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Chondrosarcoma is a rare malignant tumor that forms in bone and cartilage. The primary treatment involves surgical removal of the tumor with a margin of healthy tissue. Especially if complete surgical removal is not possible, radiation therapy and chemotherapy are used in conjunction with surgery, but with a generally low efficiency. Ongoing researches are focused on understanding the genetic and molecular basis of chondrosarcoma following high linear energy transfer (LET) irradiation, which may lead to treatments that are more effective. The goal of this study is to evaluate the differential effects of DNA damage repair inhibitors and high LET irradiation on chondrosarcoma versus chondrocyte cells and the LET-dependency of the effects. Two chondrosarcoma cell lines with different IDH mutation status and one chondrocyte cell line were exposed to low LET (X-ray) and high LET (carbon ion) irradiation in combination with an Olaparib PARP inhibitor. Cell survival and DNA repair mechanisms were investigated. High LET irradiation drastically reduced cell survival, with a biological efficiency three times that of low LET. Olaparib significantly inhibited PARylation in all the tested cells. A significant reduction in cell survival of both chondrosarcoma and chondrocyte cells was observed following the treatment combining Olaparib and X-ray. PARP inhibition induced an increase in PARP-1 expression and a reduced effect on the cell survival of WT IDH chondrosarcoma cells. No radiosensitizing effect was observed in cells exposed to Olaparib paired with high LET irradiation. NHEJ was activated in response to high LET irradiation, neutralizing the PARP inhibition effect in both chondrosarcoma cell lines. When high LET irradiation is not available, PARP inhibition could be used in combination with low LET irradiation, with significant radiosensitizing effects on chondrosarcoma cells. Chondrocytes may be affected by the treatment combination too, showing the need to preserve normal tissues from radiation fields when this kind of treatment is suggested.

Little is known regarding radiation-induced matrikines and the possible degradation of extracellular matrix following therapeutic irradiation. The goal of this study was to determine if irradiation can cut collagen proteins at specific sites, inducing potentially biologically active peptides against cartilage cells. Chondrocytes cultured as 3D models were evaluated for extracellular matrix production. Bystander molecules were analyzed in vitro in the conditioned medium of X-irradiated chondrocytes. Preferential breakage sites were analyzed in collagen polypeptide by mass spectrometry and resulting peptides were tested against chondrocytes. 3D models of chondrocytes displayed a light extracellular matrix able to maintain the structure. Irradiated and bystander chondrocytes showed a surprising radiation sensitivity at low doses, characteristic of the presence of bystander factors, particularly following 0.1 Gy. The glycine-proline peptidic bond was observed as a preferential cleavage site and a possible weakness of the collagen polypeptide after irradiation. From the 46 collagen peptides analyzed against chondrocytes culture, 20 peptides induced a reduction of viability and 5 peptides induced an increase of viability at the highest concentration between 0.1 and 1 µg/ml. We conclude that irradiation promoted a site-specific degradation of collagen. The potentially resulting peptides induce negative or positive regulations of chondrocyte growth. Taken together, these results suggest that ionizing radiation causes a degradation of cartilage proteins, leading to a functional unbalance of cartilage homeostasis after exposure, contributing to cartilage dysfunction., (© 2024. The Author(s).)

Human induced pluripotent stem cells (hiPSCs) can be differentiated into chondrocyte-like cells. However, implantation of these cells is not without risk given that those transplanted cells may one day undergo ionizing radiation (IR) in patients who develop cancer. We aimed to evaluate the effect of IR on chondrocyte-like cells differentiated from hiPSCs by determining their gene and microRNA expression profile and proteomic analysis. Chondrocyte-like cells differentiated from hiPSCs were placed in a purpose-designed phantom to model laryngeal cancer and irradiated with 1, 2, or 3 Gy. High-throughput analyses were performed to determine the gene and microRNA expression profile based on microarrays. The composition of the medium was also analyzed. The following essential biological processes were activated in these hiPSC-derived chondrocytes after IR: "apoptotic process", "cellular response to DNA damage stimulus", and "regulation of programmed cell death". These findings show the microRNAs that are primarily responsible for controlling the genes of the biological processes described above. We also detected changes in the secretion level of specific cytokines. This study demonstrates that IR activates DNA damage response mechanisms in differentiated cells and that the level of activation is a function of the radiation dose.

Introduction: Rapamycin has been considered as a potential treatment for osteoarthritis (OA). Drug carriers fabricated from liposomes can prolong the effects of drugs and reduce side effects of drugs. Low-intensity pulsed ultrasound (LIPUS) has been found to possess anti-OA effects., Materials and Methods: The anti-osteoarthritic effects of liposome-encapsulated rapamycin (L-rapa) combined with LIPUS were examined by culture of normal and OA chondrocytes in alginate beads and further validated in OA prone Dunkin-Hartley guinea pigs., Results: L-rapa with LIPUS largely up-regulated aggrecan and type II collagen mRNA in human OA chondrocytes (HOACs). L-rapa with LIPUS caused significant enhancement in proteoglycan and type II collagen production in HOACs. Large decreases in both MMP-13 and IL-6 proteins were found in the HOACs exposed to L-rapa with LIPUS. Intra-articular injection of 40 μL L-rapa at both 5 μM and 50 μM twice a week combined with LIPUS thrice a week for 8 weeks significantly increased GAGs and type II collagen in the cartilage of knee. Results on OARSI score showed that intra-articular injection of 5 μM L-rapa with LIPUS displayed the greatest anti-OA effects. Immunohistochemistry revealed that L-rapa with or without LIPUS predominantly reduced MMP-13 in vivo. The values of complete blood count and serum biochemical examinations remained in the normal ranges after the injections with or without LIPUS. These data indicated that intra-articular injection of L-rapa collaborated with LIPUS is not only effective against OA but a safe OA therapy., Conclusion: Taken together, L-rapa combined with LIPUS possessed the most consistently and effectively anabolic and anti-catabolic effects in HOACs and the spontaneous OA guinea pigs. This study evidently revealed that liposome-encapsulation collaborated with LIPUS is able to reduce the effective dose and administration frequency of rapamycin and further stably reinforce its therapeutic actions against OA., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Chen et al.)

Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair., (© 2020 Wiley Periodicals, Inc.)

The aim of this study was to evaluate the effects of low-level laser therapy using the gallium arsenide laser (λ = 830 nm) on the articular cartilage (AC) organization from knee joint in an experimental model of microcrystalline arthritis in adult male Wistar rats. Seventy-two animals were divided into three groups: A (control), B (induced arthritis), and C (induced arthritis + laser therapy). The arthritis was induced in the right knee using 2 mg of Na 4 P 2 O 7 in 0.5 mL of saline solution. The treatments were daily applied in the patellar region of the right knee after 48 h of induction. On the 7th, 14th, and 21st days of treatment, the animals were euthanized and their right knees were removed and processed for structural and biochemical analysis of the AC. The chondrocytes positively labeled for the TUNEL reaction were lower in C than in B on the 14th and 21st days. The content of glycosaminoglycans and hydroxyproline in A and C was higher than B on the 21st day. The amount of tibial TNF-α in B and C was lower than in A. The amount of tibial BMP-7 in B and C was higher than in A. The femoral MMP-13 was lower in B and C than for A. The tibial TGF-β for C was higher than the others. The femoral ADAMT-S4 content of A and C presented similar and inferior data to B on the 21st day. The AsGa-830 nm therapy preserved the content of glycosaminoglycans, reduced the cellular changes and the inflammatory process compared to the untreated group.

Introduction: Biophysical stimulation is a non-invasive therapy used in orthopaedic practice to increase and enhance reparative and anabolic activities of tissue., Methods: A sistematic web-based search for papers was conducted using the following titles: (1) pulsed electromagnetic field (PEMF), capacitively coupled electrical field (CCEF), low intensity pulsed ultrasound system (LIPUS) and biophysical stimulation; (2) bone cells, bone tissue, fracture, non-union, prosthesis and vertebral fracture; and (3) chondrocyte, synoviocytes, joint chondroprotection, arthroscopy and knee arthroplasty., Results: Pre-clinical studies have shown that the site of interaction of biophysical stimuli is the cell membrane. Its effect on bone tissue is to increase proliferation, synthesis and release of growth factors. On articular cells, it creates a strong A 2A and A 3 adenosine-agonist effect inducing an anti-inflammatory and chondroprotective result. In treated animals, it has been shown that the mineralisation rate of newly formed bone is almost doubled, the progression of the osteoarthritic cartilage degeneration is inhibited and quality of cartilage is preserved. Biophysical stimulation has been used in the clinical setting to promote the healing of fractures and non-unions. It has been successfully used on joint pathologies for its beneficial effect on improving function in early OA and after knee surgery to limit the inflammation of periarticular tissues., Discussion: The pooled result of the studies in this review revealed the efficacy of biophysical stimulation for bone healing and joint chondroprotection based on proven methodological quality., Conclusion: The orthopaedic community has played a central role in the development and understanding of the importance of the physical stimuli. Biophysical stimulation requires care and precision in use if it is to ensure the success expected of it by physicians and patients.

Purpose: Data on the response of chondrocytes differentiated from hiPSCs (hiPSC-DCHs) to ionizing radiation (IR) are lacking. The aim of present study was to assess DNA damage response (DDR) mechanisms of IR-treated hiPSC-DCHs., Methods and Materials: The following IR-response characteristics in irradiated hiPSC-DCHs were assessed: 1) the kinetics of DNA DSB formation; 2) activation of major DNA repair mechanisms; 3) cell cycle changes and 4) reactive oxygen species (ROS), level of key markers of apoptosis and senescence., Results: DNA DSBs were observed in 30% of the hiPSC-DCHs overall, and in 60% after high-dose (> 2 Gy) IR. Nevertheless, these cells displayed efficient DNA repair mechanisms, which reduced the DSBs over time until it reached 30% by activating key genes involved in homologous recombination and non-homologous end joining mechanisms. As similar to mature chondrocytes, irradiated hiPSC-DCH cells revealed accumulation of cells in G2 phase. Overall, the hiPSC-DCH cells were characterized by low levels of ROS, cPARP and high levels of senescence., Conclusions: The chondrocyte-like cells derived from hiPSC demonstrated features characteristic of both mature chondrocytes and "parental" hiPSCs. The main difference between hiPSC-derived chondrocytes and hiPSCs and mature chondrocytes appears to be the more efficient DDR mechanism of hiPSC-DCHs. The unique properties of these cells suggest that they could potentially be used safely in regenerative medicine if these preliminary findings are confirmed in future studies., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to evaluate the biostimulation (BS) effect of the gallium-aluminum-arsenide (GaAlAs) diode laser by histopathology with an experimental osteoarthritis (OA) model in the temporomandibular joints (TMJ) of rabbits, in the early period. GaAlAs diode laser is used for pain reduction in TMJ disorders. Twenty-four adult male New Zealand white rabbits were randomly divided into three equal groups: Control Group (CG), Study Group 1 (SG-1), and Study Group 2 (SG-2). Mono-iodoacetate (MIA) was administered to the right TMJs of all rabbits. The rabbits did not undergo any treatment for four weeks to allow the development of osteoarthritis. In SG-1, laser BS was applied to the rabbits at 940 nm, 5 W, and 15 J/cm2 in continuous wave mode at 48-hour intervals for 14 sessions; and in SG-2, laser BS was applied with the same parameters at 24-hour intervals for 28 sessions. Laser BS was not applied to the rabbits in CG. All rabbits were sacrificed simultaneously. The TMJ cartilage, osteochondral junction, chondrocyte appearance, and subchondral ossification were evaluated histopathologically. There was no statistically significant difference between the groups in terms of cartilage, osteochondral junction, chondrocyte appearance, and subchondral ossification values (p > 0.05). The laser BS protocol used in the study had no positive histopathological effects on TMJ OA in the early period.

The aim of this study was to compare the effects of combined treatment with chondroitin sulfate and glucosamine sulfate (CS/Gl) and photobiomodulation (PBM) on the degenerative process related to osteoarthritis (OA) in the articular cartilage in rats. Forty male Wistar rats were randomly divided into four groups: OA control group (CG); OA animals submitted to PBM treatment (PBM); OA animals submitted to CS/Gl treatment (CS/Gl); OA submitted to CS/GS associated with PBM treatments (GS/Gl + PBM). The CS/Gl started 48 h after the surgery, and they were performed for 29 consecutive days. Moreover, PBM was performed after the CS/Gl administration on the left joint. Morphological characteristics and immunoexpression of interleukin 10 (IL-10) and 1 beta (IL-1β) and collagen type II (Col II) of the articular cartilage were evaluated. The results showed that all treated groups (CS/Gl and PBM) presented attenuation signs of degenerative process (measured by histopathological analysis) and lower density chondrocytes [PBM (p = 0.0017); CS/Gl (p = 0.0153) and CS/Gl + PBM (p = 0.002)]. Additionally, CS/Gl [associated (p = 0.0089) or not with PBM (p = 0.0059)] showed significative lower values for OARSI grade evaluation. Furthermore, CS/GS + PBM decreased IL-1β protein expression (p = 0.0359) and increased IL-10 (p = 0.028) and Col II imunoexpression (p = 0.0204) compared to CG. This study showed that CS/Gl associated with PBM was effective in modulating inflammatory process and preventing the articular tissue degradation in the knees OA rats.

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO 2 ) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO 2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Background: Low-intensity pulsed ultrasound (LIPUS) is a mechanical stimulus that plays a key role in regulating the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the way in which it affects the chondrogenic differentiation of BMSCs remains unknown. In this study, we aimed to investigate whether LIPUS is able to influence TGF-β1-induced chondrogenesis of BMSCs through the integrin-mechanistic target of the Rapamycin (mTOR) signaling pathway., Methods: BMSCs were isolated from rat bone marrow and cultured in either standard or TGF-β1-treated culture medium. BMSCs were then subjected to LIPUS at a frequency of 3 MHz and a duty cycle of 20%, and integrin and mTOR inhibitors added in order to analyze their influence on cell differentiation. BMSCs were phenotypically analyzed by flow cytometry and the degree of chondrogenesis evaluated through toluidine blue staining, immunofluorescence, and immunocytochemistry. Furthermore, expression of COL2, aggrecan, SOX9, and COL1 was assessed by qRT-PCR and western blot analysis., Results: We found that LIPUS promoted TGF-β1-induced chondrogenesis of BMSCs, represented by increased expression of COL2, aggrecan and SOX9 genes, and decreased expression of COL1. Notably, these effects were prevented following addition of integrin and mTOR inhibitors., Conclusions: Taken together, these results indicate that mechanical stimulation combined with LIPUS promotes TGF-β1-induced chondrogenesis of BMSCs through the integrin-mTOR signaling pathway.

The goal of our research was demonstrated that multiple molecules in microenvironments of the early osteoarthritis (OA) joint tissue may be actively responded to extracorporeal shockwave therapy (ESWT) treatment, which potentially regulated biological function of chondrocytes and synovial cells in early OA knee. We demonstrated that shockwave treatment induced the expression of protein-disulfide isomerase-associated 3 (Pdia-3) which was a significant mediator of the 1α,25-Dihydroxyvitamin D 3 (1α,25(OH)2D3) rapid signaling pathway, using two-dimensional electrophoresis, histological analysis and quantitative polymerase chain reaction (qPCR). We observed that the expression of Pdia-3 at 2 weeks was significantly higher than that of other group at 4, 8, and 12 weeks post-shockwave treatment in early OA rat knee model. The other factors of the rapid membrane signaling pathway, including extracellular signal-regulated protein kinases 1 (ERK1), osteopontin (OPG), alkaline phosphatase (ALP), and matrix metallopeptidase 13 (MMP13) were examined and were found to be significantly increased at 2 weeks post-shockwave treatment by qPCR in early OA of the knee. Our proteomic data revealed significant Pdia-3 expression in microenvironments of OA joint tissue that could be actively responded to ESWT, which may potentially regulate the biological functions of chondrocytes and osteoblasts in the treatment of the early OA of the knee., Competing Interests: Conflicts of interest: The authors declared that they did not receive any honoraria or consultancy fees in writing this manuscript. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. One author (Chin-Jen Wang) serves as a member of the advisory committee of Sanuwave, (Alpharetta, GA, USA) and this study is performed independent of the appointment. The remaining authors declared no conflict of interest.

Collagen has shown promise as a bioink for extrusion-based bioprinting, but further development of new collagen bioink formulations is necessary to improve their printability. Screening these formulations by measuring print accuracy is a costly and time consuming process. We hypothesized that rheological properties of the bioink before, during, and/or after gelation can be used to predict printability. In this study, we investigated the effects of riboflavin photocrosslinking and pH on type I collagen bioink rheology before, during, and after gelation and directly correlated these findings to the printability of each bioink formulation. From the riboflavin crosslinking study, results showed that riboflavin crosslinking increased the storage moduli of collagen bioinks, but the degree of improvement was less pronounced at higher collagen concentrations. Dots printed with collagen bioinks with riboflavin crosslinking exhibited smaller dot footprint areas than those printed with collagen bioinks without riboflavin crosslinking. From the pH study, results showed that gelation kinetics and final gel moduli were highly pH dependent and both exhibited maxima around pH 8. The shape fidelity of printed lines was highest at pH 8-9.5. The effect of riboflavin crosslinking and pH on cell viability was assessed using bovine chondrocytes. Cell viability in collagen gels was found to decrease after blue light activated riboflavin crosslinking but was not affected by pH. Correlations between rheological parameters and printability showed that the modulus associated with the bioink immediately after extrusion and before deposition was the best predictor of bioink printability. These findings will allow for the more rapid screening of collagen bioink formulations.

Objective: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism., Methods: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm 2 power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca 2+ influx was measured by using a Fluo-4 probe., Results: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca 2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BK ca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes., Conclusion: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca 2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes., (Copyright © 2016. Published by Elsevier Ltd.)

Objective To investigate changes in nucleus pulposus cell expression and secretion of interleukin (IL)-1β and tumour necrosis factor (TNF)-α following stimulation with a low-frequency (LF) pulsed electromagnetic field (PEMF). Methods Primary rat nucleus pulposus cells were isolated and cultured in vitro, followed by stimulation with LF-PEMFs at a frequency of 2 Hz and different intensities, ranging from 0.5-3.0 A/m. Cells were observed for morphological changes, and proliferation rates were measured by cell viability counts. Expression of IL-1β and TNF-α within the nucleus pulposus cells was measured using western blotting, and levels of IL-1β and TNF-α secreted in the culture media were measured using enzyme-linked immunosorbent assay. Results Stimulation of nucleus pulposus cells with LF-PEMFs did not appear to affect cell morphology or nucleus pulposus cell IL-1β and TNF-α expression levels. LF-PEMFs did not significantly affect cell proliferation, however, levels of IL-1β and TNF-α secreted into the culture media were found to be significantly reduced in an intensity-dependent manner. Conclusion Low-frequency PEMF stimulation may inhibit secretion of IL-1β and TNF-α in cultured nucleus pulposus cells.

The deleterious effects of ionizing radiation on the growth plate continue to be cause for concern. This study evaluated the ionizing radiation effects on bone development and growth plate in the tibia of rats. All animals were submitted to ionizing radiation on the left leg. The animals were divided into two groups and euthanized 30 and 60 days after radiation. The tibiae were removed and separated into groups: control 30 days, irradiated 30 days, control 60 days and irradiated 60 days. Animals in each group (n = 7) were used for macroscopic and histological analysis. The irradiated tibiae showed arrested growth, angular deformity and limb length discrepancy when compared with nonirradiated tibiae. There was statistical difference between control and radiation groups in all the parameters analyzed, except in the lateral-medial thickness of the distal epiphysis. Histological analysis showed evident changes in the growth plate, which was thicker in the Groups irradiated for 30 days, and irradiated for 60 days, compared with their respective controls. The growth plate showed wide areas with disorganized zones of chondrocytes and severely reduced calcification zone. It was concluded that ionizing radiation damaged the growth plate, compromised the endochondral ossification process, and resulted in complete arrest of bone development., (© 2016 Wiley Periodicals, Inc.)

As a means to analyze anti-inflammatory effects by radiation and/or by cholinergic mechanisms, we found that cultured primary human osteoblasts express most cholinergic components. After X-ray irradiation, their level of acetylcholinesterase (AChE) was strongly elevated. As a 3D model, we cultured mesenchymal stem cells isolated from E11 mouse embryos as micromass nodules, and differentiated them into chondro- and osteoblasts. They were stimulated by 5 or 10 ng/ml of the inflammatory cytokine TNF-α to mimic an inflammatory condition in vitro, before exposure to 2 Gy X-rays. Effects on chondro- and osteoblasts of TNF-α, of X-rays, or both were analysed by Alcian Blue, or Alizarin Red staining, respectively. Acetylcholinesterase (AChE) activity was visualized histochemically. The results showed that treatment with TNF-α affected cartilage and bone formation in vitro, while X-rays reversed the effects of TNF-α. After irradiation, both AChE and alkaline phosphatase (ALP) activities, a marker for bone mineralization, were raised, suggesting that X-rays stimulated cholinergic mechanisms during calcification. Notably, the TNFα-effects on cultures were also counterbalanced after AChE activity was blocked by BW284c51. These findings suggest a complex crosstalk between radiation, cholinergic and inflammatory mechanisms, which could have wide significances, e.g. for understanding rheumatoid arthritis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objective To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on the extracellular matrix synthesis of human osteoarthritis (OA) chondrocytes and explore the underlying mechanism. Methods Human osteoarthritis chondrocytes were collected from abandoned articular cartilage. Then the cells were cultured and identified by toluidine blue staining and immunocytochemical staining of type 2 collagen. The passage 2 cells were randomly divided into 3 groups: control OA group, 30 mW/cm 2 LIPUS-treated OA group, 30 mW/cm 2 LIPUS combined with 5 μmol/L LY294002-treated OA group. LIPUS treatment was performed for 20 minutes per day, totally 7 days. The mRNA levels of Col2, aggrecan and matrix metalloprotease 13 (MMP-13) were determined by quantitative real-time PCR. The protein levels of Col2, aggrecan, Akt, p-Akt and MMP-13 were evaluated by Western blotting. Results Compared with the control OA group, the expressions of Col2 and aggrecan at both mRNA and protein levels significantly increased, and MMP-13 significantly reduced in the LIPUS-treated OA group. The p-Akt protein level was significantly elevated after LIPUS stimulation, but there was no significant difference in the Akt protein levels between the two groups. Moreover, LY294002, an inhibitor of PI3K/Akt, significantly suppressed the biological effect activated by LIPUS. Conclusion LIPUS enhances the synthesis and inhibits the degradation of the extracellular matrix in human osteoarthritis chondrocytes.

Objective: The aim of this study was to investigate the effects of low-dose fractionated radiotherapy on cartilage degeneration after distal femoral fresh massive osteochondral allograft transplantation., Methods: Twenty-four New Zealand White rabbits were divided into three groups of 8 rabbits each. All rabbits underwent distal femoral medial condyle fresh massive osteochondral allograft transplantation from California rabbits. The group 1 underwent transplantation without any preliminary process. The group 2 underwent fractionated local radiotherapy of 100 cGy for five days starting on the transplantation day. The group 3 included the rabbits to which the grafts transplanted after radiating in vitro by a single dose radiation of 1500 cGy. The hosts were sacrificed twelve weeks later. Anteroposterior and lateral radiographs were taken. Synovial tissue, cartilaginous tissue, and subchondral bone were assessed histopathologically., Results: Nonunion was present in three cases of group 2 and one of group 3 in which cartilage degeneration was more severe. Synovial hypertrophy and pannus formation were more obvious in non-radiated rabbits. Hypocellularity and necrosis of the subchondral bone were rare in group 2. More cartilage tissue impairment was present in group 3 compared to group 1., Conclusion: In osteochondral massive allograft transplantations, the immune reaction of the host could be precluded with radiotherapy, and the side-effects can be prevented by low-dose fractionated regimen. The total dose of fractionated radiotherapy for an immune suppression should be adjusted not to damage the cartilage tissue, but to avoid articular degeneration in the long term., (Copyright © 2016 Turkish Association of Orthopaedics and Traumatology. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Adipose-derived stem cells (ASCs) are important to homeostasis and the regeneration of subcutaneous fat. Hence, we examined the proliferation and differentiation capacity of irradiated ASCs over time., Methods: Two female pigs received a single 18 Gy dose of ionizing radiation to an 18 × 8 cm area on the dorsal body skin via a 6 MeV electron beam. After irradiation, the ASCs were cultured from adipose tissue harvested from a non-irradiated area and an irradiated area at 2, 4, and 6 weeks. The proliferation capacity of ASCs was evaluated by a colony-forming units-fibroblasts (CFUs-Fs) assay, a cholecystokinin (CCK) test with 10 % fetal bovine serum (FBS), and a 1 % FBS culture test. The senescence of ASCs was evaluated through morphological examination, immunophenotyping, and β-galactosidase activity, and the multipotent differentiation potential of ASCs was evaluated in adipogenic, osteogenic, and chondrogenic differentiation media., Results: Irradiated ASCs demonstrated significantly decreased proliferative capacity 6 weeks after irradiation. As well, the cells underwent senescence, which was confirmed by blunted morphology, weak mesenchymal cell surface marker expression, and elevated β-galactosidase activity. Irradiated ASCs also exhibited significant losses in the capacity for adipocyte and chondrocyte differentiation. In contrast, osteogenic differentiation was preserved in irradiated ASCs., Conclusions: We observed decreased proliferation and senescence of irradiated ASCs compared to non-irradiated ASCs 6 weeks after irradiation. Furthermore, irradiated ASCs demonstrated impaired adipocyte and chondrocyte differentiation but retained their osteogenic differentiation capacity. Our results could shed light on additional pathogenic effects of late irradiation, including subcutaneous fibrosis and calcinosis.

The effects of microcurrent application on the elastic cartilage defects in the outer ear of young animals were analyzed. Sixty male Wistar rats were divided into a control (CG) and a treated group (TG). An excisional lesion was created in the right outer ear of each animal. Daily treatment was started after 24h and consisted of the application of a low-intensity (20μA) continuous electrical current to the site of injury for 5min. The animals were euthanized after 7, 14 and 28 days of injury and the samples were submitted to analyses. In CG, areas of newly formed cartilage and intense basophilia were seen at 28 days, while in TG the same observations were made already at 14 days. The percentage of birefringent collagen fibers was higher in CG at 28 days. The number of connective tissue cells and granulocytes was significantly higher in TG. Ultrastructural analysis revealed the presence of chondrocytes in TG at 14 days, while these cells were observed in CG only at 28 days. Cuprolinic blue staining and the amount of glycosaminoglycans were significantly higher in TG at 14 days and 28 days. The amount of hydroxyproline was significantly higher in TG at all time points studied. The active isoform of MMP-2 was higher activity in TG at 14 days. Immunoblotting for type II collagen and decorin was positive in both groups and at all time points. The treatment stimulated the proliferation and differentiation of connective tissue cells, the deposition of glycosaminoglycans and collagen, and the structural reorganization of these elements during elastic cartilage repair., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: Particle therapy using carbon ions (C-ions) has been successfully used in the treatment of tumors resistant to conventional radiation therapy. However, the potential side effects to healthy cartilage exposed to lower linear energy transfer (LET) ions in the beam track before the tumor have not been evaluated. The aim of the present study was to assess the extent of damage after C-ion irradiation in a 3-dimensional (3D) cartilage model close to human homeostasis., Methods and Materials: Primary human articular chondrocytes from a healthy donor were cultured in a collagen scaffold to construct a physioxic 3D cartilage model. A 2-dimensional (2D) culture was used as a reference. The cells were irradiated with a single dose of a monoenergetic C-ion beam with a LET of approximatively 30 keV/μm. This LET corresponds to the entrance channel of C-ions in the shallow healthy tissues before the spread-out Bragg peak (∼100 keV/μm) during hadron therapy protocols. The same dose of X-rays was used as a reference. Survival, cell death, and senescence assays were performed., Results: As expected, in the 2D culture, C-ions were more efficient than X-rays in reducing cell survival with a relative biological effectiveness of 2.6. This correlated with stronger radiation-induced senescence (two-fold) but not with higher cell death induction. This differential effect was not reflected in the 3D culture. Both ionizing radiation types induced a comparable rate of senescence induction in the 3D model., Conclusions: The greater biological effectiveness of C-ions compared with low LET radiation when evaluated in treatment planning systems might be misevaluated using 2D culture experiments. Radiation-induced senescence is an important factor of potential cartilage attrition. The present data should encourage the scientific community to use relevant models and beams to improve the use of charged particles with better safety for patients., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The electromagnetic field (EMF) has a great impact on our body. It has been successfully used in physiotherapy for the treatment of bone disorders and osteoarthritis, as well as for cartilage regeneration or pain reduction. Recently, EMFs have also been applied in in vitro experiments on cell/stem cell cultures. Stem cells reside in almost all tissues within the human body, where they exhibit various potential. These cells are of great importance because they control homeostasis, regeneration, and healing. Nevertheless, stem cells when become cancer stem cells, may influence the pathological condition. In this article we review the current knowledge on the effects of EMFs on human adult stem cell biology, such as proliferation, the cell cycle, or differentiation. We present the characteristics of the EMFs used in miscellaneous assays. Most research has so far been performed during osteogenic and chondrogenic differentiation of mesenchymal stem cells. It has been demonstrated that the effects of EMF stimulation depend on the intensity and frequency of the EMF and the time of exposure to it. However, other factors may affect these processes, such as growth factors, reactive oxygen species, and so forth. Exploration of this research area may enhance the development of EMF-based technologies used in medical applications and thereby improve stem cell-based therapy and tissue engineering.

Background Aims: This study evaluates the biological response of adipose tissue-derived mesenchymal stromal cells (aMSCs) to ionizing radiation (IR)., Methods: Irradiated BALB/c mice aMSCs were characterized for functionality and phenotype. The clonogenic capacity of irradiated aMSCs was assessed and compared with those of metastatic breast cancer cell line (4T1) and normal mouse fibroblasts (NIH3T3-wt). We investigated the IR-induced DNA damage response, apoptosis, changes in cell cycle (CC) dynamics and protein and gene expression., Results: Irradiated and non-irradiated aMSCs were able to differentiate into adipocytes, chondrocytes and osteocytes with no significant difference. Irradiated aMSCs maintained the expression of mesenchymal stromal cells (MSCs) surface antigens and, as expected, were negative for hematopoietic stem cells (HSCs) surface antigens when tested up to 7 days after IR for all irradiation doses with no significant difference. Clonogenically, irradiated aMSCs had higher relative survival fraction and plating efficiency than 4T1 and NIH3T3-wt. Irradiated aMSCs expressed higher □H2AX and significantly showed faster and more time-efficient IR-induced DNA damage response evident by up-regulated DNA-PKcs and RAD51. Two hours after IR, most of aMSCs DNA damage/repair-related genes showed up-regulation that disappeared within 6 h after IR. Irradiated aMSCs showed a significant rise and an earlier peak of p-ATM-dependent and -independent (p84/5E10-mediated) G2/M CC arrest compared with 4T1 and NIH3T3-wt., Conclusions: After IR exposure, aMSCs showed a robust and time-efficient radiation-induced DNA damage repair response, stable phenotypical characteristics and multi-lineage differentiation potential, suggesting they may be reliable candidates for cell therapy in radiation oncology regenerative medicine., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the effect of a light-emitting diode (LED) and/or low-level laser (LLL) with or without the use of anterior bite jumping appliances (also known as functional appliances [FAs]) on mandibular growth in rats., Materials and Methods: Thirty-six 8-week-old male Sprague-Dawley rats weighing 200 g were obtained from Charles River Canada (St. Constant, QC, Canada) and were divided into six groups of six animals each. Groups were as follows: group 1: LLL; group 2: LLL + FA; group 3: LED; group 4: LED + FA; group 5: FA; and group 6: control (no treatment). Mandibular growth was evaluated by histomorphometric and micro computed tomographic (microCT) analyses., Results: The LED and LED + FA groups showed an increase in all condylar tissue parameters compared with other groups., Conclusion: The LED-treated groups showed more mandibular growth stimulation compared with the laser groups.

Background and Objective: Magnetic nanoparticles with the ability to absorb laser radiation are the perspective agents for the early diagnostics and laser therapy of degenerative cartilage. The effect of starch stabilized magnetite nanoparticles (SSNPs) on the cartilage structure components has never been studied before. The aim of the work is to establish the Erbium:glass laser effect on costal and articular cartilage impregnated with SSNPs., Materials and Methods: Porcine articular and costal cartilage disks (2.0 mm in diameter and 1.5-2 mm in thickness) were impregnated with SSNPs and irradiated using a 1.56 μm laser in therapeutic laser setting. The one sample group underwent the second irradiation after the SSNPs impregnation. The samples were analyzed by the means of histology, histochemistry and transmission electron microscopy (TEM) to reveal the alterations of cells, glycosaminoglycans and collagen network., Results: The irradiated cartilage demonstrates the higher content of cell alterations than the intact one due to the heat and mechanical affection in the course of laser irradiation. However the alterations are localized at the areas near the irradiated surfaces and not dramatic. The impregnation of SSNPs does not cause any additional cell alterations. For both costal and articular cartilage the matrix alterations of irradiated samples are not critical: there is the slight decrease in acid proteoglycan content at the irradiated areas while the collagen network is not altered. Distribution and localization of impregnated SSNPs is described: agglomerates of 150-230 nm are observed located at the borders between matrix and cell lacunas of articular cartilage; SSNPs of 15-45 nm are found in the collagen network of costal cartilage., Conclusions: It was shown that SSNPs do not appreciably affect the structural components of both articular and costal cartilage and can be safely used for the laser diagnostics and therapy. The area of structural alterations is diffuse and local as the result of the mechanical and heat effect of laser impact. SSNPs reveal the areas of the structural alterations of cartilage matrix and give information about the size of the pores and defects., (© 2015 Wiley Periodicals, Inc.)

Surface damage to articular cartilage is recognized as the initial underlying process causing the loss of mechanical function in early-stage osteoarthritis. In this study, we developed structure-modifying treatments to potentially prevent, stabilize or reverse the loss in mechanical function. Various polymers (chondroitin sulfate, carboxymethylcellulose, sodium hyaluronate) and photoinitiators (riboflavin, irgacure 2959) were applied to the surface of collagenase-degraded cartilage and crosslinked in situ using UV light irradiation. While matrix permeability and deformation significantly increased following collagenase-induced degradation of the superficial zone, resurfacing using tyramine-substituted sodium hyaluronate and riboflavin decreased both values to a level comparable to that of intact cartilage. Repetitive loading of resurfaced cartilage showed minimal variation in the mechanical response over a 7 day period. Cartilage resurfaced using a low concentration of riboflavin had viable cells in all zones while a higher concentration resulted in a thin layer of cell death in the uppermost superficial zone. Our approach to repair surface damage initiates a new therapeutic advance in the treatment of injured articular cartilage with potential benefits that include enhanced mechanical properties, reduced susceptibility to enzymatic degradation and reduced adhesion of macrophages., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chondrosarcoma, the second most common type of bone malignancy, is characterized by distant metastasis and local invasion. Previous studies have shown that treatment by pulsed electromagnetic field (PEMF) has beneficial effects on various cancer cells. In this study, we investigated the effects of PEMF applied for 3 and 7 days on the matrix metalloproteinase (MMP) levels in chondrosarcoma SW1353 cells stimulated with two different doses of IL-1β. SW1353 cells were treated with (0.5 and 5 ng/ml) IL-1β and PEMF exposure was applied either 3 or 7 days. MMP-9 and TIMP-1 levels were measured in conditioned media by enzyme-linked immunosorbent assay. The results were relative to protein levels. Statistical analyses were performed using one-way analysis of variance (ANOVA). P<0.05 was considered significant. PEMF treatment significantly decreased MMP-9 protein levels in human chondrosarcoma cells stimulated with 0.5 ng/ml IL-1β at day 7, whereas it did not show any effect on cells stimulated with 5 ng/ml IL-1β. There was no significant change in TIMP-1 protein levels either by IL-1β stimulation or by PEMF treatment. The results of this study showed that PEMF treatment suppressed IL-1β-mediated upregulation of MMP-9 protein levels in a dual effect manner. This finding may offer new perspectives in the therapy of bone cancer.

Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT.

The aim of this study was to investigate the effect of low-level laser therapy (LLLT) on short-term and long-term joint pain, synovitis, anabolic, and catabolic factors in the cartilage of a rabbit model with progressive osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). A total of 160 New Zealand white rabbits were randomly assigned into two groups (ACLT group and LLLT group). All rabbits received ACLT surgery, and 2-, 4-, 6-, and 8-week treatment after the surgery, with 20 rabbits being tested biweekly over every study period. The LLLT group received LLLT with a helium-neon (He-Ne) laser (830 nm) of 1.5 J/cm(2) three times per week, and the ACLT group received placebo LLLT with the equipment switched off. Long-term and short-term pain was tested via weight-bearing asymmetry; synovitis was assessed histologically; and knee joint cartilage was evaluated by gross morphology, histology, and gene expression analysis of anabolic and catabolic factors. The histological assessment of pain and synovitis showed that at least 6-week intermittent irradiation of LLLT could relief knee pain and control synovium inflammation. Gross morphologic inspection and histological evaluation showed that 6 weeks of LLLT could decrease cartilage damage of medical femoral condyle and 8 weeks of LLLT could decrease cartilage damage of medical and lateral femoral condyles and medical tibial plateau. Gene expression analysis revealed two results: At least 6 weeks of LLLT could decrease production of catabolic factors, for example, interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS), and MMP-3, and slow down the loss of anabolic factors, mainly TIMP-1. Eight weeks of LLLT treatment could slow down the loss of collagen II, aggrecan, and anabolic factors, mainly transforming growth factor beta (TGF-β). The study suggests that LLLT plays a protective role against cartilage degradation and synovitis in rabbits with progressive OA by virtue of the regulation of catabolic and anabolic factors in the cartilage.

Chondrocyte death and loss of extracellular matrix are the central features in articular cartilage degeneration during osteoarthritis pathogenesis. Cartilage diseases and, in particular, osteoarthritis are widely correlated to apoptosis but, chondrocytes undergoing apoptosis "in vivo" more often display peculiar features that correspond to a distinct process of programmed cell death termed "chondroptosis". Programmed cell death of primary human chondrocyte has been here investigated in micromasses, a tridimensional culture model, that represents a convenient means for studying chondrocyte biology. Cell death has been induced by different physical or chemical apoptotic agents, such as UVB radiation, hyperthermia and staurosporine delivered at both 1 and 3 weeks maturation. Conventional electron microscopy was used to analyse morphological changes. Occurrence of DNA fragmentation and caspase involvement were also investigated. At Transmission Electron Microscopy, control cells appear rounding or slightly elongated with plurilobated nucleus and diffusely dispersed chromatin. Typically UVB radiation and staurosporine induce chromatin apoptotic features, while hyperthermia triggers the "chondroptotic" phenotype. A weak TUNEL positivity appears in control, correlated to the well known cell death patterns occurring along cartilage differentiation. UVB radiation produces a strong positivity, mostly localized at the micromass periphery. After hyperthermia a higher number of fluorescent nuclei appears, in particular at 3 weeks. Staurosporine evidences a diffuse, but reduced, positivity. Therefore, DNA fragmentation is a common pattern in dying chondrocytes, both in apoptotic and "chondroptotic" cells. Moreover, all triggers induce caspase pathway activation, even if to a different extent, suggesting a fundamental role of apoptotic features, in chondrocyte cell death.

Osteoarthritis (OA) is a degenerative joint disease caused by articular cartilage loss. Many complementary and alternative medicines for OA have been reported so far, but the effectiveness is controversial. Previously, we have shown anti-inflammatory effects of low level laser therapy with static magnetic field, magnetic infrared laser (MIL), in various animal models. Therefore, the beneficial effects were examined in OA rat model. Rats were divided by six groups; no treatment controls of sham and OA model, three MIL treatment groups of OA model at 6.65, 2.66 and 1.33 J cm(-2), and Diclofenac group of OA model with 2 mg kg(-1) diclofenac sodium. The OA control exhibited typical symptoms of OA, but 4-week MIL treatment improved the functional movement of knee joint with reduced edematous changes. In addition, cartilage GAGs were detected more in all MIL treatment groups than OA control. It suggests that 4-week MIL irradiation has dose-dependent anti-inflammatory and chondroprotective effects on OA. Histopathological analyses revealed that MIL treatment inhibits the cartilage degradation and enhances chondrocyte proliferation. The fact that MIL has an additional potential for the cartilage formation and no adverse effects can be regarded as great advantages for OA treatment. These suggest that MIL can be useful for OA treatment., (© 2014 The American Society of Photobiology.)

Hydrogels are promising materials for cartilage repair, but the properties required for optimal functional outcomes are not yet known. In this study, we functionalized four materials that are commonly used in cartilage tissue engineering and evaluated them using in vitro cultures. Gelatin, hyaluronic acid, polyethylene glycol, and alginate were functionalized with methacrylic anhydride to make them photocrosslinkable. We found that the responses of encapsulated human chondrocytes were highly dependent on hydrogel type. Gelatin hydrogels supported cell proliferation and the deposition of a glycosaminoglycan rich matrix with significant mechanical functionality. However, cells had a dedifferentiated phenotype, with high expression of collagen type I. Chondrocytes showed the best redifferentiation in hyaluronic acid hydrogels, but the newly formed matrix was highly localized to the pericellular regions, and these gels degraded rapidly. Polyethylene glycol hydrogels, as a bioinert control, did not promote any strong responses. Alginate hydrogels did not support the deposition of new matrix, and the stiffness decreased during culture. The markedly different response of chondrocytes to these four photocrosslinkable hydrogels demonstrates the importance of material properties for chondrogenesis and extracellular matrix production, which are critical for effective cartilage repair., (© 2013 Wiley Periodicals, Inc.)

The effect of low-intensity pulsed ultrasound (LIPUS) on extracellular matrix (ECM) production via modulation of the integrin/focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been investigated in previous studies in normal chondrocytes, but not in osteoarthritis (OA). Therefore, we investigated the LIPUS-induced integrin β1/FAK/PI3K/Akt mechanochemical transduction pathway in a single study in rabbit OA chondrocytes. Normal and OA chondrocytes were exposed to LIPUS, and mRNA and protein expression of cartilage, metalloproteinases and integrin-FAK-PI3K/Akt signal pathway-related genes was determined by quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Compared with levels in normal chondrocytes, expression levels of ECM-related genes were significantly lower in OA chondrocytes and those of metalloproteinase-related genes were significantly higher. In addition, integrin β1 gene expression and the phosphorylation of FAK, PI3K and Akt were significantly higher in OA chondrocytes. The expression of all tested genes was significantly increased except for that of metalloproteinase, which was significantly decreased in the LIPUS-treated OA group compared to the untreated OA group. LIPUS may affect the integrin-FAK-PI3K/Akt mechanochemical transduction pathway and alter ECM production by OA chondrocytes. Our findings will aid the future development of a treatment or even cure for OA., (Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to investigate the influence of temperature on the ability of the chondrocytes to produce extracellular matrix (ECM)., Materials and Methods: Articular chondrocytes were isolated from porcine knee joints. The chondrocytes were cultured at three different temperatures: 32 °C, 37 °C, and 41 °C. The ability to produce ECM was assessed by gene expression analysis, histological, and biochemical evaluation in a pellet culture system., Results: Wet weight of the pellets generated after 21 days, was significantly heavier when cultured at lower temperatures. Picrosirius red staining, employed to evaluate collagen production, was higher at lower temperatures, and safranin-O staining, used to evaluate sulphated glycosaminoglycan (GAG), was lower at 32 °C than at 37 °C and 41 °C. Collagen type IIA1 mRNA expression was markedly up-regulated at 41 °C. However, picrosirius red staining was inhibited at 41 °C. GAG and DNA content were measured by 1,9-dimethylmethylene blue (DMMB) assay and PicoGreen® assay, respectively. The GAG content per pellet was significantly low at 41 °C compared to that at 32 °C and 37 °C. The DNA content per pellet was larger at lower temperatures. The GAG content normalised with the DNA content per pellet was significantly lower at 32 °C compared to that at 37 °C and 41 °C., Conclusion: Our results suggest that a culture temperature of approximately 41 °C inhibits ECM production by decreasing DNA content and perhaps by collagen misfolding. Taken together, the optimum temperature for ECM production in articular chondrocytes may be between 32 °C and 37 °C.

Although low-dose radiation (LDR) regulates a wide range of biological processes, limited information is available on the effects of LDR on the chondrocyte phenotype. Here, we found that LDR, at doses of 0.5-2 centiGray (cGy), inhibited interleukin (IL)-1β-induced chondrocyte destruction without causing side effects, such as cell death and senescence. IL-1β treatment induced an increase in the expression of α-, β-, and γ-catenin proteins in chondrocytes via Akt signaling, thereby promoting dedifferentiation through catenin-dependent suppression of Sox-9 transcription factor expression and induction of inflammation through activation of the NF-κB pathway. Notably, LDR blocked cartilage disorders by inhibiting IL-1β-induced catenin signaling and subsequent catenin-dependent suppression of the Sox-9 pathway and activation of the NF-κB pathway, without directly altering catenin expression. LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid. Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders., (© 2014 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.)

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production., (© 2013 Wiley Periodicals, Inc.)

Osteoarthritis (OA) is the most frequent joint disease, characterized by degradation of extracellular matrix and alterations in chondrocyte metabolism. Some authors reported that electromagnetic fields (EMFs) can positively interfere with patients affected by OA, even though the nature of the interaction is still debated. Human primary osteoarthritic chondrocytes isolated from the femoral heads of OA-patients undergoing to total hip replacement, were cultured in vitro and exposed 30 min/day for two weeks to extremely-low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF) with variable frequencies, intensities and waveforms. Sham-exposed (S.E.) cells served as control group. Cell viability was measured at days 2, 7 and 14. After two weeks, cell lysates were processed using a proteomic approach. Chondrocyte exposed to ELF and TAMMEF system demonstrated different viability compared to untreated chondrocytes (S.E.). Proteome analysis of 2D-Electrophoresis and protein identification by mass spectrometry showed different expression of proteins derived from nucleus, cytoplasm and organelles. Function analysis of the identified proteins showed changes in related-proteins metabolism (glyceraldeyde-3-phosphate-dehydrogenase), stress response (Mn-superoxide-dismutase, heat-shock proteins), cytoskeletal regulation (actin), proteinase inhibition (cystatin-B) and inflammation regulatory functions (S100-A10, S100-A11) among the experimental groups (ELF, TAMMEF and S.E.). In conclusion, EMFs do not cause damage to chondrocytes, besides stimulate safely OA-chondrocytes and are responsible of different protein expression among the three groups. Furthermore, protein analysis of OA-chondrocytes treated with ELF and the new TAMMEF systems could be useful to clarify the pathogenetic mechanisms of OA by identifying biomarkers of the disease.

Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100  μm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance.

This study provides evidence that low-intensity ultrasound directly affects nuclear processes, and the magnitude of the effect varies with frequency. In particular, we show that the transcriptional induction of first load-inducible genes, which is independent of new protein synthesis, is frequency dependent. Bovine chondrocytes were exposed to low-intensity (below the cavitational threshold) ultrasound at 2, 5 and 8 MHz. Ultrasound elevated the expression of early response genes c-Fos, c-Jun and c-Myc, maximized at 5 MHz. The phosphorylated ERK inhibitor PD98059 abrogated any increase in c-series gene expression, suggesting that signaling occurs via the MAPPK/ERK pathway. However, phosphorylated ERK levels did not change with ultrasound frequency, indicating that processes downstream of ERK phosphorylation (such as nuclear transport and chromatin reorganization) respond to ultrasound with frequency dependence. A quantitative, biphasic mathematical model based on Biot theory predicted that cytoplasmic and nuclear stress is maximized at 5.2 ± 0.8 MHz for a chondrocyte, confirming experimental measurements., (Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is the most common joint disease, characterized by matrix degradation and changes in chondrocyte morphology and metabolism. Literature reported that electromagnetic fields (EMFs) can produce benefits in OA patients, even if EMFs mechanism of action is debated. Human osteoarthritic chondrocytes isolated from femoral heads were cultured in vitro in bidimensional (2-D) flasks and in three-dimensional (3-D) alginate beads to mimic closely cartilage environment in vivo. Cells were exposed 30 min/day for 2 weeks to extremely low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic field (TAMMEF) with variable frequencies, intensities, and waveforms. Cell viability was measured at days 7 and 14, while healthy-cell density, heavily vacuolized (hv) cell density, and cluster density were measured by light microscopy only for 3-D cultures after treatments. Cell morphology was observed for 2-D and 3-D cultures by transmission electron microscopy (TEM). Chondrocyte exposure to TAMMEF enhances cell viability at days 7 and 14 compared to ELF. Light microscopy analysis showed that TAMMEF enhances healthy-cell density, reduces hv-cell density and clustering, compared to ELF. Furthermore, TEM analysis showed different morphology for 2-D (fibroblast-like) and 3-D (rounded shape) cultures, confirming light microscopy results. In conclusion, EMFs are effective and safe for OA chondrocytes. TAMMEF can positively interfere with OA chondrocytes representing an innovative non-pharmacological approach to treat OA.

Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1., Materials and Methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours., Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy., Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.

Low-frequency pulsed electromagnetic fields (PEMFs) are used for the treatment of human osteoarthritic cells in vivo without knowledge of underling principles. The authors evaluated the effect of PEMFs on human chondrocytes of the osteoarthritic knee in vitro. Biopsies of the cut femoral condyles after total knee arthroplasty were kept in a standard cell culture medium consisting of Dulbecco's modified Eagle's medium: nutrient mixture F-12, 10% fetal calf serum, PenStrept (Mediatech, Inc, Manassas, Virginia), and ascorbic acid for 4 days and randomly split into an exposed group (PEMF for 4 hours daily for 4 days at 75 Hz and 1.6 mT) and a control group. Both groups were retained for biochemical and polymerase chain reaction analysis (glycosaminoglycan and DNA levels). A P value less than .05 was considered significant.DNA analysis revealed no differences between groups and no increase in content after exposure (P=.88 and .66, respectively). The increase of glycosaminoglycans was 0.4±1.6 ng (95% confidence interval [CI], 1.4 to 0.5) and -0.5±1.8 ng (95% CI, 0.6 to -1.5) in the exposed and control groups, respectively, with no significant difference (P=.24). A smaller decrease of glycosaminoglycan and DNA levels was observed over 4 days in the exposed group compared with the control group, with no statistical significance. The authors concluded that low-frequency PEMFs do not significantly influence the biosynthetic activity of explantcultures of human osteoarthritic cells in vitro. Nevertheless, they may be suitable as an adjuvant to a larger treatment regimen., (Copyright 2013, SLACK Incorporated.)

The need for tissue-engineered constructs as replacement tissue continues to grow as the average age of the world's population increases. However, additional research is required before the efficient production of laboratory-created tissue can be realized. The multitude of parameters that affect cell growth and proliferation is particularly daunting considering that optimized conditions are likely to change as a function of growth. Thus, a generalized research platform is needed in order for quantitative studies to be conducted. In this article, an ultrasonic bioreactor is described for use in studying the response of cells to ultrasonic stimulation. The work is focused on chondrocytes with a long-term view of generating tissue-engineered articular cartilage. Aspects of ultrasound (US) that would negatively affect cells, including temperature and cavitation, are shown to be insignificant for the US protocols used and which cover a wide range of frequencies and pressure amplitudes. The bioreactor is shown to have a positive influence on several factors, including cell proliferation, viability, and gene expression of select chondrocytic markers. Most importantly, we show that a total of 138 unique proteins are differentially expressed on exposure to ultrasonic stimulation, using mass-spectroscopy coupled proteomic analyses. We anticipate that this work will serve as the basis for additional research which will elucidate many of the mechanisms associated with cell response to ultrasonic stimulation.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease of unknown etiology. Treatment of RA is very complex, and in the past years, some studies have investigated the use of low-level laser therapy (LLLT) in treatment of RA. However, it remains unknown if LLLT can modulate early and late stages of RA. With this perspective in mind, we evaluated histological aspects of LLLT effects in different RA progression stages in the knee. It was performed a collagen-induced RA model, and 20 male Wistar rats were divided into 4 experimental groups: a non-injured and non-treated control group, a RA non-treated group, a group treated with LLLT (780 nm, 22 mW, 0.10 W/cm(2), spot area of 0.214 cm(2), 7.7 J/cm(2), 75 s, 1.65 J per point, continuous mode) from 12th hour after collagen-induced RA, and a group treated with LLLT from 7th day after RA induction with same LLLT parameters. LLLT treatments were performed once per day. All animals were sacrificed at the 14th day from RA induction and articular tissue was collected in order to perform histological analyses related to inflammatory process. We observed that LLLT both at early and late RA progression stages significantly improved mononuclear inflammatory cells, exudate protein, medullary hemorrhage, hyperemia, necrosis, distribution of fibrocartilage, and chondroblasts and osteoblasts compared to RA group (p < 0.05). We can conclude that LLLT is able to modulate inflammatory response both in early as well as in late progression stages of RA.

Ultraviolet B (UVB) radiation acts as a strong apoptotic trigger in many cell types, in tumor and normal cells. Several studies have demonstrated that UVB-induced cell death occurs through the generation of reactive oxygen species. The consequent oxidative stress includes the impairment of cellular antioxidants, the induction of DNA damage and the occurrence of apoptosis. In this review, we investigated UVB apoptotic action in various cell models by using ultrastructural, molecular and cytofluorimetric techniques. Myeloid leukemia HL-60, T-lymphoblastoid Molt-4 and myelomonocytic U937 human cells, generally affected by apoptotic stimuli, were studied. Human chondrocytes and C2C12 skeletal muscle cells, known to be more resistant to damage, were also considered. All of them, when exposed to UVB radiation, revealed a number of characteristic apoptotic markers. Membrane blebbing, cytoplasm shrinkage and chromatin condensation were detected by means of electron microscopy. DNA cleavage, investigated by using agarose gel electrophoresis and TUNEL reaction, was observed in suspended cells. Differently, in chondrocytes and in skeletal muscle cells, oligonucleosomic DNA fragmentation did not appear, even if a certain TUNEL positivity was detected. These findings demonstrate that UVB radiation appears to be an ideal tool to study the apoptotic behavior.

Objective: The objectives for this study were to determine whether radical initiated photopolymerizations typically employed for cell encapsulations lead to oxidative stress incurred by chondrocytes and whether the development of a pericellular matrix (PCM) decreases this oxidative stress and has longer-term benefits on chondrocyte function., Methods: Freshly isolated bovine chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels devoid of a PCM or with a PCM, confirmed by immunocytochemistry (IC), and cultured for up to 2 weeks. Reactive oxygen species (ROS) production and damage to cell membrane by lipid peroxidation were accomplished using carboxy-2,7-difluorodihydrofluorescein diacetate (carboxy-H(2)DFFDA) and by malondialdehyde (MDA) content, respectively. Gene expression and proteoglycan synthesis were analyzed using reverse transcription (RT)-quantitative PCR (qPCR) and (35)SO(4) incorporation, respectively., Results: The photopolymerization reaction, which alone generates radicals and extracellular ROS, led to oxidative stress in chondrocytes evidenced by increased intracellular ROS and lipid peroxidation. The presence of a PCM decreased intracellular ROS and abrogated membrane lipid peroxidation, improved aggrecan, collagen II and collagen VI expression, and enhanced proteoglycan synthesis., Conclusions: The development of the PCM prior to photoencapsulation in PEG hydrogels reduces oxidative stress and improves chondrocyte anabolic activity. Our data suggest this reduction occurs by decreased ROS diffusion into the cell and decreased membrane damage. Our findings suggest that minimizing oxidative stress, such as through the presence of a PCM, may have long-term beneficial effects on tissue elaboration when employing photopolymerizations to encapsulate chondrocytes for cartilage tissue engineering applications., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to compare the radiofrequency (RF) injury effect on cartilage in the different settings that are mostly used in clinical work under rigidly controlled laboratory conditions., Methods: Twelve fresh porcine knees were used in our study. Five treatment areas were created on the femoral condyles of each knee: the control group, coagulation (setting 2) group, coagulation (setting 7) group, ablation (setting 2) group, and ablation (setting 7) group. Hematoxylin/eosin staining, dual fluorescence staining, and the GAG content were observed to evaluate the histological cartilage changes, vacuolar cell rate of chondrocytes, depth of chondrocyte death, and detection of GAG content., Results: Vacuolar cell rates of chondrocytes in each experimental group were higher than that in the control group (P < 0.05); there was no significant difference in vacuolar cell rate among experimental groups. Dual fluorescent staining showed that the ablation (setting 7) group had a smaller depth of cell death than did the coagulation (setting 2) group (P < 0.05); the other experimental groups showed no statistically significant difference (n.s.). In addition, there was no significant difference in GAG content between the experimental groups and control group (n.s.)., Conclusions: The coagulation mode results in heavier thermal radiation injury to chondrocytes than does the ablation mode. Higher-power settings in the ablation mode result in lower thermal radiation injury and may be most suitable for cartilage debridement.

Objective: To investigate the effect of linear polarized infrared light irradiation on the proliferation of primary cultured rabbit articular chondrocytes irradiation., Methods: Primary cultured rabbit articular chondrocytes were irradiated with linear polarized infrared light irradiation (Super Lizer) at the power intensity of 20% (360 mW) to 100% (1800 mW) for 5 min per day for 5 days, and CCK-8 assay was used to measure the proliferative activity of the chondrocytes after the exposures., Results: The D(450) values of the cells irradiated at the power intensity of 60% (1080 mW) and 80% (1440 mW) (1.88∓0.11 and 1.99∓0.05, respectively) were significantly greater than that of the control cells (0.95∓0.38, P<0.05). The irradiation with Super Lizer at lower (20%, 360 mW and 40%, 720 mW) or higher (100%, 1800 mW) power intensities did not cause significant increases of the D(450) values. Compared with the control group, Super Lizer irradiation at the power intensities of 20%, 40%, 60%, and 80% resulted in increment ratios of chondrocyte viability of (48.75∓15.4 )%, (67.02∓29.61)%, (97.93∓11.57)%, and (108.52∓5.81)%, respectively, but at 100% power intensity (1800 mW), the irradiation caused a lowered chondrocyte viability increment ratio of (62.84∓31.12)%., Conclusion: Linear polarized infrared light irradiation can promote the proliferation of chondrocytes in an intensity-dependent manner, and the strongest effect occurs with the irradiation power intensity of 80% (1440 mW).