RESUMO
Determination of a stem cell source with sufficient myogenic differentiation capacity that can be easily obtained in large quantities is of great importance in skeletal muscle regeneration therapies. Adipose-derived stem cells (ASCs) are readily available, can be isolated from fat tissue with high yield and possess myogenic differentiation capacity. Consequently, ASCs have high applicability in muscle regenerative therapies. However, a key challenge is their low differentiation efficiency. In this study, we have explored the potential of mimicking the natural microenvironment of the skeletal muscle tissue to enhance ASC myogenesis by inducing 3D cellular alignment and using dynamic biomimetic culture. ASCs were entrapped and 3D aligned in parallel within fibrin-based microfibers and subjected to uniaxial cyclic stretch. 3D cell alignment was shown to be necessary for achieving and maintaining the stiffness of the construct mimicking the natural tissue (12 ± 1 kPa), where acellular aligned fibers and cell-laden random fibers had stiffness values of 4 ± 1 and 5 ± 2 kPa, respectively, at the end of 21 d. The synergistic effect of 3D cell alignment and biomimetic dynamic culture was evaluated on cell proliferation, viability and the expression of muscle-specific markers (immunofluorescent staining for MyoD1, myogenin, desmin and myosin heavy chain). It was shown that the myogenic markers were only expressed on the aligned-dynamic culture samples on day 21 of dynamic culture. These results demonstrate that 3D skeletal muscle grafts can be developed using ASCs by mimicking the structural and physiological muscle microenvironment.
Assuntos
Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Biomimética , Desenvolvimento Muscular , Células-Tronco/citologia , Adipócitos/citologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Citometria de Fluxo , Humanos , Hidrogéis/química , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/metabolismo , Miogenina/metabolismo , Pressão , Regeneração , Estresse Mecânico , Células Estromais/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Osteoporosis, which is characterized by low bone mineral density and susceptibility to fracture, is caused by increased osteoclastic activity. Receptor activator of nuclear factor kappa B ligand (RANKL)/RANK signaling plays an important role in osteoclast differentiation and activation. The current treatment strategies for osteoporosis do not directly address this underlying cause and generates undesired side effects. This led to emergence of controlled delivery systems to increase drug bioavailability and efficacy specifically at the bone tissue. With better understanding of molecular pathology of bone, the use of small interfering RNA (siRNA) to inhibit translation of abnormal gene expression in cells is becoming a promising approach. In this study, we report a siRNA delivery system consisting of PEI:RANK siRNA complex entrapped in nanosized poly(lactic acid-co-glycolic acid) (PLGA) capsules intended to be used in the treatment of osteoporosis. The nanosize will enable the nanoparticles to be administered by intravenous injection. The RANK siRNA was complexed with polyethylenimine (PEI) and loaded into biodegradable PLGA nanocapsules (NCs). The PEI:RANK siRNA loaded nanocapsules significantly reduced (47%) RANK mRNA levels. The differentiation of osteoclast precursors to mature osteoclasts was significantly suppressed (â¼54%). The reduction in the osteoclastic activity of the differentiated osteoclasts (55%) was found to be statistically significant. The siRNA delivery system developed in the study is planned to be tested i.v. in mouse and has the potential to be used as a novel alternative approach for the systemic treatment of osteoporosis.
Assuntos
Nanocápsulas/química , Osteoporose/tratamento farmacológico , Polietilenoimina , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Interferente Pequeno , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Animais , Camundongos , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Polietilenoimina/química , Polietilenoimina/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Células RAW 264.7 , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismoRESUMO
Osteoporosis, a systemic skeletal disorder, occurs when bone turnover balance is disrupted. With the identification of the genes involved in the pathogenesis of the disease, studies on development of new treatments has intensified. Short interfering RNA (siRNA) is used to knockdown disease related gene expressions. Targeting siRNA in vivo is challenging. The maintenance of therapeutic plasma level is hampered by clearance of siRNA from the body. Targeted systems are useful in increasing the drug concentration at the target site and decreasing side effects. Aim of the present study was to develop an injectable siRNA delivery system to protect siRNA during systemic distribution and target the siRNA to bone tissue using a thermoresponsive, genetically engineered, elastin-like recombinamer (ELR), designed to interact with the mineral component of bone. The delivery system consisted of DNAoligo as a siRNA substitute complexed with the cationic polymer, polyethyleneimine (PEI), at N/P ratio of 20. The complex was encapsulated in poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules. PLGA capsules were characterized by SEM, TEM and XPS. FTIR was used to show the preferential attachment of ELR to HAp. Encapsulation efficiency of the complex in PLGA nanocapsules was 48%. The release kinetics of the complex fits the Higuchi release kinetics.
