chondrocytes were analyzed for protein and mRNA expression of IGF-1 as well as type II collagen. Results suggest that cell density and alginate concentration at high cell density can significantly affect the endogenous IGF-1 expression by chondrocytes. Therefore, these results indicate that construct properties can impact chondrocyte gene expression and should be considered in order to create a proper engineered articular cartilage construct.     

Immunohistochemical detection of estrogen receptor α in articular chondrocytes from cows, pigs and humans: in situ and in vitro results

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors α and β. The goal of the present study was to search for estrogen receptor α in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor α in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor α is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O₂ or with 5% O₂ positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.     

Microscopic Change in Macroscopically Normal Equine Cartilage from Osteoarthritic Joints

The objective of this study was to assess whether macroscopically normal articular cartilage taken from joints containing focal osteoarthritic lesions is histologically similar to articular cartilage taken from macroscopically normal joints. Metacarpophalangeal, proximal interphalangeal, and distal interphalangeal joints were obtained from 10 horses following euthanasia. Gross articular cartilage damage was scored and the cartilage assigned to one of two groups: (1) macroscopically normal cartilage from normal joints (control) and (2) macroscopically normal cartilage from diseased joints in which there were focal osteoarthritic lesions. Chondrocytes expressing specific cytokines and cytokine receptors were identified by immunohistochemistry. The total number of chondrocytes, and percentage of chondrocytes positive for these cytokines and receptors, was recorded in the superficial, middle, and deep cartilage zones. There was a significant increase in the expression of interleukin-1β in the superficial and middle zones and interleukin-18 receptor in the superficial zone in Group 2 compared with Group 1 control samples. A significant positive correlation also was found between the grade of osteoarthritis and the percentage of chondrocytes positive for interleukin-1β in the superficial and middle zones, and for interleukin-18 and interleukin-18R in the superficial zone. There was a significant increase in histology score for glycosaminoglycan loss in Group 2 compared with that in Group 1. In joints with focal osteoarthritis lesions, all the articular cartilage, even if macroscopically apparently normal, may have microscopic changes associated with osteoarthritis.     

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.     

ANK、ENPPl及TGF-β1基因在大鼠终板软骨细胞传代培养中的表达变化及意义

目的：通过大鼠终板软骨细胞体外自然传代的退变模型,探讨传代过程中钙化相关基因ANK、ENPPl及内源性生长因子TGF—βl表达的变化及其意义。方法：取大鼠腰椎终板软骨细胞,采用酶消化法及自然传代法分离培养,选取P1、P5及P7代,均在体外培养6d,分别用HE染色、甲苯胺蓝染色及Real—timeRT—PCR法检测软骨标志性基因Ⅱ型胶原、蛋白多糖、转录因子Sox9变化,对终板软骨细胞表型进行鉴定。用Real·timeRT—PCR及Westernblot检测钙化相关基因ANK、ENPPl的变化,Re．al—timeRT—PCR及ELISA检测生长因子TGF-βl的变化。结果：随着细胞传至P7代,细胞形态上有梭形变趋势。茜素红染色无明显变化。软骨标志性基因Ⅱ型胶原、蛋白多糖及转录因子Sox9表达均明显下调（P〈0．05）,钙化相关基因ANK、ENPPl表达均明显下调（P〈0．05）,内源性生长因子TGF-B1表达明显下调（P〈0．05）。结论：终板软骨细胞传至P7代,终板软骨细胞发生退变,终板软骨细胞内未见钙盐沉积,钙化相关基因ANK、ENPPl下调可能由内源性TGF-βl下调引起,提示调节内源性TGF-βl及钙化相关基因ANK、ENPPl在终板软骨中的表达有可能会阻止椎间盘的退变。     

Regulation of IGF-1 receptors on rabbit articular chondrocytes by inflammatory mediators

Insulin-like growth factor 1(IGF-1) is a known stimulator of proteoglycan synthesis in articular cartilage. However, arthritic cartilage shows a reduced responsiveness to IGF-1, resulting in depletion of proteoglycan moieties.A receptor binding assay for IGF-1 was set up in normal articular chondrocytes to determine whether or not growth factors and cytokines found in inflammatory tissues could influence the response of these cells to IGF-1 through modulation of target receptors.     

Mutant WISP3 triggers the phenotype shift of articular chondrocytes by promoting sensitivity to IGF-1 hypothesis of spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA)

This article introduces the hypothesis that mutant WISP3 (Wnt1 inducible secreted protein-3) triggers the phenotype shift of the chondrocytes, especially in the articular chondrocytes, by promoting sensitivity to IGF-1 (insulin-like growth factor 1), and results in chondrocytes apoptosis in SEDT-PA. SEDT-PA is also referred to as progressive pseudorheumatoid dysplasia (PPD), arthropathy progressive pseudorheumatoid of childhood (APPRC). Evidence for the hypothesis is based on the following indications: (1) SEDT-PA is caused by mutations of the WISP3 gene. WISP3 encodes a domain that bears homology to the amino-terminal domain of the insulin-like growth factor binding proteins (IGFBPs). (2) IGF-1 enhances chondrocyte hypertrophy by insulin-like actions. WISP3 can up-regulate the expression of type II collagen. When chondrocytes become hypertrophic, they reduce the expression of types II and IX collagen. (3) The chondrocytes in the normal articular cartilage maintain a stable phenotype. These cells exhibit no mitotic activity, low matrix synthesis and low degradation. But articular chondrocytes could react to certain stimuli such as IGF-1. (4) The loss of WISP3 expression alters the phenotype of the breast epithelium and promotes motility and invasion. The WISP3-deficient cells are extremely sensitive to the growth stimulatory effects of IGF-1. (5) The action of IGF-I is inhibited by IGFBPs, both in articular chondrocytes and in the normal breast epithelium. In conclusion, the mutant WISP3 lose is the function of inhibiting IGF-1 and disturbs the maintenance of a stable phenotype in articular chondrocytes. So, the articular chondrocytes undergo hypertrophic and terminal differentiation apoptosis. The precise mechanism of WISP3 function during postnatal cartilage growth and homeostasis is not clear yet. This hypothesis provides a new clue on the present mechanism study on SEDT-PA. If verified, this new concept may lead to a novel pathogenesis of SEDT-PA.     

Effect of Oxygen Tension on Chondrocyte Extracellular Matrix Accumulation

Since cartilage is mainly an avascular tissue, chondrocytes exist in a low-level oxygen environment in vivo. In the present study, we investigated the effect of oxygen tension (20%, 5% and 1% gas phase oxygen concentrations) over a 20-day period on the extracellular matrix accumulation of bovine articular chondrocytes in confluent surface cultures. Matrix accumulation was assessed by the amount of glycosaminoglycan and collagen deposited in the matrix. From initially confluent monolayers, the chondrocytes became distributed throughout a thick layer of extracellular matrix, thus forming a multicell-layer of tissue. Cells maintained their normal rounded shape, indicative of the differentiated phenotype, throughout the 20-day culture period. On a per culture and a per cell basis, the amount of collagen and glycosaminoglycan accumulation in the matrix was lower at the reduced oxygen tensions. Specifically, in 1% oxygen, matrix GAG content reached a steady-state level, with no net increase in GAG levels after two weeks, whereas in 20% oxygen, matrix GAG increased with time. It is concluded that oxygen has a significant effect on the amount of macromolecules accumulated in the extracellular matrix. The implications of these findings in growing cartilage constructs in vitro are discussed.     

The effect of interleukin-1β and transforming growth factor β on cathepsin B activity in human articular chondrocytes

Cathepsin B is a lysosomal cysteine proteinase, thought to be involved in the degradation of connective tissue breakdown products internalized by endocytosis. It has also been implicated in the extracellular matrix degradation of collagens and proteoglycans in disease states such as tumour invasion, rheumatoid arthritis and osteoarthritis. To date it is still unclear which factors can potentially modulate the release and/or activation of this enzyme.

The aim of this study was to investigate the effect of interleukin-1β and transforming growth factor β on cathepsin B activity in both cell lysates and cell supernatants, using first passage cultures of human articular chondrocytes. Enzyme activity was determined using a specific synthetic substrate for cathepsin B, in a fluorimetric assay.

After 24 h incubation, IL-1β (10–100 U/ml) significantly stimulated cathepsin B activity dose dependently in cell lysates whereas no activity could be detected in the media. Production of cathepsin B activity was unaffected by the presence of 1.4 μM indomethacin, which suggests that prostaglandins are not regulators of cathepsin B activity. TGFβ had no significant effect on basal intracellular cathepsin B levels and variable effects on IL-1β stimulated levels. In three of the five experiments carried out, TGFβ showed a down-regulation of IL-1β-induced enzyme activity. The other two experiments showed an additive effect when TGFβ and IL-1β were co-incubated. Basal and IL-1β-induced levels of cathepsin B activity in cell lysates were both abolished after E-64 (5 μM) was incorporated in the assay incubation medium, which established the specificity of the reaction.

These results suggest that IL-1β and TGFβ may be involved in the regulation of cathepsin B activity and thus cartilage breakdown associated with rheumatic diseases.

     

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-ε-caprolactone) (PLCL) with 85% porosity and 300–500 μm pore size using a gel-pressing method. The scaffold was seeded with 2 × 10⁶ chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.