Number Of Apoptotic Chondrocytes Research Articles

Effect of Danhong Injection on Articular Cartilage Degeneration in Rabbits with Knee Osteoarthritis

The aim of this research was to investigate the effect of Danhong injection nanoparticle (DIN) plus warm acupuncture (WA) on articular cartilage degeneration in knee osteoarthritis (KOA) rabbits. First, the KOA model and DIN were made. After success, the rabbits were randomly grouped: according to the treatment methods: controls, DIN, WA, and DIN+WA groups, with 10 rabbits in each group. The controls did not receive any treatment, and the other groups received corresponding treatment intervention, respectively. Through treatment, articular cartilage samples were collected. The bone morphology, LequesneMG score, Mankin score were analyzed. The safranin-fast green, toluidine blue, Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL), and immunohistochemical staining were observed. The levels of apoptosis-related factors were determined. The results showed that the KOA rabbit model and DIN were successfully constructed. Compared with other groups, the LequesneMG and Mankin scores of DIN+WA group decreased markedly (P < 0.05). Safranin-fast green and toluidine blue staining showed that the number of chondrocytes in DIN+WA group was markedly increased, which was more as against other groups. TUNEL staining revealed that the number of apoptotic chondrocytes was markedly reduced, and the apoptotic index was markedly lower in DIN+WA group as against other three groups (P < 0.05). Compared with other groups, Bax and Caspase-3 were markedly decreased, and Bcl-2 was markedly raised in DIN+WA group (P < 0.05). According to the review, the combined application of DIN and WA can promote the growth of chondrocytes, inhibit the apoptosis of chondrocytes, and effectively improve the degeneration of knee articular cartilage, which has application value.

An optical probe for detecting chondrocyte apoptosis in response to mechanical injury

Cartilage injury induced by acute excessive contact stress is common and mostly affects young adult. Although early detection of cartilage injury may prevent serious and lifelong arthritic complications, early detection and treatment is not possible due to the lack of a reliable detection method. Since chondrocyte injury and subsequent cell death are the early signs of cartilage injury, it is likely that cartilage cell apoptosis can be used to predict the extent of injury. To test this hypothesis, a near infrared probe was fabricated to have high affinity to apoptotic cells. In vitro tests show that this apoptosis probe has low toxicity, high specificity, and high affinity to apoptotic cells. In addition, there is a positive relationship between apoptotic cell numbers and fluorescence intensities. Using a mouse xiphoid injury model, we found significant accumulation of the apoptosis probes at the injured xiphoid cartilage site. There was also a positive correlation between probe accumulation and the number of apoptotic chondrocytes within the injured xiphoid cartilage, which was confirmed by TUNEL assay. The results support that the apoptosis probes may serve as a powerful tool to monitor the extent of mechanical force-induced cartilage injury in vivo.

The effect of prenatal exposure to 1800 MHz electromagnetic field on calcineurin and bone development in rats.

To investigated the effects of exposure to an 1800 MHz electromagnetic field (EMF) on bone development during the prenatal period in rats. Pregnant rats in the experimental group were exposed to radiation for six, 12, and 24 hours daily for 20 days. No radiation was given to the pregnant rats in the control group. We distributed the newborn rats into four groups according to prenatal EMF exposure as follows: Group 1 was not exposed to EMF; groups 2, 3, and 4 were exposed to EMF for six, 12, and 24 hours a day, respectively. The rats were evaluated at the end of the 60th day following birth. Increasing the duration of EMF exposure during the prenatal period resulted in a significant reduction of resting cartilage levels and a significant increase in the number of apoptotic chondrocytes and myocytes. There was also a reduction in calcineurin activities in both bone and muscle tissues. We observed that the development of the femur, tibia, and ulna were negatively affected, especially with a daily EMF exposure of 24 hours. Bone and muscle tissue development was negatively affected due to prenatal exposure to 1800 MHz radiofrequency electromagnetic field.

Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis

Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin.

Protective effect of pyrroloquinoline quinine on cartilage in rabbit osteoarthritis models

Objective To study the protective effect of pyrroloquinoline quinone (PQQ) on cartilate in rabbit osteoarthritis models.Methods Thirty-six 6-month-old Japanese white rabbits were randomly divided into experimental group,control group and sham-operation group ( 12 rabbits in each group).The osteoarthritis models at the right knee in experimental group and control group were established followed by the Huhh method.Animals in the experimental group were given intra-articular injection of 1 × 104 μmol/L PQQ (0.2 ml/kg body weight),3 days a week for 6 weeks.The same volume of saline was delivered in the control group.There was no special treatment on animals in the sham-operation group.Animals were sacrificed 6 weeks after anteroposterior X-ray evaluation.HE staining,type Ⅱ collagen immunohistochemical staining,chondrocyte apoptosis [TdT-mediated dUTP nick end labeling (TUNEL)] detection,and electron microscopy observation of mitochondrion changes in cartilage were done.Results Kellgren-Lawrance score in experimental group,control group and sham-operation group was 1.49 ±0.45,5.69 ± 1.08,and 0.28 ± 0.06,respectively,P ＜ 0.05.Degeneration changes of femoral condyle were alleviated by PQQ (JP score 1.46 ± 0.42,2.75 ± 0.60,0.08 ± 0.04,P ＜0.05 ).Mankin scores were 2.68 ± 0.86,6.95 ±1.46 and 0.25 ± 0.06,P ＜ 0.05 ; and number of apoptotic chondrocytes was ( 5.60 ± 1.20) ％,( 35.30 ±4.90) ％,(0.70 ± 0.30 ) ％,P ＜ 0.05.More mitochondrials were observed under an electron microscope (3.40 ± 0.68,1.24 ± 0.45,and 6.40 ± 1.20,P＜0.05 ).Well structured type Ⅱ collagen fibers were observed in the experimental group.Conclusion Intra-articular injection of PQQ can protect the cartilage during osteoarthritis development by alleviating the structure and function damage of the cartilage and promoting its metabolism and self-repair capacity.PQQ may take the function by protecting mitochondria in chondrocytes. Key words: Osteoarthritis; Pyrrololine quinone; Chondrocyte

The effect of therapeutic ultrasound to apoptosis of chondrocyte and caspase-3 and caspase-8 expression in rabbit surgery-induced model of knee osteoarthritis

Recent studies have shown a positive correlation between the degree of severity of OA and the number of apoptotic chondrocytes. The purpose of our study was to determine the effect of therapeutic ultrasound on apoptosis in articular cartilage of rabbits with knee osteoarthritis (KOA). Thirty 3-month New Zealand White rabbits were randomizingly divided into three groups, 10 in each group. Two groups underwent anterior cruciate ligament transaction in the right knee and another group left intact. Six weeks later, one group of the operated rabbits (OA-US) underwent ultrasound therapy (300 mW/cm(2), 1 MHz, 20% duty cycle, 10 min each day) for 2 weeks, while the other two groups (OA-Control and Normal Control) left untreated. Eight weeks after transection, all animals were killed. Microscopic morphologic grading was made for histological assessment. The caspases expressions and chondrocytes apoptosis were tested using the immunohistochemistry and TUNEL assessment, respectively. The mean grading of OA-US group was significantly higher than Normal Control group (P = 0.002), but significantly lower than OA-Control group (P = 0.002). Percentage of apoptosis and the optic density of cells expressing caspase-3 and caspase-8 in the three groups showed no statistical significances. Therapeutic ultrasound (300 mW/cm(2), 1 MHz, and 20% duty cycle) could relieve the degree of severity of induced KOA, while it had no effect on apoptosis and the expression of caspase-3 and caspase-8. These findings may provide a certain support for therapeutic ultrasound as an effective access to managing KOA.

Death and proliferation of chondrocytes in the degraded mandibular condylar cartilage of rats induced by experimentally created disordered occlusion

To investigate the effect of experimentally created disordered occlusion (ECDO) on cell death and proliferation in rat mandibular condylar cartilage. Sprague-Dawley rats were randomly assigned to experimental and control groups. In the experimental groups, ECDO was created by the dental orthodontic method. By means of histological evaluation, immunohistochemistry and TUNEL staining, we studied the histomorphological changes, the death and proliferation of chondrocytes. Time- and sex-related progressive histologic degradation was observed in the condylar cartilage of ECDO rats, accompanied with diminished chondrocyte proliferation in the female 12-week ECDO subgroup (P < 0.05). An increase in the number of apoptotic chondrocytes was seen in both the female 8- and 12-week ECDO subgroups and in the male ECDO 12-week subgroup (all P < 0.05), but not in the male ECDO 8-week subgroup (P > 0.05). ECDO induces degradation in the rat condylar cartilage accompanied by an increase in chondrocyte death.

Correlation between chondrocyte apoptosis of vertebral cartilage endplate and degeneration of intervertebral disc

To investigate the correlation between chondrocyte apoptosis of cartilage endplate and intervertebral disc degeneration. Forty adult New Zealand rabbits were randomized into 2 equal groups: experimental group, undergoing exposure of the upper and lower margins of vertebral bodies C4 and C5, lower ARFIN OF c3, and upper margin of C6, and wadding of bone cement to the depth of posterior margin to interrupt the nutrition supply to the vertebral endplate, thus establishing the models of intervertebral disc degeneration, and control group, only undergoing exposure of the cervical vertebrae. Four and 8 weeks later ten rabbits from each group were sacrificed to obtain specimens of cartilage endplate and tissues of intervertebral disc. TUNEL approach was used to detect the apoptosis of chondrocytes in the vertebral cartilage endplate and immunohistochemistry was used to determine the expression of type II collagen from the intervertebral disc tissues. Four weeks after the operation the number of apoptotic chondrocytes of the cartilage endplate in the experimental group was 9.9 +/- 0.88/HP, significantly higher than that of the control group (9.7 +/- 0.94/HP, P < 0.05). Eight weeks after the operation the number of apoptotic chondrocytes of the cartilage endplate in the experimental group was 12.4 +/- 0.71/HP, significantly higher than that of the control group (11.7 +/- 0.91/HP, P < 0.05). Four and 8 weeks after the operation, the numbers of type II collagen positive cells of the experiment group were 27.7 +/- 1.52/HP and 19.5 +/- 1.57/HP respectively, both significantly lower than those of the control group (29.6 +/- 1.54/HP and 20.6 +/- 1.40/HP respectively, both P < 0.05). Correlation analysis showed that vertebral cartilage endplate chondrocyte apoptosis was highly negatively correlated with the expression of type II collagen operation 4 and 8 weeks after the operation with the coefficient values of 0.86 and 0.82 respectively (both P < 0.05). Chondrocyte apoptosis in vertebral cartilage endplate may result in the decreased expression of type II collagen in intervertebral discs and lead to the degeneration of intervertebral discs.

Anti-apoptotic Bcl-2 gene transfection of human articular chondrocytes protects against nitric oxide-induced apoptosis

We stably transfected early passage chondrocytes with an anti-apoptotic Bcl-2 gene in vitro using a retrovirus vector. Samples of articular cartilage were obtained from 11 patients with a mean age of 69 years (61 to 75) who were undergoing total knee replacement for osteoarthritis. The Bcl-2-gene-transfected chondrocytes were compared with non-transfected and lac-Z-gene-transfected chondrocytes, both of which were used as controls. All three groups of cultured chondrocytes were incubated with nitric oxide (NO) for ten days. Using the Trypan Blue exclusion assay, an enzyme-linked immunosorbent assay and flow cytometric analysis, we found that the number of apoptotic chondrocytes was significantly higher in the non-transfected and lac-Z-transfected groups than in the Bcl-2-transfected group (p < 0.05). The Bcl-2-transfected chondrocytes were protected from NO-induced impairment of proteoglycan synthesis. We conclude that NO-induced chondrocyte death involves a mechanism which appears to be subject to regulation by an anti-apoptotic Bcl-2 gene. Therefore, Bcl-2 gene therapy may prove to be of therapeutic value in protecting human articular chondrocytes.

Influence of Maternal Nicotine Exposure on Neonatal Rat Bone: Protective Effect of Ascorbic Acid

Limited research in young adults and immature animals suggests a detrimental effect of tobacco on bone during growth. We aimed to determine the adverse effects of maternal nicotine exposure during pregnancy and lactation on neonatal rat bone development, and to determine a protective effect of ascorbic acid. Gravid rats were assigned into three groups: two experimental and one control (group I). In the first experimental group (group II), pregnant rats received 3 mg/kg/d nicotine subcutaneously during pregnancy from 1 to 21 days of gestation and lactation (until postnatal day 21). The second experimental group (group III) received nicotine and ascorbic acid (1 mg/kg body mass/d). Whole body mineral density (BMD), content (BMC), and area (BA) were measured on postnatal day 21. Histopathologic and morphologic findings of the femur were obtained. Maternal nicotine exposure decreased the body weight of the rat at the birth and postnatal day 21. The values of BMD, BA, and BMC of the groups were similar to each other. Width of the epiphyseal plate and the hypertrophic zone were higher in group III but lower in group II than in group I. Number of apoptotic chondrocytes was significantly increased in group II. The length of femur was higher in group I but lower in group II than in group III. Maternal nicotine exposure during gestation and lactation resulted in decreased body weight and bone lengthening. Ascorbic acid supplementation was found to prevent the adverse effects of maternal nicotine exposure on the growth plate.

Influence of pro-inflammatory (IL-1α, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4) cytokines on chondrocyte function

Objective: Cytokines produced by inflammatory cells play a pivotal role in synovial inflammation and joint destruction in rheumatoid arthritis. To investigate the influence of pro-inflammatory cytokines (IL-1α, IL-6, TNF-α, IFN-γ) and subsequently the possible beneficial role of an anti-inflammatory cytokine (IL-4) on chondrocyte viability (necrosis/apoptosis), proliferation and nitric oxide (NO) production. Methods: Primary bovine chondrocytes were cultured until monolayers were obtained. Cells were incubated with cytokines (IL-1α, IFN-γ, TNF-α, IL-4, IL-6) at 0.1, 1, 10 and 100 ng/mL. After 48 h, the viability of the chondrocytes was measured flow cytometrically with propidium iodide. Proliferation was determined by the incorporation of tritiated thymidine. The morphology of the chondrocytes, including presence of apoptotic nuclei, was evaluated by a May-Grünwald–Giemsa staining. In addition, the number of apoptotic chondrocytes was detected flow cytometrically with a TUNEL technique and annexin-V/propidium iodide staining. NO production was evaluated using a spectrophotometric assay, based upon the Griess reaction. Results: The viability and proliferation of bovine chondrocytes decreased after incubation with 100 ng/mL IL-1α, TNF-α or IFN-γ. In contrast, incubation of chondrocytes with IL-4 or IL-6 had no influence on the viability or the proliferation of cells. IL-1α was able to enhance NO production in a dose dependent manner. IFN-γ and TNF-α induced NO production only at the highest concentration (100 ng/mL), whereas IL-4 and IL-6 did not. There was a dose dependent increase in apoptosis of bovine chondrocytes cultured in the presence of IL-1α and TNF-α. This effect could not be prevented by preincubation with IL-4. Preincubation with IL-4 diminished IL-1α and TNF-α induced NO production and increased proliferation of chondrocytes. In an additional experiment, incubation of human chondrocytes with anti-Fas did not induce apoptosis as measured by annexin-V/propidium iodide staining. Conclusions: Pro-inflammatory cytokines are able to induce apoptosis, whereas IL-4 as an anti-inflammatory cytokine can inhibit the effect of IL-1α and TNF-α on NO production and proliferation of bovine chondrocytes.

Short-Term Exposure of Cartilage to Blood Results in Chondrocyte Apoptosis

Studies have shown that joint bleeding leads to cartilage degradation independent of concurrent synovitis. We hypothesized that the blood-induced cartilage damage is because of increased chondrocyte apoptosis after short-term exposure of whole blood or isolated mononuclear cells plus red blood cells to cartilage. Human cartilage tissue samples were co-cultured for 4 days with whole blood (50% v/v) or with mononuclear cells plus red blood cells (50% v/v equivalents). Cartilage matrix proteoglycan synthesis ((35)SO(4)(2-) incorporation) was determined after 4 days as well as at day 16 (after a 12-day recovery period in the absence of any additions). To test the involvement of apoptosis a specific caspase-3 inhibitor (acDEVDcho, 0 to 500 micro mol/L) as well as a pan-caspase inhibitor (zVADfmk, 0 to 500 micro mol/L) were added. Chondrocyte apoptosis was evaluated by immunohistochemical staining of single-strand DNA and by terminal dUTP nick-end labeling. Cartilage co-cultured with whole blood as well as mononuclear cells plus red blood cells induced a long-term inhibition of proteoglycan synthesis (74% and 78% inhibition on day 16, respectively). Immunohistochemistry showed a threefold increase in apoptotic chondrocytes in cultures with 50% whole blood as well as with mononuclear cells plus red blood cells. Both the specific caspase-3 inhibitor and the pan-caspase inhibitor partially restored proteoglycan synthesis in the cartilage after blood exposure. This effect was accompanied by a decrease in the number of apoptotic chondrocytes. These data suggest that a single joint hemorrhage (a 4-day exposure of cartilage to 50% v/v blood) results in induction of chondrocyte apoptosis, responsible for the observed inability of the chondrocytes to restore the proteoglycan synthesis during recovery from a short-term exposure to blood. This reduced restoration could eventually lead to cartilage degeneration and ultimately joint destruction.

Articular Chondrocytes from Animals with a Dermatan Sulfate Storage Disease Undergo a High Rate of Apoptosis and Release Nitric Oxide and Inflammatory Cytokines: A Possible Mechanism Underlying Degenerative Joint Disease in the Mucopolysaccharidoses

Mucopolysaccharidosis (MPS) Type VI (Maroteaux-Lamy Disease) is the lysosomal storage disease characterized by deficient arylsulfatase B activity and the resultant accumulation of dermatan sulfate-containing glycosaminoglycans (GAGs). A major feature of this and other MPS disorders is abnormal cartilage and bone development leading to short stature, dysostosis multiplex, and degenerative joint disease. To investigate the underlying cause(s) of degenerative joint disease in the MPS disorders, articular cartilage and cultured articular chondrocytes were examined from rats and cats with MPS VI. An age-progressive increase in the number of apoptotic chondrocytes was identified in the MPS animals by terminal transferase nick-end translation (TUNEL) staining and by immunohistochemical staining with anti-poly (ADP-ribose) polymerase (PARP) antibodies. Articular chondrocytes grown from these animals also released more nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) into the culture media than did control chondrocytes. Notably, dermatan sulfate, the GAG that accumulates in MPS VI cells, induced NO release from normal chondrocytes, suggesting that GAG accumulation was responsible, in part, for the enhanced cell death in the MPS cells. Coculture of normal chondrocytes with MPS VI cells reduced the amount of NO release, presumably because of the release of arylsulfatase B by the normal cells and reuptake by the mutant cells. As a result of the enhanced chondrocyte death, marked proteoglycan and collagen depletion was observed in the MPS articular cartilage matrix. These results demonstrate that MPS VI articular chondrocytes undergo cell death at a higher rate than normal cells, because of either increased levels of dermatan sulfate and/or the presence of inflammatory cytokines in the MPS joints. In turn, this leads to abnormal cartilage matrix homeostasis in the MPS individuals, which further exacerbates the joint deformities characteristic of these disorders.

Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.