Background. Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes. Methods. Normal and OA chondrocytes were incubated with various concentrations of resveratrol (1 µM, 10 µM, 25 µM and 50 µM) and cultured for 24, 48 or 72 hours or for six weeks. Cell proliferation, gene expression, and senescence were evaluated. Results. SIRT1 was significantly upregulated in normal chondrocytes with resveratrol concentrations of 25 µM and 50 µM on both two- (2D) (both p = 0.001) and three-dimensional (3D) cultures (p = 0.008 and 0.001, respectively). It was significantly upregulated in OA chondrocytes treated with 10 µM, 25 µM and 50 µM resveratrol on 2D cultures (p = 0.036, 0.002 and 0.001, respectively) and at 50 µM concentration on 3D cultures (p = 0.001). At 72 hours, the expression of collagen (COL)-10, aggrecan (AGG), and runt-related transcription factor 2 (RUNX2) was significantly greater in both 25 µM (p = 0.011, 0.006 and 0.015, respectively) and 50 µM (p = 0.019, 0.004 and 0.002, respectively) resveratrol-treated normal chondrocyte cultures. In OA chondrocytes, expression of COL10 and RUNX2 was significantly greater in 25 µM (p = 0.004 and 0.024) and 50 µM (p = 0.004 and 0.019) cultures at 72 hours on 3D cultures. Conclusions. At concentrations of 25 µM and/or 50 µM, resveratrol treatment significantly upregulates SIRT1 gene expression in normal and osteoarthritic chondrocytes. Resveratrol induces chondrocytes into a hypertrophic state through upregulation of COL1, COL10, and RUNX2. Cite this article: Bone Joint Res 2014;3:51–9

The ability of the body to constantly maintain metabolism homeostasis while fulling the heightened energy and macromolecule demand is crucial to ensure successful tissue healing outcomes. Studies investigating the local metabolic environment during healing are scarce to date. Here, using Type 2 Diabetes (T2D) as a study model, we investigate the impact of metabolism dysregulation on scaffold-guided large-volume bone regeneration. Our study treated wild-type or T2D rats with 5 mm critical-sized femoral defects with 3D-printed polycaprolactone (PCL) scaffolds with 70% porosity. Metabolomics was leveraged for a holistic view of metabolism alteration as healing progress and correlated to regenerated bone tissue volume and quality assessed using micro-computed tomography (µ-CT), histology, and immunohistology. Semi-targeted metabolomics analysis indicated dysregulation in the glycolysis and TCA cycle – the main energy production pathways, in T2D compared to healthy animals. The abundance of metabolites substrates, i.e., amino acids – for protein/ extracellular matrix synthesis was also affected in T2D. Tissue-level metabolites observations aligned with morphological observation with less newly formed bone observed in T2D than wild-type rats. This study enlightens the metabolism landscape during scaffold-guided large-volume bone regeneration in wild-type vs. T2D to further guide the personalization of the scaffold to drive successful regeneration

Aims. Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy. Methods. The human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP. Results. The S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts. Conclusion. Intracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage. Cite this article: Bone Joint Res 2022;11(5):327–341

Healthy bone metabolism is a tightly coupled dynamic process that relies on a balance between bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Traditionally, tissue-engineering approaches for non-union fracture repair employ local anabolic therapeutic delivery strategies that target mesenchymal stem cells (MSCs) and osteoblasts to induce bone formation, however, the challenge of healing non-union defects depends on the cause of defect e.g. trauma or disease, and targeting bone formation alone is often not sufficient. Our research focuses on utilising both anabolic therapeutics, including recombinant human bone morphogenic protein (rhBMP) −2 and parathyroid hormone (PTH). (1–34). , and anti-catabolic bisphosphonates (BPs) to target bone metabolism. A major challenge with harnessing a combined dosing regimen is controlling the release of the individual therapeutics to target cells. We have developed a number of polymer-ceramic based biomaterial delivery systems, including injectable and implantable scaffolds, for the controlled release of rhBMP-2 and the BP zoledronic acid (ZA) and demonstrated their efficacy in vivo. A dual therapeutic load provided a synergistic enhancement of bone regeneration, demonstrating significantly increased bone formation and remodelling compared to anabolic therapies alone. Utilising hydroxyapatite as the ceramic phase in our scaffolds further increased bone formation, demonstrating the polymer-ceramic scaffolds to be osteoconductive in the absence of therapeutics. In addition, we have demonstrated the manipulation of bone metabolism through a specific dosing regimen of PTH. (1–34). , a therapeutic traditionally used as an anabolic, to induce bone remodelling and drive healing in BP loaded fractures. Our research to date has shown that optimising the delivery and regimen of anabolic and anti-catabolic therapeutics to control bone metabolism, augments the bone regenerative potential of these therapeutics in orthopaedic applications

Previous studies have described an age-dependent distortion of bone microarchitecture for α-CGRP-deficient mice (3). In addition, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from young wildtype (WT) mice when stimulated with α-CGRP whereas loss of α-CGRP showed only little effects on bone cell metabolism of cells isolated from young α-CGRP-deficient mice. We assume that aging processes differently affect bone cell metabolism in the absence and presence of α-CGRP. To further explore this hypothesis, we investigated and compared cell metabolism of osteoblasts and bone marrow derived macrophages (BMM)/osteoclast cultures isolated from young (8–12 weeks) and old (9 month) α-CGRP-deficient mice and age matched WT controls. Isolation/differentiation of bone marrow macrophages (BMM, for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from young (8–12 weeks) and old (9 month) female α-CGRP−/− and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (crystal violet staining), proliferation (BrdU incorporation) and caspase 3/7-activity (apoptosis rate). Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and counting of multinucleated (≥ 3 nuclei), TRAP (tartrate resistant acid phosphatase) stained osteoclasts reflects osteoclast differentiation capacity. We counted reduced numbers of BMM from young α-CGRP−/− mice after initial seeding compared to young WT controls but we found no differences between old α-CGRP−/− mice and age-matched controls. Total BMM number was higher in old compared to young animals. Migration of osteoblast-like cells out of bone chips was comparable in both, young and old α-CGRP−/− and WT mice, but number of osteoblast-like cells was lower in old compared to young animals. Proliferation of old α-CGRP−/− BMM was higher when compared to age-matched WT whereas proliferation of old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation was lower. No differences in bone cell proliferation was detected between young α-CGRP−/− and age-machted WT mice. Caspase 3/7 activity of bone cells from young as well as old α-CGRP−/− mice was comparable to age-matched controls. Number of TRAP-positive multinucleated osteoclasts from young α-CGRP−/− mice was by trend higher compared to age-matched WT whereas no difference was observed in osteoclast cultures from old α-CGRP−/− mice and old WT. ALP activity, as a marker for bone formation activity, was comparable in young WT and α-CGRP−/− osteoblasts throughout all time points whereas ALP activity was strongly reduced in old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation compared to age-matched WT. Our data indicate that loss of α-CGRP results in a reduction of bone formation rate in older individuals caused by lower proliferation and reduced activity of osteogenic cells but has no profound effects on bone resorption rate. We suggest that the osteopenic bone phenotype described in aged α-CGRP-deficient mice could be due to an increase of dysfunctional matured osteoblasts during aging resulting in impaired bone formation

Intervertebral disc cells exsist in a precarious nutritional environment. Local concentrations depend on both nutritional supply and demand. Little is known about the metabolism of disc cells; existing data focuses on intact tissue, where the local metabolic environment is unknown. We have thus developed a closed chamber to study the metabolism of isolated cells under controlled conditions. Bovine disc cells were isolated from coccygeal discs and transferred to the sealed chamber, in which embedded electrodes measured pH, pO2 and glucose concentration, and a port allowed sampling and addition of metabolic reagents. Metabolic rates were assessed from concentration changes. Cell viability was assessed and intracellular ATP measured at completion of each experiment. Under standard conditions, metabolic rates were similar to those measured in tissue, with a glucose:lactic acid ratio of approximately one to two. We have also examined the effect of extracellular pH on nucleus pulposus cell metabolism. Between pH 7.4–6.8, metabolism is insensitive to extracellular pH, and lactic acid production agrees with the literature . 1. , . 2. Below pH 6.8, lactic acid production fell linearly with decreased pH. At pH 6.4, lactic acid production had fallen by 60%, and intracellular ATP by 80%. These results show a fall in lactic acid production with extracellular acidification, which in vivo arises mainly from lactic acid produced by the cells. This may be protective. However the decrease in metabolism, and hence loss of ATP, may have a detrimental effect on the cells. There is thus a complex interplay between different components of the nutritional environment. Investigating these in combination should give valuable information about disc cell metabolism, as changes in cells metabolism can affect nutrient availability and hence cellular activity and viability

Purpose of the study. To assess the ability of magnetic resonance spectroscopy (MRS) to detect changes in spinal muscle metabolism after a 4-week exercise intervention. Background. Spinal muscle atrophy is associated with back pain and exercise interventions have been shown to reduce pain and improve function. It is not always clear, however, whether improvements are due to enhanced muscle performance or occur for other reasons (e.g. psychological, neurological). MRS can be used to measure muscle metabolism and could therefore be useful for assessing the mechanisms by which exercise improves function in back pain patients. Methods. Eleven healthy participants took part in a 4-week exercise intervention to strengthen the spinal muscles. Before and after the intervention, the participants underwent an assessment that included using MRS to monitor the phosphocreatine levels in the spinal muscles around the level of L3L4 whilst they performed a modified Biering-Sorensen test to fatigue. Results. Relative to the pre-intervention assessment, the post-intervention endurance time significantly increased (mean=20 s, 95% CI 7–34 s, p=0.01). The phosphocreatine depletion, taken at an equivalent time point in both assessments, significantly decreased (mean=12%, 95% CI 5–19%, p=0.006). Even at the point of the fatigue, the phosphocreatine depletion was lower post-intervention (mean=8%, 95%CI 1–15%, p=0.045). Conclusion. MRS can be used to detect changes in the metabolism of the spinal muscles after a 4-week exercise intervention

Introduction. Some patients complain ingrown pain or discomfort after implanting Co-Cr conventional endprosthesis of the hip. Some of this complaint may be attributable for effect on cartilage metabolism. It have been reported that ceramic is bioinert for biological tissue. On the other hand, metal including cobalt-chrome (Co-Cr) have some detrimental effect on biological tissue. However, there is no report concerning acetabular cartilage metabolism after hip endprosthesis implantation. In the present study, we hypothesized that ceramic head have small detrimental effect on cartilage cell metabolism. Specific aim of the study is to compare the protein level of inflammation related cytokines, amount of hyaluronic acid (HA) in culture media, and cartilage mRNA expression in organ culture model of hip end prosthesis implanted using ceramic head and Co-Cr head. Materials and Methods. Six acetabulum of 3 matured crossbred pig (average weight: 36 +/− 3.6kg) was retrieved. Animal experiment was performed under the rules of ethical committee of animal experiment. Average diameter of pig acetabulum was 26.3 +/− 0.6 mm. Just after sacrifice, mechanical loading using Instron testing machine with 26mm diameter of Co-Cr in right hip and Ceramic heads in left hip was performed in culture media. Ten thousand cycles of cyclic compression and rotation load (1.5kN to 0.15kN of compression and 12 degrees of rotation) to cartilage was applied at 1Hz (Figure 1). Culture media was analyzed for protein levels of inflammation related cytokines and amount of HA. Relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) from acetabular cartilage was performed as previously reported using specific primer sets for type II collagen, aggrecan, TNF-alpha, Interleukine-1 and 6, and MMP-1, 3, 13. Results. IL-1 beta protein level from culture media was significantly higher in Co-Cr than that in Ceramic (155+/−25.2 pg/ml vs. 86.3+/−9.6 pg/ml respectively). MMP-3 protein level had tendency to be higher in culture media from Co-Cr than that from Ceramic (16.3+/−10.6 ng/ml vs. 10.0+/−0.1 ng/ml respectively, p<0.05), however there was no significant difference. There were no significant differences of protein levels from culture media in MMP-1, IL-1a, and TNF between two groups. Amount of HA from culture media of Co-Cr group was significantly higher than that from Ceramic group (337+/−38.4 mg/ml versus 257+/−11.1 mg/ml respectively, p<0.05). Type II collagen mRNA expression was 3 times higher in Ceramic group than that in Co-Cr group. IL-1 beta mRNA expression was 4 times higher in Co-Cr group than that in Ceramic group. Other gene expression had no significant differences. Discussion. The present study showed that Co-Cr affects cartilage metabolism than Ceramic. Co-Cr group had higher protein level and mRNA expression of inflammation related cytokine, IL-1 beta, and higher HA. Concerning the mRNA expression from cartilage, type II collagen was significantly higher in Ceramic group. It has been reported that HA level is high in osteoarthritic joint. These report and our results showed that ceramic head have small detrimental effect on cartilage cell metabolism. There are limitations of the present study. Firstly, the sample size is small. Secondly, we did not evaluate synovial membrane metabolism

Aim To determine if tissue metabolism varies in supraspinatus tendons with distance from the edge of the rotator cuff tendon tear and also with differing size of tear. Background Tissue metabolism can be assessed by measuring oxygen and nitrous oxide concentrations within the tissue. Viable tendon tissue consumes oxygen and contains nitrous oxide (used in the general anaesthetic) from the blood stream. Non-viable tendon tissue will not consume oxygen but will contain nitrous oxide. Methods Oxygen and Nitrous Oxide concentrations were measured amperometrically using silver needle microelectrodes. The needle was inserted into the supraspinatus tendon of patients with massive, large, medium and small full thickness rotator cuff tears and patients with partial thickness and no tears. Patients undergoing open stabilisation were used as controls. Measurements were made at a number of quantifiable points from the tendon edge to allow the creation of a topographical map of tissue metabolism. Oxygen consumption was calculated using measured oxygen and nitrous oxide levels at each point. Results In patients with rotator cuff tears oxygen consumption was significantly lower near to the edge of the tear. Patients with impingement syndrome but no evidence of a rotator cuff tear also showed a decreased level of oxygen consumption in the anterior part of supraspinatus, but this was significantly higher than the levels seen in the torn tendon. The control group showed no significant alteration in oxygen levels. Conclusion Patients with rotator cuff tendon tear demonstrate significantly reduced levels of tissue metabolism. This reduction in tissue viability is significantly greater at the edge of the tear and in larger tears. Patients with intact tendons and impingement syndrome also demonstrate minor reduction in tissue tendon viability compared with controls

Introduction: Kashin-Beck disease (KBD) is a special endemic osteoarthropathy whose main pathologic changes occur in growth plate cartilage and articular cartilage of human limbs and joints where it is manifested as cartilage degeneration and necrosis. Past and current research suggests that KBD, and its endemic geographic distribution in China, is due to the combined presence of fungal mycotoxins (on stored food ingested by affected populations) and a regional selenium deficiency in the environment providing local food sources. Thus, we hypothesise that the presence of fungal mycotoxins and the absence of selenium in the diet specifically affects chondrocyte metabolism in the growth plate during limb and joint development and in articular cartilage of adults, which leads to localised tissue necrosis, and the onset of degenerative joint disease. The aim of this study was to examine the effects of mycotoxins; e.g. Nivalenol (NIV), selenium and NIV in the presence of selen! ium in in vitro chondrocyte culture systems to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD. Methods: Chondrocyte tissue cultures were established using cartilage explant cultures either in the presence or absence of selenium (0.5–1.5 microg/ml) and the mycotoxin nivalenol (0.5–1.5 microg/ml) and culture for 1 to 4 days. Medium was harvested daily at day 1 through 4 and analysed for glycosaminoglycan (GAG) release and the presence of aggrecanase or MMP activity using RT-PCR for gene expression and monoclonal antibodies that detect their respective enzyme-generated neo-epitopes on cartilage aggrecan metabolites. Results: Our studies to date have shown that NIV exposure induces catabolic changes in chondrocyte metabolism with an increased expression of aggrecanase activity. Addition of selenium did not affect mRNA expression of the aggrecanases ADAMTS-4 & 5. Parallel studies involving immunohistochemical analyses of articular cartilage from KBD showed an increase in aggrecanase activity. Conclusions: These studies demonstrate that induction of aggrecanase activity as one of the molecular mechanisms involved is the pathogenesis of KBD. However, the addition of selenium does not alter aggrecanase gene expression indicating that its beneficial effects are occurring in other areas of cartilage metabolism

Introduction: Loss of nutrient supply, seen in disc degeneration, leads to low concentrations of oxygen and glucose in the centre of the disc. Here we investigate the effect of low nutrient concentrations on the metabolism and viability of the nucleus cells. Methods: Isolated bovine nucleus pulposus cells were cultured for 24–72 hrs over a range of pH levels and glucose and oxygen concentrations. Changes in metabolite concentrations with time were measured in a purpose-built chamber using embedded electrodes, or biochemically; and metabolic rates determined. On completion, cell numbers were counted and viability assessed. Results: Metabolic rates varied both with oxygen concentration and with pH. At low oxygen (2% pO. 2. ) and low pH (pH 6.2) for example, oxygen consumption rates and lactic acid production rates were 10–30% those in air at pH 7.4. Low pH in air saturated medium, or low pO2 in neutral medium, reduced metabolism but not as drastically. Glucose concentrations in the range 0.5–5mM in contrast did not affect cellular metabolism. Cells could survive with zero oxygen, although metabolism was seriously dimished; but after 24 hours at low (< 0.5mM) glucose, cell death was observed. Discussion: Regulation of the concentrations of nutrients in vivo is complex, and depends on both supply and demand. Little is known about cellular demand, and studies such as this could give insight into the situation in the disc in vivo and help determine the cellular consequences of a fall in nutrient supply. Our results, apart from showing the deleterious effects of low nutrient concentrations, also indicate that isolated cells may metabolise differently from cells in the tissue; at low pO. 2. we observed a fall in lactate production, the opposite effect to that seen in tissue previously. The mechanism for this difference is as yet unknown

Previously, we have demonstrated reduced biomechanical bone strength and matrix quality in Tachykinin (Tac)1-deficient mice lacking the sensory neuropeptide substance P (SP). A similar distortion of bone microarchitecture was described for α-calcitonin gene-related pepide (α-CGRP)-deficient mice. In previous studies we observed alterations in cell survival and differentiation capacity of bone cells isolated from wildtype mice when stimulated with SP and α-CGRP. We assume that changes in sensory neurotransmitter balance modulate bone cell metabolism thereby possibly contributing to inferior bone quality. In order to explore this hypothesis, we investigated and compared metabolic parameters in osteoblasts and osteoclasts isolated from SP- and α-CGRP-deficient mice and wildtype (WT) controls. Bone marrow-derived macrophages (BMMs) and osteoblast-like cells from female C57Bl/6J (WT-control), Tac1-deficient (Tac1-/−) and α-CGRP-deficient (α-CGRP-/−) mice were isolated and differentiated according to established protocols (Niedermair et al., 2014). Cell metabolism studies were performed for enzyme activity and cell survival. We observed reduced numbers of BMM from Tac1-/− and α-CGRP-/− mice after initial seeding compared to WT but no changes in viability. Osteoblast-like cells from Tac1-/− mice tend to migrate out faster from bone chips compared to WT-controls whereas migration of osteoblast-like cells from α-CGRP-/− mice was not affected. Osteoblasts and osteoclast/BMM cultures from WT mice endogenously synthesize and secrete SP as well as α-CGRP at a picomolar range. We found no changes regarding BMM or osteoblast proliferation from both, Tac1-/− and α-CGRP-/− mice when compared to WT-controls. Caspase 3/7-activity was reduced by trend in osteoclast/BMM cultures of α-CGRP-/− mice and significantly reduced in osteoclast/BMM cultures of Tac1-/− mice compared to WT-controls. We found significantly higher Caspase 3/7-activity in osteoblasts of Tac1-/− mice after 14 days of osteogenic culture conditions when compared to WT-controls whereas osteoblasts of α-CGRP-/− mice were unaffected. Cathepsin K enzyme activity was significantly reduced in osteoclast/BMM cultures of Tac1-/− and α-CGRP-/− mice compared to WT-controls. ALP activity of Tac1-/− osteoblasts was higher after 7 days and reduced after 21 days of osteogenic culture compared to WT-controls whereas ALP activity of osteoblasts of α-CGRP-/− mice was unchanged. Acccording to our in vitro observations, we suggest some reduction in bone resorption rate but concomitantly a reduction in bone formation rate in Tac1-/− mice compared to WT-controls resulting in a net bone loss in these mice as bone resorption is faster than bone formation. Furthermore, we assume that bone resorption rate is slightly reduced in α-CGRP-/− mice but bone formation rate seems to be unchanged. Therefore we hypothesize that additional conditions present in vivo might contribute to the inferior bone properties of α-CGRP-/− mice

It was aimed to investigate the isolated effect of hydrostatic pressure on chondrocyte metabolism. Chondrocytes obtained from bovine metatarso-phalangeal joints were cultured in cylindrical 2% agarose gels. A special apparatus which was designed and constructed, allowed the application of hydrostatic pressure of either 2 MPa or 5 MPa on the chondrocytes for 4 hours either in a pulsatile (1Hz) or a static manner. Changes in the syntheses of glycosaminoglycan (GAG) and DNA during and after the application of the hydrostatic pressure were analysed with . 35. S-sulphate and . 3. H-thymidine incorporation, respectively. Radiolabelling was carried out for the following conditions: (a) 4 hours during the application of hydrostatic pressure; (b) 4 hours and (c) 20 hours immediately after the application of load. In addition, the experiments were carried out at 2 days, 7 days and 14 days after embedding the chondrocytes in agarose gels. Static hydrostatic pressure of 5 MPa caused a significant increase by 13% on average in the GAG synthesis during the load application on Day 2 7 and 14 (p < 0.05). On the contrary, pulsatile pressure of 2 MPa caused a significant decrease by 17% in the GAG synthesis measured at 20 hours after the loading on Day 14 (p < 0.01). In addition, there was a significant decrease by 29% in the DNA synthesis measured at 4 hours after the pulsatile loading of 5 MPa on Day 7 (p < 0.01). The results suggest that hydrostatic pressure alone, which causes no cell deformation, can affect the GAG synthesis and proliferation of chondrocytes. In addition, the effect of hydrostatic pressure on the chondrocyte metabolism varies according to the regimes of loading and with the period of cell culture

In the past the prevailing view believed that there was an inverse relationship between osteoarthritis and osteoporosis; a recent study showed that elderly women with advanced osteoarthritis requiring total hip replacement had an evidence of osteoporosis and vitamin-D deficiency. An altered metabolic bone status as induced by low level of vitamin D could be one of the major causes of aseptic bone loosening and consequently failure of the implant. We studied the bone mineral metabolism of thirty elderly women with osteoarthritis undergoing total hip replacement in order to identify whether or not there were a bone metabolic alterations. All the subjects included in the study were over than 70 years old (mean age 74 ± 2.5). The results showed that six (20%) subjects had a hypovitaminosis D status and eighteen (60%) had a vitamin D deficiency status. Five subjects (16%) had a secondary iperparathyroidism. The bone mineral metabolism of elderly women with osteoarthritis undergoing total hip replacement is characterised by a high prevalence of vitamin D deficiency and in a less percentage of the cases by a secondary iperparathyroidism. Both of these metabolic conditions could compromise the bone integration of the implant and lead to aseptic bone loosening

Objective: To develop and use a closed chamber to study the metabolism of isolated disc cells under controlled conditions such as reduced pH. Design: Disc cells were incubated in the chamber for four hours, while embedded electrodes measured pH and pO2. A port allowed sampling. Subjects: Nucleus pulposus cells were isolated from the coccygeal discs of 33 steers (18–24 months old), within three hours of slaughter. Outcome measures: Metabolic rates were calculated from concentration changes. Cell viability was assesed on completion. Results: At pH 7.4, metabolic rates were similar to those measured in tissue [. 1. , . 2. ] with lactic acid production and oxygen consumption rates of 157 and 12 nmol/million cells/hour respectively, and a 1: 2 ratio of glucose consumption: lactic acid production. Lactic acid production and oxygen consumption fell with extracellular pH, to 89 and 65 nmol/million cells/hour (lactate) and 8 and 5 nmol/million cells/hour (oxygen), at pH 6.7 and pH 6.2 respectively. Conclusions: These results show a fall in lactic acid production and oxygen consumption with extracellular acid-ification. There is a complex interplay between different components of the nutritional environment. Investigating these in combination should give valuable information about disc cell metabolism, as changes can affect nutrient availability and hence cellular activity, viability, and matrix production rates

Introduction: The mitochondrial Translocator Protein 18 kDa (TSPO, previously named as the peripheral benzodiazepine receptor - PBR) is involved in cellular respiration, steroidogenesis and apoptosis. In our recent study we reported on the role of the synthetic pharmacological ligands to the TSPO in enhancing human osteoblast catabolism. There is also a previous evidence of the existence of an endogenous ligands to the TSPO, but their role in the human osteoblast physiology hasn’t been verified yet. Porphyrine IX has been found having affinity to the TSPO. Therefore we hypothesize that human osteoblast metabolism might be mediated by the porphyrine IX and the mode of its action is similar the synthetic ligand to the TSPO. Methods: Cell cycle of the cultured human derived osteoblast- like cells, following exposure to Porphyrine IX, endogenous ligand to TSPO, and N,N-di-n-hexyl 2-(4- fluorophenyl)indole-3-acetamide (FGIN-1–27), synthetic ligand to the TSPO, was determined by flow cytometry (FACS). These ligands’ affect on cell number, metabolic activity, i.e. cellular fluorodeoxyglucose ([. 18. F]-FDG) incorporation and alkaline phosphatase activity, and cell death rate, i.e. LDH activity in the culture media, were assayed. The semi-quantitative response of TSPO to exposure to these ligands was estimated by Western blotting. Six samples of cultured cells for each condition were used. The t test was implemented for the statistical analyses. P values below.05 considered as statistically significant. Results: Cell count significantly decreased following exposure to FGIN-1–27 or porphyrine IX. Cellular [. 18. F]-FDG incorporation and alkaline phosphatase activity were suppressed by both ligands. Cell cycle analysis showed a significant decrease in the fraction of cells in the G1 and G2/M phases when exposed to each ligand with a higher proportion of necrotic and apoptotic cells. Western blotting showed a decrease in TSPO abundance following treatment by both ligands. LDH activity in culture media significantly increased following exposure to FGIN-1–27 or porphyrine IX. Discussion: We show that FGIN-1–27 and porphyrine IX have a similar cell death inducing affect on human osteoblast-like cell in vitro. This affect is parallel to the inhibition of the cellular metabolism. Since both ligands similarly reduce the availability of TSPO we postulate that their mode of action is similar by affecting this mitochondrial structure with sub sequential induction of cell death, i.e. apoptosis and necrosis. Therefore we suggest that human osteoblast metabolism and cell cycle are mediated through TSPO and that porphyrine IX might be an active endogenous ligand to the TSPO having a regulatory affect on the human bone cell cycle

Introduction: Previous studies have demonstrated that exposure of normal bovine and human osteoarthritic cartilage to n-3 polyunsaturated fatty acids (PUFAs) such as those present in fish oils can modulate the expression and activity of the degradative and inflammatory factors that are responsible for cartilage destruction [. 1. ,. 2. ]. In these studies, supplementation of cartilage explant cultures with n-3 PUFAs resulted in an abrogation of aggrecanase activity as well as mRNA expression of mediators of inflammation. To date, few studies have examined the effect of PUFAs on the metabolism of other tissues within the musculoskeletal system, therefore the present work examines the effect of n-3 PUFA supplementation on tendon metabolism. Methods: Bovine deep digital flexor tendon explants were obtained from the compressed region of young metacarpophalangeal joints (2-week-old) and supplemented with eicosapentaenoic acid (EPA), as previously described [. 2. ]. Release of proteoglycan metabolites was analysed using Western blotting whilst RT-PCR analysis was used to examine the mRNA expression patterns of matrix proteases and inflammatory agents. Results: Exposure to the n-3 fatty acid, EPA, markedly changed the overall lipid composition profile of the tendon with major changes occurring in the supplemented fatty acid (i.e., EPA), with a concomitant percentage reduction in other polyunsaturated fatty acids. Aggrecanase activity was present in the media from control cultures, as expected [. 3. ]. However, supplementation with EPA had no effect on this activity, in contrast to articular cartilage where aggrecanase catabolites were absent from the conditioned media following treatment with n-3 PUFAs [. 1. ,. 2. ]. mRNA expression for the inflammatory mediators (COX-2, IL-1β, TNF), ADAMTS-5, MMPs and TIMPs was also unchanged following supplementation with EPA, again contrasting with articular cartilage where mRNA expression was abolished. Discussion: This study demonstrates that exposure of bovine tendon explant cultures to an n-3 PUFA, EPA, had no effect on the mRNA expression or activity of aggrecanases; similarly, expression of the inflammatory mediators was also unaffected. Importantly, within this musculoskeletal tissue, aggrecanases are constitutively active and appear to be involved in normal, everyday turnover of aggrecan, in contrast to non-pathological articular cartilage where aggrecanase-generated metabolites are only detected following treatment with catabolic agents. Similarly, COX-2 mRNA expression is present constitutively within tendons whereas in cartilage it is absent under basal (unstimulated) conditions. These data demonstrate that the incorporation of n-3 PUFAs have a differential effect on the regulatory mechanisms which control gene expression within articular cartilage versus tendon

Purpose: There is interest in biologic strategies that can potentially treat degenerative disc disease (DDD). A new deacetylated derivative of a marine diatomic glycosaminoglycan (DEAC) was developed and incorporated into two sulphated hydrogel formulations; Gel 1 and 2. These materials were proposed to have a reparative effect on damaged tissue. Biochemical studies were conducted using primary human disc cell (HDC) cultures. Method: HDCs were isolated from surgical specimens by sequential enzymatic digestion (pronase and collagenase). Time-course in-vitro studies were conducted on cell cultures treated with DEAC, Gel 1 or Gel 2 (28 day period). Proteoglycan content (alcian blue), cellular viability/proliferation (MTT assay), and type collagen II, aggrecan expression (RT-PCR, immunohistochemistry) was assessed. Results: When compared to controls, the DEAC, Gel 1 and 2 treated HDC groups showed significant increases in proteoglycan content as early as day 12. The greatest effect was observed with Gel 1 (78.4±1.9 fold greater optical density compared to control, p < 0.05). The amount of proteoglycan quantified on DEAC treated HDCs on day 28 was 27.7±0.09 times higher than control (p< 0.05). MTT results demonstrated that Gel 1 group showed the highest viability over the study period (mean optical density 0.13+.01 versus 0.039+0.01 in controls). There were no significant differences in cell proliferation of Gel 2, DEAC and untreated control groups. RT-PCR and immunohistochemistry demonstrated expression of type II collagen and aggrecan consistent with the disc phenotype. Conclusion: The results of this study demonstrates that formulations derived from poly-N-acetyl glucosamine (pGLcNAc) have positive effects of disc cell metabolism as quantified by proteoglycan content, cellular viability and proliferation, and the expression of key extra-cellular matrix molecules. The sulphated formulation of deacetylated pGLcNAc (Gel 1) appeared to have the greatest in-vitro effect followed by DEAC and the short fiber construct of Gel 2. It is possible that the pGlcNAc fibers in Gel 2 were not as soluble to the extent of DEAC due to their inability to form strong hydrogen bonds. This study shows promise towards ongoing evaluation of novel biomaterials for the potential DDD treatment through tissue regenerative or reparative schemes

Problems of vertebral growth plate metabolism regulation at different stages of ontogenesis are insufficiently covered in the literature. However, the study of function mechanism of provisional cartilage of vertebral growth plate is a practical and theoretical basis of pathogenesis model of idiopathic scoliosis and Scheuermann’s disease both associated with growth disorders. Objective: To investigate the function mechanism of vertebral growth plate structural components during formation and growth. Materials and methods: Fifty vertebral body specimens of children at the age from 1 to 14 years obtained from the forensic medicine department were studied by methods of morphohistochemistry, biochemistry, and ultra-structural analysis. The expression of five proteoglycan genes and their albuminous products was investigated by RT-PCR method. Results: The process of growth represents a sequence of morphogenetic movements ongoing up to the achievement of sexual maturity. But morphofunctional organization and regulation of growth are different in different periods of ontogenesis. Early postnatal growth of vertebral bodies is governed by a radially located zone of growth. The cell population in a just-formed cartilage growth plate is non-uniform: from poorly differentiated chondroblast through the form of highly differentiated ones to degrading chondrocyte. This period of the spine development is characterised by the presence of vessels in provisional cartilage tissue. The concept of “chondro/hematic barrier” suggested and validated by A.M Zaidman explains a conservation of homeostasis at a stage of vertebral bodies differentiation. The process of chondrogenic differentiation of prechondroblasts in the early postnatal period is inducted by the chorda influence. In the late postnatal period (12–14 years) the laws of structural and functional organization of cartilage growth plate of vertebral body remain the same: phenotypic heterogeneity, polarity, and zonality of cells. A metabolic centre of complex architectonics of cartilage tissue is chondroblast. Chondroblast is functioning at the level of chondron which is a functional unit of vertebral growth plate. Chondroblast (chondrocyte) is located in the centre of chondron and surrounded by pericellular matrix presented by diffuse aggrecan molecules, or growth plate aggregates. Due a peculiar architectonics, growth plate molecules have inner spaces comparable in size with Golgi’s vesicles. Metabolites, small molecules, and water freely penetrate through these molecules. Diffuse molecules together with type II thin collagenic fibres, minor collagenes, and structure-forming growth plates perform barrier function. Besides barrier function, diffuse molecules perform information function inside a chondron, forming a kind of information field. Signals of this field are perceived by chondroblast receptors, and the cell gene apparatus expression is carried out through second messengers. Thus, either stimulation of proliferative activity with subsequent differentiation during intensive growth, or interruption of these processes (period of growth delay) occurs. Single chondrons unite into chains in proliferation zones. Cell interaction inside chondron occurs due transmembrane structures, as a contact coordination of functions of cells with inherent high specificity. Concentration of diffuse molecules of growth plate (aggrecan) in proliferation zones is the highest on evidence of histochemical and ultrastructural assays. Besides, diffuse molecules are the short-distance regulators of DNA synthesis the mechanism of action of which is realised through the system of receptors on a cellular membrane. Hence, contact intercellular interactions are one of the mechanisms controlling cell division. These are so-called extracellular factors of chondroblast proliferation regulation. Thus, the process of growth represents a complex two-stage mechanism of proliferation and differentiation of chondroblasts, and adequate osteogenesis. All three processes provide harmonious spine formation, and disturbance of one of them results in pathology development

Introduction: Kashin-Beck disease (KBD) is an endemic osteoarthropathy with pathological changes occurring in growth plate and articular cartilage in humans. It manifests as cartilage degeneration and necrosis. It is postulated that KBD is due to fungal mycotoxins infiltrating the diet and a regional selenium deficiency in the environment providing food sources in a broad belt across China. Previous work has established an in vitro system in which chondrocytes are cultured and an ex vivo cartilage graft is produced. Subjecting these chondrocytes to either selenium (SEL), Nivalenol (NIV) or in combination during the growth of the graft was found to alter the morphology of the cartilage graft. In addition, the quantity of the large aggregating proteoglycan, was significantly reduced in a dose dependent manner in the presence of Nivalenol. This study aimed to examine the composition of aggrecan from grafts grown in the presence of NIV or SEL alone, or in combination to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD. Methods: Chondrocytes (from 7 day old bovine cartilage) were seeded at high density in MilliCell filter inserts (12mm diameter; Millipore, MA). Cultures were maintained for 4 weeks in DMEM supplemented with 20% heat–inactivated FBS, ascorbate (100μg/ml) and TGFß2 (5ng/ml) or additionally supplemented with either SEL , NIV or both at concentrations of 0.01, 0.05 and 0.1μg/ml. Media was refreshed thrice weekly and later analysed. At 4 weeks the cartilage grafts were harvested, weighed and extracted in 4M guanidium chloride (with an inhibitor cocktail) for biochemical analysis of matrix molecules. Residues were papain digested. Glycosaminoglycan concentration was determined using the DMMB assay in all media samples, guanidine extracts and papain digests. Aggrecan and GAG composition was determined using Western blotting with a panel of antibodies recognising chondroitin sulphate (CS), keratan sulphate (KS) and protein core epitopes present in aggrecan. Results: The total GAG synthesised in a 4week period was substantially reduced in chondrocytes cultured in the presence of NIV at 0.05 and 0.1μg/ml and to a lesser extent in those cultures exposed to the highest dose of SEL. However, the amount of GAG released into the media remained fairly constant within the treatment groups, but a marked reduction was apparent in the guanidine extracts of the cartilage grafts. Western blot analysis with a series of antibodies on guanidine extracted aggrecan showed no substantial changes in the core protein molecular weights however analysis demonstrated that KS was reduced in NIV treated cultures. Results also indicated that NIV treated cultures appeared to contain less CS substitutions on the aggrecan core protein. Discussion: The GAG concentration data indicates that there is an inability of the GAG to remain within the cartilage grafts extracellular matrix. when treated with NIV. Western blot analysis indicates minor changes in the composition of the aggrecan in relation to protein core length and CS/KS side chain substitutions or length. Further work will investigate the proportion of aggrecan able to form high molecular weight aggregates, the metabolism of link protein and hyaluronan