The chondrotoxic and apoptotic effects of levobupivacaine and bupivacaine on the rabbit knee joint / Os efeitos condrotóxicos e apoptóticos de levobupivacaína e bupivacaína na articulação do joelho de coelhos

Abstract Background: A single dose injection or continuous infusion of local anesthetics into the joint space is considered to be a well-defined analgesia technique. The aim of this study was to investigate the chondrotoxic and apoptotic effects of single-dose intra-articular injection of levobupivacaine and bupivacaine on rabbit knee joint tissues. Materials and methods: The animals were allocated into two groups each containing 20 rabbits. 0.5% levobupivacaine (Group L) and 0.5% bupivacaine (Group B) were applied intra-articularly to the left posterior joints of rabbits. At the same time, normal saline was applied to the right posterior leg knee joints of rabbits in both groups and used as a control (Group S). At the end of the 7th and 28th days after the intraarticular injections, ten randomly chosen rabbits in each group were killed by applying intraperitoneal thiopental. Sections of cartilage tissue samples were stained for light microscopic examinations and the TUNEL method was used to investigate apoptotic cells. Results: As a result of immunofluorescence microscopic examination, the number of apoptotic cells in Group B at day 7 and day 28 were both significantly higher than Group L and S (p < 0.05). Also, the number of apoptotic cells in Group L at day 7 and day 28 were both significantly higher than Group S (p < 0.05). Conclusions: We found that bupivacaine is more chondrotoxic than other anesthetic agent and increases the number of apoptotic cells. These results indicated that bupivacaine caused high chondrotoxic damage and it led to more apoptotic activation than levobupivacaine.

Resumo Justificativa: Uma injeção em dose única ou infusão contínua de anestésicos locais no espaço articular é considerada uma técnica de analgesia bem definida. O objetivo deste estudo foi investigar os efeitos condrotóxicos e apoptóticos da injeção intra-articular com dose única de levobupivacaína e bupivacaína em tecidos articulares do joelho de coelho. Material e métodos: Os animais foram alocados em dois grupos, cada um contendo 20 coelhos. Levobupivacaína a 0,5% (Grupo L) e bupivacaína a 0,5% (Grupo B) foram aplicadas intra-articularmente nas articulações posteriores esquerdas de coelhos. Ao mesmo tempo, solução salina normal foi aplicada nas articulações do joelho da perna posterior direita de coelhos em ambos os grupos e usada como controle (Grupo S). Ao fim do 7° e 28° dias após as injeções intra-articulares, 10 coelhos escolhidos aleatoriamente em cada grupo foram mortos por aplicação de tiopental intraperitoneal. Seções de amostras de tecido cartilaginoso foram coradas para exames de microscopia de luz, e o método TUNEL foi usado para investigar células apoptóticas. Resultados: Como resultado do exame microscópico de imunofluorescência nos dias 7 e 28, o número de células apoptóticas no Grupo B foi significativamente maior que nos grupos L e S (p < 0,05). Além disso, o número de células apoptóticas nos dias 7 e 28 foi significativamente maior no Grupo L do que no Grupo S (p < 0,05). Conclusões: Demonstramos que a bupivacaína é mais condrotóxica do que o outro agente anestésico e aumenta o número de células apoptóticas. Esses resultados indicaram que a bupivacaína causou intensa lesão condrotóxica e levou a uma ativação apoptótica maior do que a levobupivacaína.

Priming stem cells with protein kinase C activator enhances early stem cell-chondrocyte interaction by increasing adhesion molecules

BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)- induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.

Withaferin A-Caused Production of Intracellular Reactive Oxygen Species Modulates Apoptosis via PI3K/Akt and JNKinase in Rabbit Articular Chondrocytes

Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

[ effect of soybean meal on the maintenance of cartilage in animal knee joints]

The soybean is one of the most important diet protein resources and induces a reduction of joint pain. The aim of the present study was to determine the incidence of joint disorders and arthritis, especially in the knee joint. According to the feed regimens, 36 female mice [balb/c; 3 weeks old] were divided into 3 groups including Group 1: Low protein for 6 months; Group 2: Low protein for 3 months and then followed by complete [or full] protein without soybean meal for the next 3 months and Group 3: Low protein for the first 3 months and then followed by complete protein with 20% soybean meal for the next 3 months. Finally the serum alkaline phosphatase [ALP] was measured and sections from the tibial cartilage were histomorphometrically studied. The thickness of the middle part of the tibial cartilage and the number of chondrocytes in the group used soybean meal [148/84 +/- 14/05, 12/77 +/- 1/57, respectively] showed a significant difference compared with the other groups [p<0.001]. Meanwhile, serum ALPlevels [76/402L/U] and the concentrations of the inter-cellular substance in the 3th group showed no significant difference compared with the other groups. It is concluded that soybean may effect on the growth of the knee joint cartilage in animals and also may induce stability of the knee joint cartilage in individuals with protein deficiency since childhood

The metabolic dynamics of cartilage explants over a long-term culture period

INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

2-Deoxy-D-glucose regulates dedifferentiation through beta-catenin pathway in rabbit articular chondrocytes

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.

[Study of differentiation potential of the dedifferentiated chondrocytes from rat articular cartilage into skeletal cell lineages]

Dedifferentiation of the chondrocyte from rat articular cartilage with multiple subcultures and study of the redifferentiation potential of the cells into bone, cartilage and fat cell lineages. In this experimental study, chondrocytes from rat articular cartilage were isolated and expanded through several successive subcultures during which the expression levels of cartilage-specific genes including aggreacan and type II collagen were measured by using real-time PCR to determine the cell dedifferentiation [the time in which cartilage genes ceased their expression]. Furthermore, during the culture period, the chondrocyte was examined morphologically by scanning electron microscopy [SEM]. At the end, the dedifferentiated cells were subjected to osteogenic, adipogenic and chondrogenic culture condition to investigate whether or not they are able to redifferentaite into specialized progenies. Differentiation state was examined by specific staining and RT-PCR analysis. Based on the findings by real time PCR, the expression levels of the both studied genes were high at passage 2 and dramatically decreased at passage 4. Aggreacan expression ceased at passage 10 and collagen II stopped expressing at passage 6. SEM images indicated the flattened morphology of the cells at early passages and the fibroblastic appearance at late passages. Differentiation examination revealed that the dedifferentiated cells were readily differentiated into bone, adipose and cartilage cell lineages. Considering all aspects together, this concluded that articular chondrocyte gradually lost their differentiated state during the long-term culture and changed into multipotent cells capable of differentiating into skeletal cell lineages

Ectopic expression of cyclooxygenase-2-induced dedifferentiation in articular chondrocytes

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.

ERK-1/-2 and p38 Kinase Oppositely Regulate 15-deoxy-delta(12,14)-prostaglandinJ2-Induced PPAR-gamma Activation That Mediates Dedifferentiation But Not Cyclooxygenase-2 Expression in Articular Chondrocytes

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-gamma, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-gamma activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-gamma. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-gamma activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-gamma activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-gamma-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-gamma-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-gamma and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.

Extracellular matrix of ostrich articular cartilage

The composition and organization of the extracellular matrix of ostrich articular cartilage was investigated, using samples from the proximal and distal surfaces of the tarsometatarsus. For morphological analysis, sections were stained with toluidine blue and analyzed by polarized light microscopy. For biochemical analysis, extracellular matrix components were extracted with 4 M guanidinium chloride, fractionated on DEAE-Sephacel and analyzed by SDS-PAGE. Glycosaminoglycans were analyzed by electrophoresis in agarose gels. Structural analysis showed that the fibrils were arranged in different directions, especially on the distal surface. The protein and glycosaminoglycan contents of this region were higher than in the other regions.SDS-PAGE showed the presence of proteins with molecular masses ranging from 17 to 121 kDa and polydisperse components of 67, 80-100, and 250-300 kDa in all regions. The analysis of glycosaminoglycans in agarosepropylene diamine gels revealed the presence of only chondroitin-sulfate. The electrophoretic band corresponding to putative decorin was a small proteoglycan containing chondroitin-sufate and not dermatan-sulfate, unlike other cartilages. The higher amounts of proteins and glycosaminoglycans and the multidirectional arrangement of fibrils seen in the distal region may be correlated with the higher compression normally exerted on this region

The relationship of the expression of estrogen receptor in cartilage cell and osteoarthritis induced by bilateral ovariectomy in guinea pig / 华中科技大学学报（医学）（英德文版）

To investigate the estrogen receptor (ER) expression in cartilage cell in the development of osteoarthritis induced by bilateral ovariectomy in guinea pig and to find their relationship. 30 two-month-old female guinea pigs were randomly divided into two groups (n = 15 each): sham operation (control) group and ovariectomized group (OVX); Scanning electorne microscope (SEM) and transmission electron microscope (TEM) were obtained to analysis the cartilage degeneration of the hind limb knee joint after 6 and 12 weeks of ovariectomy. Dextran-Coated-Charcoal (DCC) was taken to quantitively detect the expression of ER. The serum levels of estrogen and gestone were detected by immune contest assay. The results showed that ER do exist in the cartilages of the guinea pigs, with higher expression in the control group than in OVX group at the same time point (P < 0.05). It was increased also at 12 th week after operation than that of preoperation. The blood serum levels of estrogen and gestone showed a similar tendency to the expression of ER. Joint cartilage degeneration detected by SEM and TEM could be found at 6 th week, but severe degenerative lesions at 12 th week in the OVX group compared with the control group (P < 0.01). The data suggested that bilateral ovariectomy in guinea pig lead to severe osteoarthritis which mighgt be related to the lower serum level of estrogen and the downregulation of the expression of ER in the cartilage also.

The Modulation of Integrin Expression by the Extracellular Matrix in Articular Chondrocytes

Normal articular cartilage is composed of chondrocytes embedded within an extracellular matrix (ECM). The patterns of integrin expression determine the adhesive properties of cells by modulating interactions with specific ECMs. Our hypothesis is that chondrocyte integrin expression changes in response to changes in their microenvironment. Porcine articular chondrocytes were encapsulated in alginate beads with several ECMs (collagen type I, collagen type II and fibronectin) for 7 days, subjected to RT-PCR, western blot analysis and immunofluorescence staining. It was found that chondrocytes in different ECMs showed different patterns of integrin expression. Integrin alpha5 and beta1 were strongly expressed in all groups, but integrin alpha1 was strongly expressed only in collagen type I and fibronectin conjugated alginate beads, and integrin alpha2 was strongly expressed only in collagen type II conjugated alginate beads. These findings suggest that the addition of different ECMs to chondrocytes can modulate the patterns and levels of integrin expression possibly through a feedback mechanism. These finding suggest that the modulation of ECM interactions may play a critical role in the pathogenesis of osteoarthritis.

Transforming growth factor-beta 1 responsiveness of human articular chondrocytes in vitro: normal versus osteoarthritis

The transforming growth factor-beta 1 was known as having the most important influence on chondrocytes among various growth factors, being abundant in articular chondrocytes and osteocytes. We performed in vitro monolayer cultures of human articular chondrocytes from normal and osteoarthritic patients and studied the transforming growth factor-beta 1 responsiveness of those chondrocytes. The cell-growth curve indicated that the primary osteoarthritic chondrocyte culture with transforming growth factor-beta 1 showed a more rapid growth pattern than normal chondrocytes with or without TGF-beta 1 and osteoarthritic chondrocytes without TGF-beta 1. The osteoarthritic group showed a sharp decline in growth pattern with subsequent culture. The shape of osteoarthritic chondrocytes was bigger and more bizarre compared to those of normal chondrocytes. With subsequent culture, this change became prominent. The transforming growth factor-beta 1 increased the [3H]-TdR uptake in each group. The phenotypes of chondrocytes were more clearly expressed in the normal group. The chondrocytes lost their phenotype (production of collagen type II) following subculture in each group. The transforming growth factor-beta 1 could not inhibit or delay the dedifferentiation process (loss of phenotype).

Isolation & cryo-preservation of human foetal articular chondrocytes.

An attempt to determine the ideal temperature and duration of storage of human foetal chondrocytes yielded highly cellular preparations with no alteration in morphology or loss of viability. Initial digestion with activated papain was followed by incubation in 0.5 per cent collagenase. Trypan blue exclusion test revealed a viability count of 95-99 per cent and radioactive thymidine uptake a corresponding labelling index. On TEM no subcellular damage was evident. The isolated viable chondrocytes were further banked at varying temperatures of +4 degrees, -4 degrees, -30 degrees, -79 degrees and -196 degrees C, in Eagles MEM with 10 per cent dimethyl sulfoxide. Post storage morphology and viability of these cells, thawed after durations of 20 h, 1 wk, 1, 2, 3, 4 and 6 months, were compared with prestorage readings in an attempt to define the ideal temperature for banking. Storage in liquid nitrogen at -196 degrees C demonstrated excellent preservation even at the end of six months with minimal subcellular change. Electron microscopy and labelling index were found to be superior to Trypan blue exclusion test in assessing the stored chondrocytes for retention of their functions.