RESUMO
BACKGROUND: The human body has an extensive capacity to regenerate bone tissue after trauma. However large defects such as long bone fractures of the lower limbs cannot be restored without intervention and often lead to nonunion. Therefore, the aim of the present study was to assess the pool and biological functions of human mesenchymal stromal cells (hMSCs) isolated from different bone marrow locations of the lower limbs and to identify novel strategies to prime the cells prior to their use in bone fracture healing. Following, bone marrow from the ilium, proximal femur, distal femur and proximal tibia was aspirated and the hMSCs isolated. Bone marrow type, volume, number of mononuclear cells/hMSCs and their self-renewal, multilineage potential, extracellular matrix (ECM) production and surface marker profiling were analyzed. Additionally, the cells were primed to accelerate bone fracture healing either by using acoustic stimulation or varying the initial hMSCs isolation conditions. RESULTS: We found that the more proximal the bone marrow aspiration location, the larger the bone marrow volume was, the higher the content in mononuclear cells/hMSCs and the higher the self-renewal and osteogenic differentiation potential of the isolated hMSCs were. Acoustic stimulation of bone marrow, as well as the isolation of hMSCs in the absence of fetal bovine serum, increased the osteogenic and ECM production potential of the cells, respectively. CONCLUSION: We showed that bone marrow properties change with the aspiration location, potentially explaining the differences in bone fracture healing between the tibia and the femur. Furthermore, we showed two new priming methods capable of enhancing bone fracture healing.
Assuntos
Regeneração Óssea/fisiologia , Consolidação da Fratura/fisiologia , Fraturas Ósseas/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Terapia por Ultrassom/métodos , Estimulação Acústica/métodos , Regeneração Óssea/efeitos da radiação , Terapia Combinada/métodos , Consolidação da Fratura/efeitos da radiação , Humanos , Células-Tronco Mesenquimais/classificação , Resultado do TratamentoRESUMO
One of the ligaments most often damaged during sports-the anterior cruciate ligament (ACL)-has poor healing capacity. On damage, reconstructive surgery is performed to restore the mechanical stability of the knee and to reduce the inflammatory milieu otherwise present in the joint. A return to normal activities, however, takes between 9 and 12 months. Thus, strategies capable of improving ACL graft healing are needed. Embryonic development of tendon and ligament (T/L) is regulated by a crosstalk between different cell types. We hypothesized that terminally differentiated skeletal-derived cells such as osteoblasts, chondrocytes, and myoblasts modulate T/L healing. Using an indirect coculture system, we discovered that myoblast-secreted signals-but not osteoblasts, chondrocytes, or stromal-secreted signals-are capable of upregulating classical T/L markers such as scleraxis and tenomodulin on human hamstring tendon-derived cells (hTC), which contribute to ACL graft healing. Transcriptome analysis showed that coculturing hTC with myoblasts led to an upregulation of extracellular matrix (ECM) genes and resulted in enhanced ECM deposition. In vivo, using a rat model of ACL reconstruction showed that conditioned media derived from human muscle tissue accelerated femoral tunnel closure, a key step for autograft integration. Collectively, these results indicate that muscle-secreted signals can be used to improve ACL graft healing in a clinical setting where muscle remnants are often discarded.
Assuntos
Reconstrução do Ligamento Cruzado Anterior/métodos , Ligamento Cruzado Anterior/citologia , Cicatrização/fisiologia , Animais , Ligamento Cruzado Anterior/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados , Matriz Extracelular/metabolismo , Humanos , Masculino , Camundongos , Mioblastos/citologia , Ratos , Ratos Sprague-Dawley , Tendões/citologiaRESUMO
Modulating the bone morphogenetic protein 2 (BMP-2) and transforming growth factor-ß1 (TGF-ß1) signaling pathways is essential during tendon/ligament (T/L) healing. Unfortunately, growth factor delivery in situ is far from trivial and, in many cases, the necessary growth factors are not approved for clinical use. Here we used a BMP-2 and a TGF-ß1 reporter cell line to screen a library of 1280 Food and Drug Administration-approved small molecules and identify modulators of both signaling pathways. We identified four compounds capable of modulating BMP and TGF signaling on primary human tendon-derived cells (huTCs) and describe their effects on proliferation and differentiation of these cells.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Colágeno/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Camundongos , Vison , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos Testes , Proteínas Smad/metabolismo , Tendões/citologiaRESUMO
Anterior cruciate ligament (ACL) reconstruction involves the replacement of the torn ligament with a new graft, often a hamstring tendon (HT). Described as similar, the ACL and HT have intrinsic differences related to their distinct anatomical locations. From a cellular perspective, identifying these differences represents a step forward in the search for new cues that enhance recovery after the reconstruction. The purpose of this study was to characterize the phenotype and multilineage potential of ACL- and HT-derived cells. ACL- and HT-derived cells were isolated from tissue harvest from patients undergoing total knee arthroplasty (TKA) or ACL reconstruction. In total, three ACL and three HT donors were investigated. Cell morphology, self-renewal potential (CFU-F), surface marker profiling, expression of tendon/ligament-related markers (PCR) and multilineage potential were analysed for both cell types; both had fibroblast-like morphology and low self-renewal potential. No differences in the expression of tendon/ligament-related genes or a selected set of surface markers were observed between the two cell types. However, differences in their multilineage potential were observed: while ACL-derived cells showed a high potential to differentiate into chondrocytes and adipocytes, but not osteoblasts, HT-derived cells showed poor potential to form adipocytes, chondrocytes and osteoblasts. Our results demonstrated that HT-derived cells have low multilineage potential compared to ACL-derived cells, further highlighting the need for extrinsic signals to fully restore the function of the ACL upon reconstruction. Copyright © 2015 John Wiley & Sons, Ltd.