Regenerative Medicine Applied to Musculoskeletal Diseases in Equines: A Systematic Review.

Musculoskeletal injuries in horses have a great economic impact, predominantly affecting tendons, ligaments, and cartilage, which have limited natural regeneration. Cell therapy, which uses mesenchymal stem cells due to their tissue differentiation properties and anti-inflammatory and immunoregulatory effects, aims to restore damaged tissue. In this manuscript, we performed a systematic review using the Parsifal tool, searching the PubMed and Web of Science databases for articles on regenerative medicine for equine musculoskeletal injuries. Our review covers 17 experimental clinical studies categorized by the therapeutic approach used: platelet-rich plasma, conditioned autologous serum, mesenchymal stem cells, and secretome. These therapies reduce healing time, promote regeneration of fibrocartilaginous tissue, improve cellular organization, and improve joint functionality and sustainability. In conclusion, regenerative therapies using platelet-rich plasma, conditioned autologous serum, equine mesenchymal stem cells, and the emerging field of the secretome represent a promising and highly effective approach for the treatment of joint pathologies in horses, implying a valuable advance in equine healthcare.

Application of adipose-derived mesenchymal stem cells in an in vivo model of peripheral nerve damage.

Background: Neuropathic pain is one of the most difficult to treat chronic pain syndromes. It has significant effects on patients' quality of life and substantially adds to the burden of direct and indirect medical costs. There is a critical need to improve therapies for peripheral nerve regeneration. The aim of this study is to address this issue by performing a detailed analysis of the therapeutic benefits of two treatment options: adipose tissue derived-mesenchymal stem cells (ASCs) and ASC-conditioned medium (CM). Methods: To this end, we used an in vivo rat sciatic nerve damage model to investigate the molecular mechanisms involved in the myelinating capacity of ASCs and CM. Furthermore, effect of TNF and CM on Schwann cells (SCs) was evaluated. For our in vivo model, biomaterial surgical implants containing TNF were used to induce peripheral neuropathy in rats. Damaged nerves were also treated with either ASCs or CM and molecular methods were used to collect evidence of nerve regeneration. Post-operatively, rats were subjected to walking track analysis and their sciatic functional index was evaluated. Morphological data was gathered through transmission electron microscopy (TEM) of sciatic nerves harvested from the experimental rats. We also evaluated the effect of TNF on Schwann cells (SCs) in vitro. Genes and their correspondent proteins associated with nerve regeneration were analyzed by qPCR, western blot, and confocal microscopy. Results: Our data suggests that both ASCs and CM are potentially beneficial treatments for promoting myelination and axonal regeneration. After TNF-induced nerve damage we observed an upregulation of c-Jun along with a downregulation of Krox-20 myelin-associated transcription factor. However, when CM was added to TNF-treated nerves the opposite effect occurred and also resulted in increased expression of myelin-related genes and their corresponding proteins. Conclusion: Findings from our in vivo model showed that both ASCs and CM aided the regeneration of axonal myelin sheaths and the remodeling of peripheral nerve morphology.

Extracellular vesicle and soluble fractions of adipose tissue-derived mesenchymal stem cells secretome induce inflammatory cytokines modulation in an in vitro model of discogenic pain.

BACKGROUND CONTEXT: Mesenchymal stem cells (MSCs) secretome or conditioned medium (CM) is a complex cocktail of different molecules, some of which, particularly those contained in extracellular vesicles, already have proven therapeutic applications. PURPOSE: CM may well represent promising therapy for discogenic pain and the intention of this work is to assess its therapeutic potential using an in vitro model of this condition. STUDY DESIGN: This is an experimental study. METHODS: Our in vitro model comprised nucleus pulposus (NP) and annulus fibrosus (AF) cells inflamed with TNF. To assess the potential therapeutic value of CM and its components, extracellular vesicles (EVs) and soluble culture fraction (SF), cell inflammation took place under 3 different conditions: either in the presence of whole CM, isolated EVs or SF, and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and neurotrophic factors produced in all 3 cases were compared. RESULTS: In the presence of whole CM, both in vitro gene expression by the NP and AF test cells and analysis of their protein content showed high modulatory effects on inflammation and MMP inhibition. The presence of EVs and SF showed similar but much smaller effects, and this was particularly marked in the case of NP cells. CONCLUSIONS: Our results show that, compared to EVs and SF, the presence of whole CM has the greatest positive effect on the modulation of pro-inflammatory and catabolic factors. These observations suggest that CM could protect against inflammation and the resulting intervertebral disc (IVD) degeneration that leads to discogenic pain. CLINICAL SIGNIFICANCE: Many patients' expectations are not met by current non-operative and surgical treatments for discogenic low back pain. We propose the use of the MSCs secretome for assessing its potential as cell-free therapy to treat degenerative disc disease modulating the inflammatory response.

Isolation and morphological characterization of equine mesenchymal stem cells from harvested adipose tissue and bone marrow and stably transfected with green fluorescent protein.

OBJECTIVE: To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. SAMPLE: MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. PROCEDURES: Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characteristics of transfected and nontransfected AT-MSCs and BM-MSCs were compared with electron microscopy. RESULTS: The protocols for transfection and subsequent isolation of transfected cells with use of G418 were suitable for obtaining transfected MSCs. Transfection efficiency was 5% in AT-MSCs and 4% in BM-MSCs. Characterization of transfected and nontransfected MSCs revealed that they share immunocytochemical and morphological profiles. Expression of CD90 was significantly higher for transfected versus nontransfected AT-MSCs (97% vs 92%). Expression of CD105 was significantly lower for transfected versus nontransfected BM-MSCs (85% vs 94%). Transfected BM-MSCs had differences in organelles, compared with the other cell types, specifically including most commonly the rough endoplasmic reticulum with dilated cisternae and mitochondria. CONCLUSION AND CLINICAL RELEVANCE: These findings contribute to the knowledge base of the characteristics of equine AT-MSCs and BM-MSCs and of transfected versus nontransfected equine MSCs. The data provided a valuable starting point for researchers wishing to further study the morphological characteristics of equine MSCs.

Effect of mesenchymal stem cells combined with chondroitin sulfate in an in vitro model of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease affecting the whole joint structure. The specific molecules responsible for the inflammatory processes involved in the development of OA have been the focus of many studies. Adipose tissue-derived mesenchymal stem cells (ASCs) constitute a promising cell-based therapy which could be used as an alternative to or in combination with drug therapies. Chondroitin sulfate (CS) plays a protective role in the joint by decreasing concentrations of pro-inflammatory cytokines and therefore has an important part in moderating chondrocyte metabolism. The aim of this study is to use an in vitro model of OA to evaluate the combined effectiveness of CS and ASCs as a treatment. We give a detailed discussion of the roles of cytokines and other key molecules involved in OA. In addition, we report the effects of treating inflamed chondrocytes with ASCs and CS on the expression of specific cartilage genes. Findings show that both treatments reduced expression of all genes associated with the pro-inflammatory cytokines we analyzed. However, we saw no increase in the expression of the specific genes encoding for cartilage matrix proteins, such as collagen type II and aggrecan. This study shows the effectiveness of combining ASCs and CS in the treatment of OA.

Inflamación en la hernia del disco intervertebral / Inflammation in the intervertebral disc herniation

Hasta no hace muchos años, se consideró que el dolor ciático, en la hernia del disco intervertebral lumbar, estaba originado por la compresión sobre la raíz nerviosa. Sin embargo, la hernia del disco intervertebral lumbar presenta cuadros muy heterogéneos inexplicables con el simple compromiso mecánico. En los últimos años, numerosos estudios de inmuhistoquímica y de biología molecular han demostrado que el tejido herniado no es un material inerte sino, por el contrario, biológicamente muy activo con capacidad de expresar una serie de mediadores de inflamación entre los que destacan citoquinas proinflamatorias como la interleuquina1, interleuquina 6, interleuquina 8 y el factor de necrosis tumoral. La inflamación parece estar no solo inducida por la irritación química de las sustancias bioactivas liberadas por el núcleo pulposo, sino también mediante una respuesta autoinmune contra el mismo. Por tanto, además del factor mecánico, la mediación bioquímica tiene un papel importante en la fisiopatología del dolor ciático y de la radiculopatía. A través de una extensa revisión sistemática de la literatura se han investigado los mediadores celulares y moleculares que intervienen en dicho proceso inflamatorio alrededor de la hernia del disco intervertebral lumbar y su implicación en el dolor ciático (AU)

Up until fairly recently, it was thought that sciatic pain in the lumbar herniated disc was caused by compression on the nerve root. However, the lumbar herniated disc shows mixed pictures which are difficult to explain by simple mechanical compromise. In recent years various immunology, immunohistochemistry and molecular biology studies have shown that the herniated tissue is not an inert material, but rather it Is biologically very active with the capability of expressing a series of inflammatory mediators: cytokines such as interleukin-1, interleukin-6, interleuquin-8 and tumor necrosis factor being the ones which stand out. The inflammation is not only induced by the chemical irritation of the bioactive substances released by the nucleus pulposus but also by an autoimmune response against itself. Thus, in addition to the mechanical factor, the biomechanical mediation plays an important role in the pathophysiology of sciatic pain and of radiculopathy. Through a review of a wide range of literature, we researched the cellular molecular mediators involved in this inflammatory process around the lumbar herniated disc and its involvement in sciatic pain (AU)

Access to Information and Degree of Community Awareness of Preventive Health Measures in the Face of COVID-19 in Spain.

The COVID-19 pandemic is posing a major health crisis. Spanish legislation establishes the mandatory use of masks and the implementation of hygienic measures such as hand washing and physical distancing. The aim of this study is to describe access to information and the level of community knowledge/adoption about the preventive measures proposed by the Spanish health authorities in response to the COVID-19 pandemic and to analyze the influence of socio-demographic factors in compliance among people over 18 years of age resident in Spain. An observational, descriptive and transversal study was conducted. Data was collected on sociodemographic variables, access to information and the degree of knowledge/adoption about the preventive measures: use of masks, hand hygiene and physical distancing. A total of 1811 people participated. The average age was 45.1 ± 15.1 years, predominantly female (69.3%), from an urban geographical area (74%), with a higher education level of 53.2%. Most of the respondents (57.5%) are or live with people at risk. The main access to information on preventive measures was from secondary sources (49.2%), with television being the main medium; 72.3% think that there are some difficulties in accessing information, while 8.7% of the participants do not consider the use of masks to be useful. As regards the choice of type of mask, the majority of people (44.8%) opt for the surgical variety; 88.5% of respondents believe that the physical distancing established is at least 1.5 m. This study confirmed that socio-demographic factors influence compliance with or the degree of knowledge/adoption of the preventive measures proposed to combat the COVID-19 pandemic and has made it possible to ascertain the sectors of the population with the greatest deficiencies in this respect. It shows the importance of implementing health information and education systems in the community, and it is advisable to promote specific programs aimed at men, people living in rural areas and people with a low level of education.

Isolation and characterization of exosomes from adipose tissue-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are the subject of intense research as they are a potential therapeutic tool for several clinical applications. The new MSCs action models are focused on the use of MSC-derived secretome which contains several growth factors, cytokines, microRNAs, and extracellular vesicles such as exosomes. Exosomes have recently emerged as a component with great potential involved as mediators in cellular communication. The isolation and identification of exosomes has made it possible for them to be used in cell-free therapies. The purposes of this study are: (i) to detect exosomes released into adipose-derived MSC conditioned cell culture medium, (ii) to identify exosome morphology, and (iii) to carry out a complete characterization of said exosomes. Moreover, it is aimed at determining which method for exosome isolation would be best to use. Precipitation has been identified as a highly useful method of exosome isolation since it provides higher efficiency and purity values than other methods. A broad characterization of the exosomes present in the MSC-conditioned medium was also carried out. This work fills a gap in the existing literature on bioactive molecules which have attracted a great deal of interest due to their potential use in cellular therapies.

Inflammation in the intervertebral disc herniation. / Inflamación en la hernia del disco intervertebral.

Up until fairly recently, it was thought that sciatic pain in the lumbar herniated disc was caused by compression on the nerve root. However, the lumbar herniated disc shows mixed pictures which are difficult to explain by simple mechanical compromise. In recent years various immunology, immunohistochemistry and molecular biology studies have shown that the herniated tissue is not an inert material, but rather it Is biologically very active with the capability of expressing a series of inflammatory mediators: cytokines such as interleukin-1, interleukin-6, interleuquin-8 and tumor necrosis factor being the ones which stand out. The inflammation is not only induced by the chemical irritation of the bioactive substances released by the nucleus pulposus but also by an autoimmune response against itself. Thus, in addition to the mechanical factor, the biomechanical mediation plays an important role in the pathophysiology of sciatic pain and of radiculopathy. Through a review of a wide range of literature, we researched the cellular molecular mediators involved in this inflammatory process around the lumbar herniated disc and its involvement in sciatic pain.

Immunomodulation of mesenchymal stem cells in discogenic pain.

BACKGROUND CONTEXT: Back pain is a highly prevalent health problem in the world today and has a great economic impact on health-care budgets. Intervertebral disc (IVD) degeneration has been identified as a main cause of back pain. Inflammatory cytokines produced by macrophages or disc cells in an inflammatory environment play an important role in painful progressive degeneration of IVD. Mesenchymal stem cells (MSCs) have shown to have immunosuppressive and anti-inflammatory properties. Mesenchymal stem cells express a variety of chemokines and cytokines receptors having tropism to inflammation sites. PURPOSE: This study aimed to develop an in vitro controlled and standardized model of inflammation and degeneration of IVD with rat cells and to evaluate the protective and immunomodulatory effect of conditioned medium (CM) from the culture of MSCs to improve the conditions presented in herniated disc and discogenic pain processes. STUDY DESIGN: This is an experimental study. METHODS: In this study, an in vitro model of inflammation and degeneration of IVD has been developed, as well as the effectiveness of CM from the culture of MSCs. RESULTS: Conditioned medium from MSCs downregulated the expression of various proinflammatory cytokines produced in the pathogenesis of discogenic pain such as interleukin (IL)-1ß, IL-6, IL-17, and tumor necrosis factor (TNF). CONCLUSION: Mesenchymal stem cells represent a promising alternative strategy in the treatment of IVD degeneration inasmuch as there is currently no treatment which leads to a complete remission of long-term pain in the absence of drugs.

Assessment of regeneration in meniscal lesions by use of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

OBJECTIVE To assess the ability to regenerate an equine meniscus by use of a collagen repair patch (scaffold) seeded with mesenchymal stem cells (MSCs) derived from bone marrow (BM) or adipose tissue (AT). SAMPLE 6 female Hispano-Breton horses between 4 and 7 years of age; MSCs from BM and AT were obtained for the in vitro experiment, and the horses were subsequently used for the in vivo experiment. PROCEDURES Similarities and differences between MSCs derived from BM or AT were investigated in vitro by use of cell culture. In vivo assessment involved use of a meniscus defect and implantation on a scaffold. Horses were allocated into 2 groups. In one group, defects in the medial meniscus were treated with MSCs derived from BM, whereas in the other group, defects were treated with MSCs derived from AT. Defects were created in the contralateral stifle joint but were not treated (control samples). RESULTS Both types of MSCs had universal stem cell characteristics. For in vivo testing, at 12 months after treatment, treated defects were regenerated with fibrocartilaginous tissue, whereas untreated defects were partially repaired or not repaired. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSCs derived from AT could be a good alternative to MSCs derived from BM for use in regenerative treatments. Results also were promising for a stem cell-based implant for use in regeneration in meniscal lesions. IMPACT FOR HUMAN MEDICINE Because of similarities in joint disease between horses and humans, these results could have applications in humans.

c-Jun N-terminal kinase 1 is required for Toll-like receptor 1 gene expression in macrophages.

The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.

Effects of extracellular matrix on the morphology and behaviour of rabbit auricular chondrocytes in culture.

Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, a sugar extract, and hyaluronan to the medium of previously dedifferentiated chondrocytes. When protein extract was added, many cartilaginous nodules appeared. Hyaluronan produced changes in cell phenotype and behaviour, but not nodule formation. Protein extract has positive effects on the differentiation of previously proliferated chondrocytes and permits nodule formation and the extensive production of type-II collagen. A comparison with incompletely dissociated chondrocyte cultures suggests that the presence of some living cells anchored to their natural extracellular matrix provides some important additional factors for the phenotypical stability of chondrocytes on plastic surfaces. In order to elucidate if it is possible that the incidence of apoptosis is related to the results, we also characterized the molecular traits of apoptosis.