Chronic lateral ankle instability (CLAI) is treated operatively, whereas acute ligament injury is usually treated nonoperatively. Such treatments have been widely validated. Apoptosis is known to cause ligament degeneration; however, few reports have focused on the possible role of apoptosis in degeneration of ruptured lateral ankle ligaments. The aim of our study is to elucidate the apoptosis that occurs within anterior talofibular ligament (ATFL) to further validate current CLAI treatments by adducing molecular and cellular evidence. Between March 2019 and February 2021, 50 patients were prospectively enrolled in this study. Ruptured ATFL tissues were collected from 21 CLAI patients (group C) and 17 acute ankle fracture patients (group A). Apoptotic cells were counted using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Western blotting for caspases 3, 7, 8, and 9 and cytochrome c, was performed to explore intrinsic and extrinsic apoptotic pathways. Immunohistochemistry was used to detect caspases 3, 7, 8, and 9 and cytochrome c, in ligament vessel endothelial cells. More apoptotic cells were observed in group C than group A in TUNEL assay. Western blotting revealed that the apoptotic activities of group C ligaments were significantly higher than those of group A (all p < 0.001). Immunohistochemistry revealed increased expression of caspases 3, 7, 8, and 9, and cytochrome c, in group C compared to group A. The ATFL apoptotic activities of CLAI patients were significantly higher than those of acute ankle fracture patients, as revealed biochemically and histologically. Our data further validate current CLAI treatments from a molecular and cellular perspective. Efforts should be made to reverse or prevent ATFL apoptosis in CLAI patients

Introduction: Apoptosis has been observed following experimental contusive and transective spinal cord injury, but it is not known whether this is related to secondary excitotoxic injury or other factors. This study examines apoptosis after a purely excitotoxic injury and the relationship between apoptosis and syrinx formation. Methods: Twenty-four male Sprague-Dawley rats were divided into six groups. Twenty rats received four 0.5 μL injections of 24 mg/mL quisqualic acid and 1% Evans blue between the rostral C8 and caudal T1 level. Ten microliters of 250 mg/mL kaolin were then injected into the subarachnoid space. Animals were sacrificed at 1, 5, 10, 20 and 50 days following the injections. There were 4 control animals. Spinal cord tissue was frozen and sectioned, and damaged DNA was detected immunohistochemically by using anti-single-stranded DNA monoclonal antibody. The area and density of single strand DNA were semi-quantitated. Results: No significantly damaged DNA was found in the 1 day group. Light staining of single-stranded DNA was observed at C6, C7, T1 and T2 levels in 30% of the section area in the 5 and10 day groups. Moderate staining of damaged DNA occurred at C7 and T1 levels in 25–30% of the section area at 20 day group. Syrinxes formed in this group. Heavy staining and larger syrinxes were noted in the 50 day group. Discussion: Apoptosis increased with time after excito-toxic injury. These findings suggest that apoptosis may play a pivotal role in syrinx pathogenesis

INTRODUCTION: Apoptosis has been observed following experimental contusive and transective spinal cord injury, but it is not known whether this is related to secondary excitotoxic injury or other factors. This study examines apoptosis after a purely excitotoxic injury and the relationship between apoptosis and syrinx formation. METHODS: Twenty-four male Sprague-Dawley rats were divided into six groups. Twenty rats received four 0.5 μL injections of 24 mg/mL quisqualic acid and 1% Evans blue between the rostral C8 and caudal T1 level. Ten microliters of 250 mg/mL kaolin were then injected into the subarachnoid space. Animals were sacrificed at 1, 5, 10, 20 and 50 days following the injections. There were four control animals. Spinal cord tissue was frozen and sectioned, and damaged DNA was detected immunohistochemically by using anti-single-stranded DNA monoclonal antibody. The area and density of single strand DNA were semi-quantitated. RESULTS: No significantly damaged DNA was found in the 1 day group. Light staining of single-stranded DNA was observed at C6, C7, T1 and T2 levels in 30% of the section area in the 5 and 10 day groups. Moderate staining of damaged DNA occurred at C7 and T1 levels in 25–30% of the section area at 20 day group. Syrinxes formed in this group. Heavy staining and larger syrinxes were noted in the 50 day group. DISCUSSION: Apoptosis increased with time after excitotoxic injury. These findings suggest that apoptosis may play a pivotal role in syrinx pathogenesis

Apoptosis of articular chondrocytes may play an important role in the pathogenesis of osteoarthritis (OA). The aim of this study was to investigate the incidence of chondrocyte apoptosis in equine articular cartilage (AC) specimens and examine the relationship between the process of cell death and the degree of cartilage degradation. The study comprised 2 populations of equine cartilage taken from the left forelimb. Population 1 (n=10) consisted of full depth cartilage from weight-bearing regions of equine metacarpophalangeal (MCP), proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints. Population 2 (n=9) comprised cartilage from 6 different regions of the MCP joint: dorsomedial, dorsolateral, centromedial, centrolateral, palmarome-dial and palmarolateral areas. Cartilage from each horse for each of the joints and joint regions was not always available. Seven micrometre cryostat sections were obtained. Haematoxylin and Eosin with Safranin-O stained sections were used to score structural differences between samples for features of cartilage pathology using a ‘modified’ Mankin scoring system. Two methods were used to quantify apoptotic chondrocytes: a direct method in which chondrocytes were assessed for morphological features of apoptosis using a light microscope and an immunohistochemical staining technique to detect the expression of active caspase-3 using a commercially available monoclonal antibody. Apoptosis assessed by the direct method did not show any association with increasing severity of OA (r=0.11, p=0.7205). Overall there was a positive correlation between caspase-3 expression and cartilage damage (r= 0.44, p=0.0043). Caspase-3 expression was found to increase linearly with increasing severity of OA in the superficial, middle and deep zones of AC (r=0.36, p=0.0198; r=0.49, p=0.0011 and r=0.37, p=0.0237 respectively). Moreover, caspase-3 expression was higher in the superficial and middle zones than in the deep zone (p< 0.001). In the superficial, middle and deep zones the expression of caspase-3 was higher in the MCP joint than the PIP joint (p< 0.05, p< 0.01 and p< 0.05 respectively). The significant positive correlation between disease severity and chondrocyte apoptosis, suggests that this process plays an important role in the pathogenesis of OA. The differences in the extent of apoptosis observed in different joints could be explained by the biomechanical environment of the joints

Cells of the intervertebral disc exist in an unusual environment compared to those of other tissues. Within the disc there are low levels of nutrients available, low oxygen levels and it is an acidic environment due to high lactate levels. Apoptosis (programmed or controlled cell death) has been reported in intervertebral discs, as well as necrosis (uncontrolled cell death). This study has focused on examining the sensitivity of nucleus pulpo-sus (NP) cells to several stimuli, in comparison to two other cells types. Ultra violet (UV) irradiation, serum starvation (with no foetal calf serum) and treatment with 2mM hydrogen peroxide were used to induce apoptosis in cultured bovine NP cells, HeLa (cancer cell line) and 293T cells (human embryo kidney derived) cells. Apoptosis was identified by nuclear morphology following staining with fluorescent Hoechst 33342 dye and propidium iodide; the incidence was measured at 24, 48 and 72 hours. Untreated controls were used for each treatment and at each time point. The incidence of apoptosis increased with time for all treatments. After 72 hours, UV treatment produced the highest levels of apoptosis with levels of apoptosis occurring in the order of HeLa (94%) > NP cells (29%) > 293T cells (15%). Treatment with hydrogen peroxide and serum starvation induced apoptosis at lower levels in all three cell types (maximum of 30%). Serum starvation induced apoptosis in only 10% of NP cells at 72 hours, compared to 20% in HeLa cells. None of the controls contained apoptotic cells. NP cells are stimulated to apoptose in response to UV irradiation, hydrogen peroxide and serum starvation. However, levels of apoptosis are much lower after UV treatment in comparison to HeLa cells (3 times lower), suggesting that they may have a protective mechanism to this apoptotic stimulus, compared to HeLa cells. The low levels of apoptosis observed in NP cells with serum starvation may be due to the low nutrient environment that they exist in normally

Introduction: Apoptosis, or secondary cell death, has been demonstrated in a number of neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and brain ischaemia. It is well established from studies of acute spinal cord injury that apoptosis seems an important factor in secondary cell death and irreversible neurological deficit. It is only recently that studies have emerged analysing secondary cell death in chronic injury to the cord. In this study, the spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathies due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). The study aimed to demonstrate apoptosis in compressive spinal cord injury and to analyse the spatial and temporal distribution of apoptosis in acute and chronic myelopathy. Method: Archival material from 21 spinal cords of patients with documented myelopathy during life and definitive evidence on post mortem examination were available for study. The spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathy due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). Immunohistochemical analysis of each specimen was conducted using markers of apoptosis, as well as the biochemical apoptotic marker TUNEL. A total of 1800 histopathological slides were analysed. Specimens were also analysed using confocal microscopy to identify the immunopositive cell type. A combination of morphological, immunohistochemical and in situ end-labelling techniques were used to investigate the mechanism of cell death in this experiment. The analytical techniques employed were aimed at showing firstly the presence of apoptosis and secondly the size and position of the damaged regions. Results: Positivity for active Caspase-3, DNA-PKCS, PARP, TUNEL and active Caspase-9 was found in glia (oligodendrocytes and microglia) axons and neurons in both acute and chronic compression above, below and at the site of compression. In chronic compression, the severity of positivity for apoptotic immunological markers was positively correlated with the severity of white matter damage, as measured by APP immunostaining for axonal injury, and Wallerian degeneration. There was no correlation between the duration of chronic compression and immunopositivity for apoptotic markers. In acute SCI, axonal swellings were consistently positive for Caspases −9 and -3, suggesting mitochondrial activation of apoptotic pathways. Conclusion: Apoptosis occurs in both acute and chronic spinal cord injury. In acute compression, axonal injury is associated with apoptotic immunopositivity of glia and neurons. In chronic compression, apoptosis of oligodendrocytes and microglia correlates with demyelination of axons within the white matter

INTRODUCTION: Apoptosis, or secondary cell death, has been demonstrated in a number of neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and brain ischaemia. It is well established from studies of acute spinal cord injury that apoptosis seems an important factor in secondary cell death and irreversible neurological deficit. It is only recently that studies have emerged analysing secondary cell death in chronic injury to the cord. In this study, the spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathies due to meta-static tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). The study aimed to demonstrate apoptosis in compressive spinal cord injury and to analyse the spatial and temporal distribution of apoptosis in acute and chronic myelopathy. METHOD: Archival material from 21 spinal cords of patients with documented myelopathy during life and definitive evidence on post mortem examination were available for study. The spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathy due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). Immunohistochemical analysis of each specimen was conducted using markers of apoptosis, as well as the biochemical apoptotic marker TUNEL. A total of 1800 histopathological slides were analysed. Specimens were also analysed using confocal microscopy to identify the immunopositive cell type. A combination of morphological, immunohistochemical and in situ end-labelling techniques were used to investigate the mechanism of cell death in this experiment. The analytical techniques employed were aimed at showing firstly the presence of apoptosis and secondly the size and position of the damaged regions. RESULTS: Positivity for active Caspase-3, DNA-PKCS, PARP, TUNEL and active Caspase-9 was found in glia (oligodendrocytes and microglia) axons and neurons in both acute and chronic compression above, below and at the site of compression. In chronic compression, the severity of positivity for apoptotic immunological markers was positively correlated with the severity of white matter damage, as measured by APP immunostaining for axonal injury, and Wallerian degeneration. There was no correlation between the duration of chronic compression and immunopositivity for apoptotic markers. In acute SCI, axonal swellings were consistently positive for Caspases −9 and -3, suggesting mitochon-drial activation of apoptotic pathways. CONCLUSION: Apoptosis occurs in both acute and chronic spinal cord injury. In acute compression, axonal injury is associated with apoptotic immunopositivity of glia and neurons. In chronic compression, apoptosis of oligodendrocytes and microglia correlates with demyelination of axons within the white matter

Introduction: Anteromedial gonarthrosis (AMG) is a distinct phenotype of osteoarthritis (OA), with a specific pattern of disease. There is full thickness cartilage loss anteromedially, progressing to an area of damaged cartilage, and then to an area of macroscopically and histologically normal cartilage posteriorly. It can be considered to be a spatial model of OA progression. Apoptosis, or chondrocyte cell death, has been shown to be a feature of OA cartilage, however the triggers are poorly understood; similarly, reactive oxygen species (ROS) have been implicated in OA. They have never been studied in a replicable topographical model of OA. This study characterises the regional levels of cell death and implicated ROS in AMG using a number of immunohistochemical studies. Method: Ten tibial resection specimens were obtained from patients undergoing unicompartmental knee arthroplasty. Eight above knee amputations (from patients with peripheral vascular disease) were used as age matched controls. Cross sections taken through all regions were paraffin embedded. Routine histology was performed and immunohistochemical studies were conducted for Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Active Caspase 3, Cytochrome C, Active Bax, Bim, 3-Nitrotyrosine and Forkhead Box O3A (FOXO 3A). Results: Cell death, as detected by TUNEL appeared predominantly in the surface layer of chondrocytes of damaged cartilage (p< 0.001). Median values were 23% in superficial cartilage (range 0 – 51) compared to 0% in deeper cartilage (range 0 – 15). There was a significant difference in TUNEL staining between regions (p=0.001). This ranged from 26% (most damaged) to 4% (undamaged). There was a good correlation with degree of cartilage damage (ρ=0.66, p< 0.001) asdefined by histological grade and TUNEL was significantly higher (p< 0.001) in AMG compared to the control samples which showed an average of 2% TUNEL overall. Upstream markers of apoptosis (Active Caspase 3, Cytochrome C, Active Bax), assessed qualitatively, were present in a similar distribution to that of TUNEL staining. 3-Nitrotyrosine was also shown to be a predominantly surface phenomenon. There was a significant difference (p< 0.001) between regions, ranging from 58% (most damaged) to 10% (undamaged). Again, this was significantly higher that the control samples (p< 0.001). In line with indicators of ROS mediated damage, Bim and FOXO3A were also detected. Discussion: The mechanism of apoptosis in OA cartilage has not been studied in depth, and understanding the biochemical and molecular responses of ‘stressed’ chondrocytes may provide invaluable information about the specific causes of cell death. Such cellular responses may provide targets for disease modification, thus delaying or preventing the need for joint arthroplasty. We conclude that AMG is a phenotype demonstrating cartilage at progressive stages of disease. Apoptosis involves the intrinsic mitochondrial pathway and ROS appear to be implicated. Further work is needed to provide evidence of what lies further upstream of markers demonstrated in this study

Introduction: The pathogenesis of chronic tendinopathy is unclear. A role for increased apoptosis of tenocytes has been suggested. Nitric oxide is thought to be a mediator of apoptosis and nitric oxide synthase (NOS) isoforms have been shown to be up regulated in rotator cuff tendons as a result of chronic overuse. We found, the same up regulation of NOS in the Achilles tendon in non-insertional Achilles tendinopathy in a previous study. The purpose of this study was to investigate whether apoptotic cells were present in these tissues with raised endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) levels. Methods: Consent was obtained preoperatively from all patients and the research and ethics committee granted ethical approval. Samples were obtained from the Achilles Tendons of patients with non-insertional Achilles tendinopathy who had failed conservative treatment for at least six months and were undergoing a surgical procedure. Several biopsies were taken of the visibly abnormal tendon tissue. Control samples were taken from macroscopically normal tendon correlating with areas of normal tissue on MRI. Standard immunohistochemical techniques were used to identify the expression of eNOS and iNOS. Apoptotic cells were identified using terminal deoxynucleotidyl transferase-mediated dUTP neck end labelling (TUNEL reaction) with TdT-FragEL and the demonstration of Caspase-3 activation. Results: Significant differences were found between the diseased tendon and the controls for all of the parameters measured. The mean Caspase-3 cell count for diseased tendon was 51.9 compared to 28.3 for the controls (p=0.000001). The mean TUNEL cell count for diseased tendon was 24.1 compared to 14.8 (p=0.00014). iNOS densitometry revealed a mean of 26.1 for the diseased tissue verses 15.0 for the controls (p=0.000009) and the values for eNOS were 48.3 and 23.7 respectively (p=0.015). Conclusions: Apoptosis clearly plays a role in the development of non-insertional Achilles tendinopathy and appears to be related to the presence of raised eNOS and iNOS levels. It is possible that, by blocking the apoptotic pathway, the tendinopathic process could be halted. This may lead to the development of treatments strategies for early Achilles tendinopathy

The aim of this study was to understand the role tissue hypoxia and apoptosis have on a human model of rotator cuff failure. We studied twenty seven patients with no tear mild impingment (3), no tear moderate impingment (3), no tear severe impingment (3), partial tear (3), small tear (3), moderate tear (3), large tear (3), massive tear (3) and control (3). A supraspinatus tendon biopsy was taken during debridement/repair in all cases (ethics no. C01.071). Control tendon was obtained from the subscapularis tendon of patients undergoing stabilization surgery. Biopsies were analysed using two immunocytological techniques. A monoclonal antibody against BNIP-III (a marker of hypoxia) and TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling – an apoptotic detection process). An immunoflorescent counterstain DAPI (4′,6-diamidino-2-phenylindol) was used to stain all cells. Positive cells and total cell number were then counted in 10 high powered fields. The results showed a significant increase in BNIP-III expression in the cuff tears compared with intact tendons. This increase was least in the massive tears. Apoptosis increases from mild impingment to massive cuff tears (mean 7.3% to 21%) In conclusion, as tear size increases, the viability of the tendon reduces with increasing hypoxia and apoptosis

Introduction: The pathogenesis of chronic tendinopathy is unclear. The role of the increased apoptosis of tenocytes has been suggested by high intratendinous levels of glutamate being demonstrated in patients with tendinosis. Nitric oxide is a known mediator of apoptosis and nitric oxide synthase (NOS) isoforms have been shown to be upregulated in rotator cuff tendons as a result of chronic overuse. We found, the same upregulation of NOS in the Achilles tendon in non-insertional Achilles tendinopathy in a previous study. The purpose of this study was to investigate whether apoptotic cells were present in these tissues with raised eNOS and iNOS levels. Methods: Samples were obtained from the Achilles Tendons of patients with in non-insertional Achilles tendinopathy who had failed conservative treatment for at least six months and were undergoing a surgical procedure. Consent was obtained preoperatively from all patients and ethical approval was granted by the research ethics committee. Several biopsies were taken of the visibly abnormal tendon tissue. Control samples were taken from macroscopically normal tendon correlating with areas of normal tissue on MRI. Standard immunohistochemical techniques were used to identify the expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Apoptotic cells were identified using terminal deoxynucleotidyl transferase-mediated dUTP neck end labelling (TUNEL reaction) with TdT-FragEL and the demonstration of Caspase-3 activation. A power calculation was performed which showed that 14 patients in each group would be required to show a 50% difference between the two groups using a level of significance of 5%. Results: Significant differences were found between the diseased tendon and the controls for all of the parameters measured. The mean Caspase-3 cell count for diseased tendon was 51.9 compared to 28.3 for the controls (p=0.000001). The mean TUNEL cell count for diseased tendon was 24.1 compared to 14.8 (p=0.00014). iNOS densitometry revealed a mean of 26.1 for the diseased tissue verses 15.0 for the controls (p=0.000009) and the values for eNOS were 48.3 and 23.7 respectively (p=0.015). Conclusions: Apoptosis clearly plays a role in the development of non-insertional Achilles tendinopathy and appears to be related to the presence of raised eNOS and iNOS levels. It is possible that, by blocking the apoptotic pathway, the tendinopathic process could be halted. This may lead to the development of treatments strategies for early Achilles tendinopathy

Excessive apoptosis has been found in torn supraspinatus tendon1 and mechanically loaded tendon cells2. Following oxidative and other forms of stress, one family of proteins that is often unregulated are Heat Shock Proteins (HSPs). The purpose of this study was to determine if HSPs were unregulated in human and rat models of tendinopathy and to determine if this was associated with increased expression of regulators of apoptosis (cFLIP, Caspases 3& 8). A running rat supraspinatus tendinopathy overuse model 3 was used with custom microarrays consisting of 5760 rat oligonucleotides in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. Rat Microarray: Upregulation of HSP 27 (×3.4) & 70 (×2.5) and cFLIP (×2.2) receptor was noted in degenerative rat supraspinatus tendon subjected to daily treadmill running for 14 days compared to tendons of animals subject to cage activity only. Histological analysis: All torn human supraspinatus tendons exhibited changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Apoptosis mRNA expression: The expression levels of caspase 3 & 8 and HSPs 27 & 70 were significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and control tendon (p< 0.01). cFLIP showed significantly greater (p< 0.001) expression in matched subscapularis compared to supraspinatus and control tendon. Immunohistochemical analysis: cFLIP, Caspase 3 & 8 and HSP 27 and 70 was confirmed in all samples of torn supraspinatus tendon. Significantly increased immunoactivity of Caspase 3& 8 and HSP 27 & 70 were found in torn supraspinatus (p< 0.001) compared to matched and normal subscapularis. The proteins were localized to tendon cells. The finding of significantly increased levels of Heat Shock Proteins in human and rat models of tendinopathy with the co-expression of other regulators of apoptosis suggests that Heat Shock Proteins play a role in the cascade of stress activated-programmed cell death and degeneration in tendinopathy

Due to recent advances in diagnostic technology and an increased awareness among clinicians, osteochondral damage is being detected more frequently. Thus, there is a need to preserve and store articular cartilage for the repair of joint surfaces. Chondrocytes, embedded within extracellular matrix must remain viable during storage for successful tissue transplantation. We have been able to store osteochondral tissue for over a month and maintain high chondrocyte viability. Apoptosis can be minimized in articular cartilage during hypothermic storage if biopreservation media (XVIVO-10) is used. Cadaveric osteochondral dowels are a potential source of tissue for banking and allogeneic transplantation. The purpose of this study was to:. Establish a timeline and optimal conditions for storing human articular cartilage (AC). Determine a suitable source of AC for banking and transplantation. Distinguish apoptosis from necrosis in human AC following hypothermic storage. Forty fresh human AC samples from femoral condyle notchplasties were used to determine a storage timeline. Each sample was divided into three portions:. initial chondrocyte viability,. stored in phosphate buffered saline,. stored in Biowhitaker XVIVO-10. All samples were randomly allocated to one of five time intervals (2–10 weeks). Following each time period final viability assays were conducted. Secondly, osteochondral dowels were drilled from eight cadaveric femoral condyles. Five dowels were obtained from each joint: one for initial viability/annexin V assays, the others were stored in PBS or XVIVO-10 for four and six weeks. Following storage, final viability, annexin V, and TUNEL assays were preformed. Notchplasty samples stored in XVIVO-10 for four weeks had an average final viability of 68%, but an average loss in viability of only 6%. By one month the viability of samples stored in PBS had dropped to 5%. Osteochondral dowels stored in XVIVO-10 not only had greater chondrocyte viability, but less apoptosis. Cadaveric dowels are a suitable source of osteochon-dral tissue for hypothermic storage and in turn allogeneic transplantation. An osteochondral tissue bank would provide a reliable source of articular cartilage for repairing joint surfaces for patients who are not suitable candidates for total joint replacements

The pathogenesis of frozen shoulder remains unclear. Fibroblast proliferation has been implicated in the pathogenesis with subsequent fibrosis of the capsule. We studied patients undergoing manipulation under anaesthesia for frozen shoulder. All fitted Codman’s criteria for the diagnosis. Normal saline was injected and then aspirated from 14 patients undergoing manipulation under anaesthesia for treatment of frozen shoulder and from 15 patients undergoing shoulder arthroscopy for other pathology. Human fibroblasts were cultured from sections of human anterior abdominal wall obtained from patients undergoing elective surgery. The effect of frozen shoulder aspirate versus normal control on human fibroblast proliferation and apoptosis was measured. Cellular proliferation was determined using the Promega celltitre 96TM non-radioactive cell proliferation assay. Results: Proliferation of human fibroblasts was significantly increased in the cells treated with aspirate obtained from frozen shoulder patients versus both negative control (growth medium only) and control (normal shoulder aspirate) at concentrations of 105, 25% and 50%. This increase in proliferation was in a dose dependent manner, with the most significant increase seen in cells treated with a 505 concentration of frozen shoulder aspirate. Apoptosis was unregulated at all concentrations of shoulder aspirate, but only achieves statistical significance at 255 and 505 concentrations. Conclusion: This study supports the hypothesis that frozen shoulder results from alteration in fibroblast regulation. Pharmacological modulation of fibroblast proliferation may be a potential therapeutic option

The pathogenesis of frozen shoulder remains unclear. Fibroblast proliferation has been implicated in the pathogenesis with subsequent fibrosis of the capsule. We studied patients undergoing manipulation under anaesthesia for frozen shoulder. All fitted Codman’s criteria for the diagnosis. Normal saline was injected and then aspirated from 15 patients undergoing manipulation under anaesthesia for treatment of frozen shoulder and from 15 patients undergoing shoulder arthroscopy for other pathology. Human fibroblasts were cultured from sections of human anterior abdominal wall obtained from patients undergoing elective surgery. The effect of frozen shoulder aspirate versus normal control on human fibroblast proliferation and apoptosis was measured. Cellular proliferation was determined using the Promega celltitre 96TM non-radioactive cell proliferation assay. Proliferation of human fibroblasts was significantly increased in the cells treated with aspirate obtained from frozen shoulder patients versus both negative control (growth medium only) and control (normal shoulder aspirate) at concentrations of 105, 25% and 50%. This increase in proliferation was in a dose dependent manner, with the most significant increase seen in cells treated with a 505 concentration of frozen shoulder aspirate. Apoptosis was upregulated at all concentrations of shoulder aspirate, but only achieves statistical significance at 255 and 505 concentrations. This study supports the hypothesis that frozen shoulder results from alteration in fibroblast regulation

Background: Low molecular weight heparins (LMWH) are of undoubted efficacy as thromboprophylaxis in orthopaedic surgical practice. However, prolonged dosage inhibits bone nodule formation in vitro and we have previously reported that daily dosing significantly delays fracture healing. To further investigate these phenomena we hypothesised that LMWH’s would reduce osteoblast survival and thus bone formation by inducing programmed cell death (apoptosis). Methods: Primary human osteoblasts were isolated from femoral heads excised during hip arthoplasty and cultured to passage 3–5. These were examined for VEGF receptor expression using a biotinylated binding assay on flow cytometry. Osteoblasts were grown to confluence and then incubated for 24 hours in control medium or medium treated with enoxaparin (200 – 2X10(−4) IU/mL) or combination of enoxaparin (200 – 2X10 (−4) IU/mL) and VEGF (1ng/ml). Apoptosis was determined by measuring cytosolic histone-associated DNA fragmentation using an enzyme linked immunosorbant assay. Results were confirmed by DNA fragmentation analysis on agarose gel electrophoresis. Cell functional viability was measured by a tetrazolium bioreduction colorimetric assay. Results: Data is expressed as percentage of control apoptosis or viability, illustrates mean ± s.e.m. and n=4 experiments in each case. ANOVA was employed for statistical analysis; *versus control, #versus enoxaparin treated; p< 0.05 was considered significant. Conclusions: Therapeutic doses of LMWH attenuate osteo-blast survival by inducing significant apoptosis. This effect is partly abrogated by VEGF, which independently enhances osteoblast viability, thus delaying spontaneous and enoxaparin induced apoptosis. These findings may explain the bone resorptive effects of prolonged LMWH therapy and suggest a potential therapeutic role for VEGF in conditions of delayed bone formation

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development.

Abstract

The purpose of this study was to evaluate whether AGEs induce annulus fibrosus (AF) cell apoptosis and to further explore the mechanism by which this process occurs.

AF cells were treated with various concentrations of AGEs for 3 days. Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8) and EdU incorporation assays. Cell apoptosis was examined by the Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3 and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by RT-PCR. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by JC-1 staining and DCFH-DA fluorescent probes, respectively.

Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced MMP decrease and AF cell apoptosis.

These results suggest that AGEs induce rabbit AF cell apoptosis and mitochondrial pathways may be involved in AGE-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration.

Aseptic loosening is the single most important long-term complication of total joint arthroplasty. Wear debris induced inflammation stimulates osteoclastic resorption of bone. Cellular mechanisms involved in osteoblast viability in PWD induced inflammation is poorly understood.

Wear induced inflammation increases osteoblast necrosis and susceptibility to death by apoptosis. PMMA cement has a detrimental effect on osteoblast resistance to apoptosis, and that this is via an receptor mediated pathway. Osteoblast cell cultures (Human and MG63) were grown with and without PMMA cement and assessed for apoptosis and necrosis. TNF-α or Fas antibody simulated inflammation. Viability and apoptosis with PI exclusion, flow cytometry and western blotting assessed response.

Cement induced osteoblast necrosis up to 1 hour. This effect was negated after 24 hours. Culture of osteob1asts on cement had no direct effect on spontaneous apoptosis but susceptibility to inflammation was increased.

Polymerised cement has no direct effect on osteoblast cell death. Effects are mediated by inhibiting expression of anti-apoptotic protein (Bcl-2), and increasing susceptibility to inflammatory. Osteoblast resistance to death may represent a novel and important factor in aseptic loosening. The role of gene therapy is explored.