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

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

Summary Statement

Survivin is a member of the inhibitor of apoptosis family, which may contribute to the progression of human MFH via inhibiting the mitochondrial apoptosis, and may be considered as a potent therapeutic target for the treatment of human MFH.

The growth plates of the femoral head of Japanese white rabbits aged 5, 10, 15 and 20 weeks were stained for apoptotic and proliferating chondrocytes using the TUNEL and PCNA antibody staining techniques. Both TUNEL- and PCNA-positive chondrocytes were detected in all of the specimens. The positive ratios of both stainings were calculated for the whole plate and for the resting, proliferating and hypertrophic zones. The highest ratios in both stainings occurred in the hypertrophic zone in all age groups. With growth, the TUNEL-positive ratio increased whereas the proliferating ratio decreased.

We suggest that the increase in chondrocytic death by apoptosis and the decrease in cell proliferation potential led to closure of the growth plate.

Osteonecrosis of the femoral head usually affects young individuals and is responsible for up to 12% of total hip arthroplasties. The underlying pathophysiology of the death of the bone cells remains uncertain. We have investigated nitric oxide mediated apoptosis as a potential mechanism and found that steroid- and alcohol-induced osteonecrosis is accompanied by widespread apoptosis of osteoblasts and osteocytes. Certain drugs or their metabolites may have a direct cytotoxic effect on cancellous bone of the femoral head leading to apoptosis rather than purely necrosis.

Using in situ hybridisation and the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling (TUNEL) reaction in rats with osteonecrosis of the femoral head we have studied the effect of ischaemia on the gene expression of the stress proteins oxygen-regulated protein 150 (ORP150) and haemoxygenase 1 (HO1) and the death mechanism of the cells involved in osteonecrosis. Both ORP150 and HO1 have been reported to have important roles in the successful adaptation to oxygen deprivation.

ORP150 and HO1 mRNA expression was induced by ischaemia in osteoblasts and osteocytes. In proliferative chondrocytes, these signals were detected constitutively. During the development of ischaemic osteonecrosis, the mechanism of cell death was apoptosis as indicated by DNA fragmentation and the presence of apoptotic bodies in osteocytes, chondrocytes and bone-marrow cells. After the initial ischaemic event, expression of ORP150 and HO1 mRNA, the TUNEL-positive reaction and empty lacunae were found sequentially. These findings were exclusive and may be considered to be markers for each stage in the development of osteonecrosis.

Mitochondrial dysfunction has been demonstrated in aging and osteoarthritic tissues. We investigated knee joints of prematurely aging mitochondrial DNA mutator mice (PolgD275A) to evaluate a relationship between mitochondrial dysfunction and osteoarthritis. Cartilage damage was evaluated using OARSI histopathology grading and osteoclast numbers were quantified by tartrate-resistant acid phosphatase staining in wild type, heterozygous and homozygous PolgD275A mice. Subchondral cortical plate and epiphyseal trabecular bone structures were determined by micro-computed tomography. Apoptosis in cartilage and subchondral bone tissues was studied using an indirect TUNEL method. Homozygous mutants displayed osteopenia of the epiphyseal trabecular bone and subchondral cortical plate in comparison to wild type and heterozygous mutants. Subchondral osteopenia was associated with a strong increase of osteoclast numbers (0.88±0.30/mm bone perimeter) compared to heterozygous (0.25±0.03/mm) and wild type mice (0.12±0.04/mm). Wild type mice as well as hetero- and homozygous mutants displayed low-grade cartilage degeneration due to loss of cartilage proteoglycans. In contrast, chondrocyte hypertrophy was more abundant in the homozygous mice. There were no differences in chondrocyte apoptosis rates between groups. Prematurely ageing mtDNA mutator mice with or without further mechanic or metabolic stimuli might serve as a valuable model for further experimental studies on aging-induced osteoporotic OA phenotype

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff. We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis). The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears. These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes. We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic. We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain. Cite this article: Bone Joint J 2014; 96-B:989–94

Osteoarthritis (OA) is a degenerative disease with a strong inflammatory component. Intra-articular (IA) injections of mesenchymal stem cells (MSCs) modulate local inflammation, although the lack of engraftment suggests that they undergo apoptosis. The aim of this study is to investigate the fate of IA-delivered MSCs in an animal model of OA and to assess the role of apoptosis in vitro. Collagenase-induced OA (CIOA) was performed on C57BL/6 mice and 2×10∧5 GFP+ MSCs were IA-injected in the animals. 3 days later, knee joints were digested into a single-cell suspension and MSCs retrieved by cell sorting. Conditioned medium (CM) of retrieved cells was tested on murine macrophages and cytokine secretion was measured. Apoptosis of MSCs was induced in vitro with staurosporine (STS) and evaluated by Annexin V/Sytox Blue staining; activation of caspases was measured by FLICA assays. Murine lymphocytes were cocultured with apoptotic MSCs and their proliferation measured by quantification of Cell Trace Violet. 1.63% of injected cells were retrieved and proliferated in culture. Their CM significantly modulated activation of macrophages, with greater effects from OA-induced MSCs. STS induced apoptosis with activation of Caspase 3/7. Apoptotic MSCs significantly prevented the proliferation of murine lymphocytes. MSCs can be administered and retrieved from murine knees. Retrieval yield is low, consistent with previous studies. MSCs were licensed from the OA joint to produce an immunosuppressive milieu that modulated macrophages ex vivo. In vitro, apoptosis increased the immunomodulatory potential of MSCs. This suggests that apoptosis may contribute to the therapeutic effects of MSCs in OA

Summary Statement. The effects of local glucocorticoid on tendon appear broadly negative and this supports the emerging clinical evidence which points toward significant long term harms associated with this treatment modality. Introduction. The use of locally administered glucocorticoid is widespread in the treatment of painful tendinopathy. Despite evidence of short term benefit, the emerging evidence points toward significant long term harms associated with this method of treatment, including an increased risk of recurrence, rupture and worsened clinical outcomes (1, 2). Our primary purpose was to summarise the known effects of locally administered glucocorticoid on tendon tissue and tendon cells. Methods. We conducted a systematic review of the scientific literature using the PRISMA and Cochrane guidelines of the Medline database using specific search criteria. Only studies analysing the effects of locally administered glucocorticoid on tendon tissue or tendon cells with adequate controls were included. Specific attention was paid to histological and biomechanical findings. Inclusion was agreed upon by two independent researchers after review of abstracts or full text. The search yielded 4424 results, of which 42 met the inclusion criteria. The final 42 articles consisted of 13 human in vitro studies, 15 animal in vivo studies and 14 animal in vitro studies. Results. Due to study heterogeneity, statistical pooling or meta-analysis of data was not possible. The results are therefore described qualitatively. Histologically, there was a loss of collagen structure (5 studies) and an increase in collagen necrosis (4 studies). The proliferation and viability of fibroblasts was reduced (11 studies). An increased inflammatory cell infiltrate was shown in 3 animal in vivo studies, while an increased fibroblast infiltrate was seen in 2 studies. Fibroblast migration was reduced in 2 in vitro studies. Collagen synthesis was reduced in 13 studies. An increased ratio of type 3 to type 1 Collagen was shown in 2 studies. Apoptosis was unaffected in 2 studies. 19 studies investigated the mechanical properties of tendon. Of these 7 showed deterioration in mechanical properties, 4 showed an improvement and 8 showed no difference. Discussion/Conclusion. Overall it is clear that the local administration of glucocorticoid has significant negative effects on tendon cells in vitro, such as reduced cell viability, cell proliferation and collagen synthesis. There is increased collagen disorganisation and necrosis as shown by animal in vivo studies. The mechanical effects are equivocal. This review supports emerging clinical evidence showing significant long term harms associated with glucocorticoid injections. There is clearly a significant need for better designed human trials with appropriate blinding and control arms to investigate the effects of glucocorticoid on both clinical outcomes and characteristics of tendon tissue

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect.

Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1.

Objectives

The cytotoxicity induced by cobalt ions (Co²⁺) and cobalt nanoparticles (Co-NPs) which released following the insertion of a total hip prosthesis, has been reported. However, little is known about the underlying mechanisms. In this study, we investigate the toxic effect of Co²⁺ and Co-NPs on liver cells, and explain further the potential mechanisms.

We attempted to repair full-thickness defects in the articular cartilage of the trochlear groove of the femur in 30 rabbit knee joints using allogenic cultured chondrocytes embedded in a collagen gel. The repaired tissues were examined at 2, 4, 8, 12 and 24 weeks after operation using histological and histochemical methods. The articular defect filling index measurement was derived from safranin-O stained sections. Apoptotic cellular fractions were derived from analysis of apoptosis in situ using TUNEL staining, and was confirmed using caspase-3 staining along with quantification of the total cellularity. The mean articular defect filling index decreased with time. After 24 weeks it was 0.7 (sd 0.10), which was significantly lower than the measurements obtained earlier (p < 0.01). The highest mean percentage of apoptotic cells were observed at 12 weeks, although the total cellularity decreased with time. Because apoptotic cell death may play a role in delamination after chondrocyte transplantation, anti-apoptotic gene therapy may protect transplanted chondrocytes from apoptosis.

Objectives

To investigate the appropriate dose and interval for the administration of triamcinolone acetonide (TA) in treating tendinopathy to avoid adverse effects such as tendon degeneration and rupture.

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.

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups.

These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic.

Post-traumatic arthritis is a frequent consequence of articular fracture. The mechanisms leading to its development after such injuries have not been clearly delineated. A potential contributing factor is decreased viability of the articular chondrocytes. The object of this study was to characterise the regional variation in the viability of chondrocytes following joint trauma. A total of 29 osteochondral fragments from traumatic injuries to joints that could not be used in articular reconstruction were analysed for cell viability using the fluorescence live/dead assay and for apoptosis employing the TUNEL assay, and compared with cadaver control fragments.

Chondrocyte death and apoptosis were significantly greater along the edge of the fracture and in the superficial zone of the osteochondral fragments. The middle and deep zones demonstrated significantly higher viability of the chondrocytes. These findings indicate the presence of both necrotic and apoptotic chondrocytes after joint injury and may provide further insight into the role of chondrocyte death in post-traumatic arthritis.