RESUMO
Cultivation of autologous human tenocytes in a cell-free xenogenic extracellular tendon matrix (xECM) could present an approach for tendon reconstruction. The aim of this study was to achieve tendon-like tissue formation by implanting decellularized porcine Achilles tendons recellularized with human hamstring tendon-derived tenocytes into nude mice. The structure of decellularized xECM was histologically monitored before being dynamically reseeded with human tenocytes. After 6â»12 weeks in vivo, construct quality was monitored using macroscopical and histological scoring systems, vitality assay and quantitative DNA and glycosaminoglycan (GAG) assays. For comparison to tendon xECM, a synthetic polyglycolic acid (PGA) polymer was implanted in a similar manner. Despite decellularized xECM lost some GAGs and structure, it could be recellularized in vitro with human tenocytes, but the cell distribution remained inhomogeneous, with accumulations at the margins of the constructs. In vivo, the xECM constructs revealed in contrast to the PGA no altered size, no inflammation and encapsulation and a more homogeneous cell distribution. xECM reseeded with tenocytes showed superior histological quality than cell-free implanted constructs and contained surviving human cells. Their DNA content after six and 12 weeks in vivo resembled that of native tendon and xECM recellularized in vitro. Results suggest that reseeded decellularized xECM formed a tendon-like tissue in vivo.
Assuntos
Tendão do Calcâneo/metabolismo , Matriz Extracelular/transplante , Xenoenxertos/metabolismo , Tenócitos/transplante , Tendão do Calcâneo/citologia , Animais , Células Cultivadas , Glicosaminoglicanos/metabolismo , Xenoenxertos/citologia , Humanos , Camundongos Nus , Ácido Poliglicólico/metabolismo , Suínos , Traumatismos dos Tendões/cirurgia , Tenócitos/citologia , Engenharia Tecidual/métodos , Transplante HeterólogoRESUMO
Tissue engineering of an anterior cruciate ligament (ACL) implant with ACL cells requires detailed analysis of the tissue characteristics that should be mimicked. Therefore, we studied the histological and biochemical properties of rabbit derived ACLs in comparison to other knee-associated tendons that are used as autografts in men. Rabbit derived ACLs and Musculus (M.) semimembranosus, M. semitendinosus tendons and patellar ligaments were explanted from adult New Zealand white rabbits and analyzed histologically for tissue organization (e.g. cellularity, nuclear shapes, elastic fibers), total collagen and sulfated glycosaminoglycan (sGAG) contents. Gene expression analysis was performed for the main extracellular matrix (ECM) components type I collagen, decorin and the glycoprotein tenomodulin. The ACLs had a dimension of 1.39x0.39x0.1 cm in situ. They were characterized by high sGAG content in comparison to the other tendons/ligaments, whereas the total collagen content did not differ. ACLs possessed higher cellularity and lower feret diameter of the cell nuclei compared with the investigated rabbit-derived tendons. In ACLs long elastic fibers were observed. Concerning the gene expression level, lower transcription of tenomodulin was detected in the ACL compared with the other tendons, without significant difference in the decorin gene expression. The M. semitendinosus tendon had a significantly higher type I collagen expression than the ACL and the other investigated tendons. This phenotypical characterization of the lapine ACL presented in this study provides some key standards to evaluate tissue engineered ACL constructs to be tested in the rabbit model.
Assuntos
Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/metabolismo , Engenharia Tecidual , Animais , Autoenxertos , Fenômenos Biomecânicos , Modelos Animais de Doenças , Humanos , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Transplante AutólogoRESUMO
Hypothesizing that the implantation of non-articular (heterotopic) chondrocytes might be an alternative approach to support articular cartilage repair, we analyzed joint cartilage defect healing in the rabbit model after implantation of autologous auricle-derived (auricular) chondrocytes. Autologous lapine articular and auricular chondrocytes were cultured for 3 weeks in polyglycolic acid (PGA) scaffolds before being implanted into critical sized osteochondral defects of the rabbit knee femoropatellar groove. Cell-free PGA scaffolds and empty defects served as controls. Construct quality was determined before implantation and defect healing was monitored after 6 and 12 weeks using vitality assays, macroscopical and histological score systems. Neo-cartilage was formed in the PGA constructs seeded with both articular and auricular chondrocytes in vitro and in vivo. At the histological level, cartilage repair was slightly improved when using autologous articular chondrocyte seeded constructs compared to empty defects and was significantly superior compared to defects treated with auricular chondrocytes 6 weeks after implantation. Although only the immunohistological differences were significant, auricular chondrocyte implantation induced an inferior healing response compared with the empty defects. Elastic auricular chondrocytes might maintain some tissue-specific characteristics when implanted into joint cartilage defects which limit its repair capacity.
Assuntos
Cartilagem Articular/lesões , Transplante de Células/métodos , Condrócitos/transplante , Pavilhão Auricular/citologia , Pavilhão Auricular/transplante , Animais , Fenômenos Biomecânicos , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Células Cultivadas , Colágeno/metabolismo , Feminino , Membro Posterior/patologia , Articulações/patologia , Coelhos , Alicerces TeciduaisRESUMO
Minipigs are widely used as a large animal model for cartilage repair. However, many in vitro studies are based on porcine chondrocytes derived from abundantly available premature hybrid pigs. It remains unclear whether pig line-dependent differences exist which could limit the comparability between in vitro and in vivo results using either hybrid or miniature pig articular chondrocytes. Porcine knee joint femoral cartilage was isolated from 3- to 5-month-old hybrid pigs and Göttingen minipigs. Cartilage from both pig lines was analysed for thickness, zonality, cell content, size and proteoglycan deposition. Cultured articular chondrocytes from both pig lines were investigated for gene and/or protein expression of cartilage-specific proteins such as type II collagen, aggrecan, the chondrogenic transcription factor Sox9, non-specific type I collagen and the cell-matrix receptor ß1-integrin. Cartilage was significantly thinner in the miniature pig compared to the hybrid pig, but the differences between the medial and lateral femur condyles did not reach a significant level. Knee joint cartilage zone formation started only in the minipig, whereas cellularity and cell diameters were comparable in both pig lines. Blood vessels could be detected in the hybrid pig but not the minipig cartilage. Sulphated proteoglycan deposition was more pronounced in cartilage zones II-IV of both pig lines. Minipig chondrocytes expressed type II and I collagen, Sox9 and ß1-integrin at a higher level than hybrid pig chondrocytes. These distinct line-dependent differences should be considered when using hybrid pig-derived chondrocytes for tissue engineering and Göttingen minipigs as a large animal model.
Assuntos
Cartilagem/anatomia & histologia , Condrócitos/metabolismo , Porco Miniatura/anatomia & histologia , Suínos/anatomia & histologia , Agrecanas/metabolismo , Animais , Cartilagem/citologia , Técnicas de Cultura de Células , Tamanho Celular , Condrócitos/citologia , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Hibridização Genética , Integrina beta1/metabolismo , Articulação do Joelho/anatomia & histologia , Articulação do Joelho/citologia , Modelos Animais , Fatores de Transcrição SOX9/metabolismo , Especificidade da Espécie , Engenharia TecidualRESUMO
Tendon ruptures and defects remain major orthopaedic challenges. Tendon healing is a time-consuming process, which results in scar tissue with an altered biomechanical competence. Using a xenogeneic tendon extracellular matrix (ECM) as a natural scaffold, which can be reseeded with autologous human tenocytes, might be a promising approach to reconstruct damaged tendons. For this purpose, the porcine Achilles (AS) tendons serving as a scaffold were histologically characterized in comparison to human cell donor tendons. AS tendons were decellularized and then reseeded with primary human hamstring tenocytes using cell centrifuging, rotating culture and cell injection techniques. Vitality testing, histology and glycosaminoglycan/DNA quantifications were performed to document the success of tendon reseeding. Porcine AS tendons were characterized by a higher cell and sulfated glycosaminoglycan content than human cell donor tendons. Complete decellularization could be achieved, but led to a wash out of sulfated glycosaminoglycans. Nevertheless, porcine tendon could be recellularized with vital human tenocytes. The recellularization led to a slight increase in cell number compared to the native tendon and some glycosaminoglycan recovery. This study indicates that porcine tendon can be de- and recellularized using adult human tenocytes. Future work should optimize cell distribution within the recellularized tendon ECM and consider tendon- and donor species-dependent differences.
Assuntos
Tendão do Calcâneo , Matriz Extracelular , Tendões/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Tendão do Calcâneo/anatomia & histologia , Adulto , Animais , Sobrevivência Celular , Glicosaminoglicanos/análise , Humanos , Suínos , Traumatismos dos Tendões , Tendões/química , Transplante Heterólogo , CicatrizaçãoRESUMO
Implantation of non-articular (heterotopic) chondrocyte-based implants might be an alternative approach to articular cartilage repair. This strategy could be helpful in cases in which there are no or too few articular chondrocytes available. Therefore, this study was undertaken to compare joint cartilage defect healing in the minipig model after implantation of heterotopic auricular and orthotopic articular chondrocytes. Poly-glycolic acid (PGA) associated three-dimensional (3D) constructs were prepared culturing autologous minipig-derived articular and auricular chondrocytes for 7 days in a dynamic culture system. Chondrocyte PGA constructs were implanted into 8mm diameter and â¼1.1mm deep chondral defects within the medial and lateral condyles of the minipig knee joints. Empty defects served as controls for assessment of the intrinsic healing response. Defect healing was monitored 6 months post implantation using a macroscopic and microscopic score system and biomechanical analysis. Neo-cartilage formation could be observed in the PGA constructs seeded with articular and auricular chondrocytes in vivo. The defect healing did not significantly differ at the macroscopic and histological level in response to implantation of either autologous articular or auricular chondrocytes seeded constructs compared with the empty defects. Although the differences were not significant, the auricular chondrocytes-based implants led to a slightly inferior repair quality at the macroscopic level, but a histologically superior healing response when compared with the empty defect group. However, biomechanical analysis revealed a higher stiffness in repair tissues produced by auricular chondrocyte implantation compared with the other groups. Deduced from these results, articular chondrocytes represent the preferable cell source for implantation.
Assuntos
Doenças das Cartilagens/cirurgia , Cartilagem Articular/citologia , Transplante de Células/métodos , Condrócitos/transplante , Transplante Heterotópico/métodos , Animais , Artrite/patologia , Fenômenos Biomecânicos , Biópsia , Doenças das Cartilagens/fisiopatologia , Células Cultivadas , Corantes , Marcha , Imuno-Histoquímica , Articulações/patologia , Ácido Poliglicólico/farmacologia , Postura/fisiologia , Suínos , Porco MiniaturaRESUMO
Tendon healing is generally a time-consuming process and often leads to a functionally altered reparative tissue. Using degradable scaffolds for tendon reconstruction still remains a compromise in view of the required high mechanical strength of tendons. Regenerative approaches based on natural decellularized allo- or xenogenic tendon extracellular matrix (ECM) have recently started to attract interest. This ECM combines the advantages of its intrinsic mechanical competence with that of providing tenogenic stimuli for immigrating cells mediated, for example, by the growth factors and other mediators entrapped within the natural ECM. A major restriction for their therapeutic application is the mainly cell-associated immunogenicity of xenogenic or allogenic tissues and, in the case of allogenic tissues, also the risk of disease transmission. A survey of approaches for tendon reconstruction using cell-free tendon ECM is presented here, whereby the problems associated with the decellularization procedures, the success of various recellularization strategies, and the applicable cell types will be thoroughly discussed. Encouraging in vivo results using cell-free ECM, as, for instance, in rabbit models, have already been reported. However, in comparison to native tendon, cells remain mostly inhomogeneously distributed in the reseeded ECM and do not align. Hence, future work should focus on the optimization of tendon ECM decellularization and recolonization strategies to restore tendon functionality.
RESUMO
Although rabbits are commonly used as tendon repair model, interpretative tools are divergent and comprehensive scoring systems are lacking. Hence, the aim was to develop a multifaceted scoring system to characterize healing in a partial Achilles tendon defect model. A 3 mm diameter defect was created in the midsubstance of the medial M. gastrocnemius tendon, which remained untreated or was filled with a polyglycolic-acid (PGA) scaffold + fibrin and either left cell-free or seeded with Achilles tenocytes. After 6 and 12 weeks, tendon repair was assessed macroscopically and histologically using self-constructed scores. Macroscopical scoring revealed superior results in the tenocyte seeded PGA + fibrin group compared with the controls at both time points. Histology of all operated tendons after 6 weeks proved extracellular matrix (ECM) disorganization, hypercellularity and occurrence of irregular running elastic fibres with no significance between the groups. Some inflammation was associated with PGA implantation and increased sulphated proteoglycan deposition predominantly with the empty defects. After 12 weeks defect areas became hard to recognize and differences between groups, except for the increased sulphated proteoglycans content in the empty defects, were almost nullified. We describe a partial Achilles tendon defect model and versatile scoring tools applicable for characterizing biomaterial-supported tendon healing.
Assuntos
Tendão do Calcâneo/citologia , Tendão do Calcâneo/patologia , Materiais Biocompatíveis/metabolismo , Implantes Experimentais , Cicatrização , Tendão do Calcâneo/fisiologia , Animais , Materiais Biocompatíveis/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Fibrina/química , Humanos , Masculino , Ácido Poliglicólico/química , Coelhos , Alicerces Teciduais/químicaRESUMO
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.
RESUMO
Injured articular cartilage is limited in its capacity to heal. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but it requires articular cartilage biopsies for sufficient autologous chondrocyte expansion in vitro. Hence, ACT is restricted by donor-site morbidity and autologous articular chondrocytes availability. The use of nonarticular heterotopic chondrocytes such as auricular, nasoseptal, or costal chondrocytes for ACT might overcome these limitations: heterotopic sources show lesser donor-site morbidity and a comparable extracellular cartilage matrix synthesis profile to articular cartilage. However, heterotopic (h)ACT poses a challenge. Particular tissue characteristics of heterotopic cartilage, divergent culturing peculiarities of heterotopic chondrocytes, and the advantages and drawbacks related to these diverse cartilage sources were critically discussed. Finally, available in vitro and in vivo experimental (h)ACT approaches were summarized. The quality of the cartilage engineered using heterotopic chondrocytes remains partly controversy due to the divergent methodologies and culture conditions used. While some encouraging in vivo results using (h)ACT have been demonstrated, standardized culturing protocols are strongly required. However, whether heterotopic chondrocytes implanted into joint cartilage defects maintain their particular tissue properties or can be adapted via tissue engineering strategies to fulfill regular articular cartilage functions requires further studies.
Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/patologia , Condrócitos/transplante , Transplante Heterotópico , Cicatrização , Animais , Condrócitos/citologia , Humanos , Engenharia Tecidual , Transplante AutólogoRESUMO
Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.