Tenogenic differentiation of equine adipose-tissue-derived stem cells under the influence of tensile strain, growth differentiation factors and various oxygen tensions
Abstract:Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation t… Show more
“…On one hand, ASCs isolated from a variety of animal species have been reported to upregulate tenogenic markers in vitro under specific treatment, 13,14,18,20,51 suggesting the tenogenic potential of ASCs. On the other hand, Eagan et al questioned the suitability of hASCs for tendon tissue engineering and reported the lack of any significant and consistent upregulation in the expression of COL I, TNC, or SCX, in hASCs treated for up to 4 weeks with TGF-b1 or IGF-1.…”
Because of the limited and unsatisfactory outcomes of clinical tendon repair, tissue engineering approaches using adult mesenchymal stem cells are being considered a promising alternative strategy to heal tendon injuries. Successful and functional tendon tissue engineering depends on harnessing the biochemical cues presented by the native tendon extracellular matrix (ECM) and the embedded tissue-specific biofactors. In this study, we have prepared and characterized the biological activities of a soluble extract of decellularized tendon ECM (tECM) on adult adipose-derived stem cells (ASCs), on the basis of histological, biochemical, and gene expression analyses. The results showed that tECM enhances the proliferation and transforming growth factor (TGF)-b3-induced tenogenesis of ASCs in both plate and scaffold cultures in vitro, and modulates matrix deposition of ASCs seeded in scaffolds. These findings suggest that combining tendon ECM extract with TGFb3 treatment is a possible alternative approach to induce tenogenesis for ASCs.
“…On one hand, ASCs isolated from a variety of animal species have been reported to upregulate tenogenic markers in vitro under specific treatment, 13,14,18,20,51 suggesting the tenogenic potential of ASCs. On the other hand, Eagan et al questioned the suitability of hASCs for tendon tissue engineering and reported the lack of any significant and consistent upregulation in the expression of COL I, TNC, or SCX, in hASCs treated for up to 4 weeks with TGF-b1 or IGF-1.…”
Because of the limited and unsatisfactory outcomes of clinical tendon repair, tissue engineering approaches using adult mesenchymal stem cells are being considered a promising alternative strategy to heal tendon injuries. Successful and functional tendon tissue engineering depends on harnessing the biochemical cues presented by the native tendon extracellular matrix (ECM) and the embedded tissue-specific biofactors. In this study, we have prepared and characterized the biological activities of a soluble extract of decellularized tendon ECM (tECM) on adult adipose-derived stem cells (ASCs), on the basis of histological, biochemical, and gene expression analyses. The results showed that tECM enhances the proliferation and transforming growth factor (TGF)-b3-induced tenogenesis of ASCs in both plate and scaffold cultures in vitro, and modulates matrix deposition of ASCs seeded in scaffolds. These findings suggest that combining tendon ECM extract with TGFb3 treatment is a possible alternative approach to induce tenogenesis for ASCs.
“…Only limited cross-reactivity of monoclonal antibodies between species has been demonstrated in an earlier study [Ibrahim et al, 2007], in which only 14 of 379 monoclonal antibodies against human CD molecules showed cross-reactivity with equine leukocytes. Different techniques such as gene transfection [Wang et al, 2005a], gene transduction [Murray et al, 2010] and application of tensile strain, GDFs and oxygen tension [Raabe et al, 2013] have been attempted for the induction of tenogenic differentiation in MCSs. We evaluated the effect of exogenous supplementation of BMP-12 on tenogenic differentiation of equine AF-MSCs and observed that equine AF-MSCs could be differentiated into tenocytes by day 14 following supplementation of BMP-12 (50 ng/ml) to the growth medium, similar to observations by Violini et al [2009] for equine bone marrow, while 10 ng/ml BMP-12 supplementation in- which regulates the assembly of collagen fibrils and acquisition of biomechanical properties during tendon development [Zhang et al, 2006].…”
Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry, but did not express haematopoietic markers (CD34, CD45 and CD14). In flow cytometry, the expression of CD29, CD44, CD73 and CD90 was observed in 68.83 ± 1.27, 93.66 ± 1.80, 96.96 ± 0.44 and 93.7 ± 1.89% of AF-MSCs, respectively. Osteogenic, chondrogenic and adipogenic differentiation of MSCs was confirmed by von Kossa and Alizarin red S, Alcian blue and oil red O staining, respectively. Upon supplementation of MSC growth media with 50 ng/ml bone morphogenetic protein (BMP)-12, AF-MSCs differentiated to tenocytes within 14 days. The differentiated cells were more slender, elongated and spindle shaped with thinner and longer cytoplasmic processes and showed expression of tenomodulin and decorin by RT-PCR and immunocytochemistry. In flow cytometry, 96.7 ± 1.90 and 80.9 ± 6.4% of differentiated cells expressed tenomodulin and decorin in comparison to 1.6 and 3.1% in undifferentiated control cells, respectively. Our results suggest that AF is an easily accessible and effective source of MSCs. On BMP-12 supplementation, AF-MSCs can be differentiated to tenocytes, which could be exploited for regeneration of ruptured or damaged tendon in race horses.
“…At first, there was the belief that tissue repair was obtained through the in vivo differentiation of the MSCs implanted to the prevailing cellular type in the tissue, i.e., when the MSCs were implanted in the tendon they would become cells similar to the tenocytes, thus favoring the synthesis of extracellular matrix (ECM) components and establishing the tissue repair. However, so far, it is known that the MSCs have tenogenic differentiation potential in vitro (RAABE et al, 2013). Evidences that the implanted MSCs may differentiate from the tendon cells in vivo still lack (CARVALHO et al, 2014b).…”
Section: The Action Mechanism Of Mscsmentioning
confidence: 99%
“…The adipose tissue is another important source, which stands out in equine tendinitis therapy (MUTTINI et al, 2012;REED;LEAHY, 2013). Recent studies show the tenogenic differentiation potential of mesenchymal stem cells from adipose tissue (AdMSCs), and they evidence the therapeutic potential of this source for equine tendinitis treatment (RAABE et al, 2013).…”
Tendinitis is an important high-relapse-rate disease, which compromises equine performance and may result in early athletic life end to affected animals. Many therapies have been set to treat equine tendinitis; however, just few result in improved relapse rates, quality of extracellular matrix (ECM) and increased biomechanical resistance of the treated tissue. Due to advances in the regenerative medicine, promising results were initially obtained through the implantation of mesenchymal stem cells (MSC) derived from the bone marrow in the equine tendon injury. Since then, many studies have been using MSCs from different sources for therapeutic means in equine. The adipose tissue has appeared as feasible MSC source. There are promising results involving equine tendinitis therapy using mesenchymal stem cells from adipose tissue (AdMSCs). Keywords: Tendon. Therapy. Stem cells. Regenerative medicine.
ResumoA tendinite é uma importante afecção com alta taxa de recidivas que compromete o desempenho de equinos e pode resultar no término precoce da vida atlética dos animais acometidos. Diversas terapias têm sido estabelecidas para o tratamento da tendinite equina, no entanto poucas resultam na melhora da taxa de recidiva, melhora da qualidade da matriz extracelular (MEC) e aumento da resistência biomecânica do tecido tratado. Com o advento da medicina regenerativa resultados promissores foram inicialmente obtidos com o implante de células tronco mesenquimais (CTMs) derivadas da medula óssea na lesão tendínea de equinos. Desde então, diversos estudos têm sido conduzidos utilizando CTMs de diferentes fontes para esta finalidade terapêutica em equinos. O tecido adiposo tem se mostrado como uma fonte viável de CTMs e também há resultados promissores envolvendo a terapia da tendinite equina utilizando as células tronco mesenquimais derivadas do tecido adiposo (AdCTMs). Neste artigo, dados relacionados ao uso das AdCTMs na terapia da tendinite equina serão revisados. Palavras-chave: Tendão. Terapia. Células tronco. Medicina regenerativa.
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