Ionizing radiation exposure of stem cell-derived chondrocytes affects their gene and microRNA expression profiles and cytokine production.

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.

Steady Augmentation of Anti-Osteoarthritic Actions of Rapamycin by Liposome-Encapsulation in Collaboration with Low-Intensity Pulsed Ultrasound.

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.

Pulsed electromagnetic fields promote repair of focal articular cartilage defects with engineered osteochondral constructs.

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.

Effects of low-level laser therapy on the organization of articular cartilage in an experimental microcrystalline arthritis model.

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 Na4P2O7 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-ß 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.

Biophysical stimulation of bone and cartilage: state of the art and future perspectives.

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 A2A and A3 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.

Chondrocytes differentiated from human induced pluripotent stem cells: Response to ionizing radiation.

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.

Short term histopathological effects of GaAlAs laser on experimentally induced TMJ osteoarthritis in rabbits.

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.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

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 (CO2) 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 CO2 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.

Chondroitin sulfate and glucosamine sulfate associated to photobiomodulation prevents degenerative morphological changes in an experimental model of osteoarthritis in rats.

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-1ß) 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-1ß 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.

Short term histopathological effects of GaAlAs laser on experimentally induced TMJ osteoarthritis in rabbits

Abstract 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.

TGF-ß1-induced chondrogenesis of bone marrow mesenchymal stem cells is promoted by low-intensity pulsed ultrasound through the integrin-mTOR signaling pathway.

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-ß1-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-ß1-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-ß1-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-ß1-induced chondrogenesis of BMSCs through the integrin-mTOR signaling pathway.

Extracorporeal Shockwave Therapy Enhances Expression of Pdia-3 Which Is a Key Factor of the 1α,25-Dihydroxyvitamin D 3 Rapid Membrane Signaling Pathway in Treatment of Early Osteoarthritis of the Knee.

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.

Correlating rheological properties and printability of collagen bioinks: the effects of riboflavin photocrosslinking and pH.

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.

Effect of a low-frequency pulsed electromagnetic field on expression and secretion of IL-1ß and TNF-α in nucleus pulposus cells.

Objective To investigate changes in nucleus pulposus cell expression and secretion of interleukin (IL)-1ß 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-1ß and TNF-α within the nucleus pulposus cells was measured using western blotting, and levels of IL-1ß 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-1ß and TNF-α expression levels. LF-PEMFs did not significantly affect cell proliferation, however, levels of IL-1ß 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-1ß and TNF-α in cultured nucleus pulposus cells.

Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.

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/cm2 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, Ca2+ 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, Ca2+ 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 BKca 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 Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

The effects of low-dose radiotherapy on fresh osteochondral allografts: An experimental study in rabbits.

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.

[Low-intensity pulsed ultrasound promotes extracellular matrix synthesis of human osteoarthritis chondrocytes].

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/cm2 LIPUS-treated OA group, 30 mW/cm2 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.

The effects of ionizing radiation on the growth plate in rat tibiae.

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.

Serial changes in the proliferation and differentiation of adipose-derived stem cells after ionizing radiation.

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 ß-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 ß-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.

Effects of microcurrent therapy on excisional elastic cartilage defects in young rats.

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.