RESUMEN
Chondrogenic differentiated mesenchymal stromal cells (MSCs) are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D) culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL)-10 and Tumor Necrosis Factor (TNF)α on chondrogenesis by MSCs in 3D high-density (H-D) culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA) scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls.
Asunto(s)
Condrocitos/citología , Condrogénesis , Interleucina-10/farmacología , Células Madre Mesenquimatosas/citología , Factor de Necrosis Tumoral alfa/metabolismo , Agrecanos/genética , Agrecanos/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ácido Poliglicólico/farmacología , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Interplay between complement factors, regulatory proteins, anaphylatoxins and cytokines could be involved in tendon healing and scar formation. The expression and regulation of complement factors by cytokines or anaphylatoxins are completely unclear in tendon. Hence, the gene expression of the anaphylatoxin receptors C3aR, C5aR and cytoprotective complement regulatory proteins (CRPs) was analysed in human tendon, cultured primary tenocytes and to directly compare the general expression level, additionally in human leukocytes. Time-dependent regulation of complement by cytokines and the anaphylatoxin C3a was assessed in cultured tenocytes. Gene expression of the anaphylatoxin receptors C3aR, C5aR and the CRPs CD46, CD55 and CD59 was detected in tendon, cultured tenocytes and leukocytes, whereas CD35 could only be found in tendon and leukocytes. Compared with cultured tenocytes, complement expression was higher in tendon and compared with leukocytes C3aR, C5aR, CD35 and CD55, but not CD46 and CD59 gene expression levels were lower in tendon. C3aR mRNA was up-regulated by both TNFα and C3a in cultured tenocytes in a time-dependent manner whereby C5aR gene expression was only induced by C3a. IL-6 or C3a impaired the CRP gene expression. C3a stimulation lead to an up-regulation of TNFα and IL-1ß mRNA in tenocytes. Degenerated tendons revealed an increased C5aR and a reduced CD55 expression. The expression profile of the investigated complement components in tendon and cultured tenocytes clearly differed from that of leukocytes. Tenocytes respond to the complement split fragment C3a with CRP suppression and enhanced pro-inflammatory cytokine gene expression suggesting their sensitivity to complement activation.
Asunto(s)
Complemento C3/farmacología , Células del Tejido Conectivo/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptor de Anafilatoxina C5a/genética , Receptores de Complemento/genética , Tendones/efectos de los fármacos , Adulto , Antígenos CD55/genética , Antígenos CD55/inmunología , Antígenos CD59/genética , Antígenos CD59/inmunología , Activación de Complemento/efectos de los fármacos , Complemento C3/inmunología , Células del Tejido Conectivo/citología , Células del Tejido Conectivo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/inmunología , Interleucina-1beta/farmacología , Interleucina-6/inmunología , Interleucina-6/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/inmunología , Especificidad de Órganos , Cultivo Primario de Células , ARN Mensajero/inmunología , Receptor de Anafilatoxina C5a/inmunología , Receptores de Complemento/inmunología , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/inmunología , Tendones/citología , Tendones/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures. Subsequently, tenocytes gene expression of extra-cellular matrix (ECM) components (type I collagen, decorin, fibronectin), the cell-ECM receptor ß1-integrin, the angiogenic factor myodulin, ECM degrading matrix-metalloproteinase (MMP)1 and pro-inflammatory cytokines (interleukin [IL]-1ß, tumour necrosis factor [TNFα] and IL-6) was analysed. The only significant effect of leukocytes on tenocytes ECM genes expression was a suppression of type I collagen by neutrophils combined with TNFα stimulation. The same effect could be observed analysing the ß1-integrin and myodulin gene expression. However, PBMCs up-regulated significantly cytokine and MMP1 gene expression in tenocytes. These in vitro results suggest that mononuclear cells could present an exogenic stimulus for the induction of pro-inflammatory and catabolic mediators in tendon.