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.

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.

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.

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.

Adipose mesenchymal stromal cells response to ionizing radiation.

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.

Laser radiation effect on chondrocytes and intercellular matrix of costal and articular cartilage impregnated with magnetite nanoparticles.

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.

Cell death in human articular chondrocyte: a morpho-functional study in micromass model.

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.

Low-dose γ-radiation inhibits IL-1ß-induced dedifferentiation and inflammation of articular chondrocytes via blockage of catenin signaling.

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)-1ß-induced chondrocyte destruction without causing side effects, such as cell death and senescence. IL-1ß treatment induced an increase in the expression of α-, ß-, 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-1ß-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.

Optimum temperature for extracellular matrix production by articular chondrocytes.

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.

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

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.

Effects of low-level laser therapy on joint pain, synovitis, anabolic, and catabolic factors in a progressive osteoarthritis rabbit model.

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 1ß (IL-1ß), 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-ß). 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.

Proteomics of human primary osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF EMFs) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF).

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.

Human osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF) and therapeutic application of musically modulated electromagnetic fields (TAMMEF) systems: a comparative study.

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.

Low-level laser therapy in different stages of rheumatoid arthritis: a histological study.

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.

The role of the PCM in reducing oxidative stress induced by radical initiated photoencapsulation of chondrocytes in poly(ethylene glycol) hydrogels.

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.

The effect of low-level laser to apoptosis of chondrocyte and caspases expression, including caspase-8 and caspase-3 in rabbit surgery-induced model of knee osteoarthritis.

The purpose of this study was to observe the effect of 810-nm low-level laser to apoptosis of chondrocyte and related proteins, including caspase-3 and caspase-8, in rabbit surgery-induced model of knee osteoarthritis. A total of 24 New Zealand White rabbits were randomly assigned into 3 groups: test group, model group, and normal group. The rabbits in test group and model group received anterior cruciate ligament transection in the right knee. Six weeks after transection, the rabbits in test group were given 10-session 810-nm laser illumination. Eight weeks after transection, all animal were killed. Modified Mankin Score was made for histological assessment. The caspases expressions and chondrocytes apoptosis were tested using the immunohistochemistry and TUNEL assessment, respectively. The modified Mankin Score of test group was significantly lower than model group (P < 0.01) and higher than normal group (P < 0.01). The caspase-8 expression of test group was lower than model group and higher than normal group, but no significant difference was found (P > 0.05). This study revealed that the 810-nm low-level laser can improve cartilage structure, prevent articular cartilage degradation and significantly decrease the expression of caspase-3 in this surgery-induced OA model. Further studies are needed.

Effects of radiofrequency energy on porcine articular cartilage: higher-power settings in ablation mode show lower thermal radiation injury.

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.