RESUMO
Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E(2) were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.
Assuntos
Reatores Biológicos , Diferenciação Celular , Técnicas de Cultura/métodos , Células-Tronco Mesenquimais/citologia , Osteogênese , Fluxo Pulsátil , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Láctico/farmacologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteogênese/efeitos dos fármacos , Osteopontina/genética , Osteopontina/metabolismo , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Alicerces TeciduaisRESUMO
Lumbar intervertebral body fusion devices (L-IBFDs) are intended to provide stability to promote fusion in patients with a variety of lumbar pathologies. Different L-IBFD designs have been developed to accommodate various surgical approaches for lumbar interbody fusion procedures including anterior, lateral, posterior, and transforaminal lumbar interbody fusions (ALIF, LLIF, PLIF, and TLIF, respectively). Due to design differences, there is a potential for mechanical performance differences between ALIF, LLIF, PLIF, and TLIF devices. To evaluate this, mechanical performance and device dimension data were collected from 124 Traditional 510(k) submissions to the FDA for L-IBFDs cleared for marketing from 2007 through 2016. From these submissions, mechanical test results were aggregated for seven commonly performed tests: static and dynamic axial compression, compression-shear, and torsion testing per ASTM F2077, and subsidence testing per ASTM F2267. The Kruskal-Wallis test and Wilcoxon signed-rank test were used to determine if device type (ALIF, LLIF, PLIF, TLIF) had a significant effect on mechanical performance parameters (static testing: stiffness and yield strength; dynamic testing: runout load; subsidence testing: stiffness [Kp]). Generally, ALIFs and LLIFs were found to be stiffer, stronger, and had higher subsidence resistance than PLIF and TLIF designs. These results are likely due to the larger footprints of the ALIF and LLIF devices. The relative mechanical performance and subsidence resistance can be considered when determining the appropriate surgical approach and implant for a given patient. Overall, the mechanical performance data presented here can be utilized for future L-IBFD development and design verification.
Assuntos
Vértebras Lombares/cirurgia , Fenômenos Mecânicos , Projetos de Pesquisa , Fusão Vertebral/instrumentação , United States Food and Drug Administration , Feminino , Humanos , Teste de Materiais , Pressão , Estados UnidosRESUMO
Segmented polyurethanes have been used extensively in implantable medical devices, but their tunable mechanical properties make them attractive for examining the effect of biomaterial modulus on engineered musculoskeletal tissue development. In this study, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized from 1,4-diisocyanatobutane, a poly(epsilon-caprolactone) (PCL) macrodiol soft segment and a tyramine-1,4-diisocyanatobutane-tyramine chain extender. By systematically increasing the PCL macrodiol molecular weight from 1100 to 2700Da, the storage modulus, crystallinity and melting point of the PCL segment were systematically varied. In particular, the melting temperature, T(m), increased from 21 to 61 degrees C and the storage modulus at 37 degrees C increased from 52 to 278MPa with increasing PCL macrodiol molecular weight, suggesting that the crystallinity of the PCL macrodiol contributed significantly to the mechanical properties of the polymers. Bone marrow stromal cells were cultured on rigid polymer films under osteogenic conditions for up to 21 days. Cell density, alkaline phosphatase activity, and osteopontin and osteocalcin expression were similar among PEUURs and comparable to poly(d,l-lactic-coglycolic acid). This study demonstrates the suitability of this family of PEUURs for tissue engineering applications, and establishes a foundation for determining the effect of biomaterial modulus on bone tissue development.
Assuntos
Materiais Biocompatíveis/química , Elastômeros/química , Poliuretanos/química , Engenharia Tecidual/métodos , Ureia/química , Fosfatase Alcalina/metabolismo , Animais , Materiais Biocompatíveis/síntese química , Osso e Ossos/citologia , Contagem de Células , Técnicas de Cultura de Células , Células Cultivadas , Elastômeros/síntese química , Masculino , Estrutura Molecular , Osteocalcina/metabolismo , Osteopontina/metabolismo , Poliuretanos/síntese química , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células Estromais/citologia , TemperaturaRESUMO
Segmented polyurethanes (PURs), consisting of degradable poly(a-hydroxy ester) soft segments and aminoacid-derived chain extenders, are biocompatible elastomers with tunable mechanical and degradative properties suitable for a variety of tissue-engineering applications. In this study, a family of linear PURs synthesized from poly(ϵ-caprolactone) (PCL) diol, 1,4-diisocyanobutane and tyramine with theoretical PCL contents of 65-80 wt% were processed into porous foam scaffolds and evaluated for their ability to support osteoblastic differentiation in vitro. Differential scanning calorimetry and mechanical testing of the foams indicated increasing polymer crystallinity and compressive modulus with increasing PCL content. Next, bone marrow stromal cells (BMSCs) were seeded into PUR scaffolds, as well as poly(lactic-co-glycolic acid) (PLGA) scaffolds, and maintained under osteogenic conditions for 14 and 21 days. Analysis of cell number indicated a systematic decrease in cell density with increasing PUR stiffness at both 14 and 21 days in culture. However, at these same time points the relative mRNA expression for the bone-specific proteins osteocalcin and the growth factors bone morphogenetic protein-2 and vascular endothelial growth factor gene expression were similar among the PURs. Finally, prostaglandin E2 production, alkaline phosphatase activity and osteopontin mRNA expression were highly elevated on the most-crystalline PUR scaffold as compared to the PLGA and PUR scaffolds. These results suggest that both the modulus and crystallinity of the PUR scaffolds influence cell proliferation and the expression of osteoblastic proteins.
Assuntos
Elastômeros/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Poliésteres/metabolismo , Poliuretanos/metabolismo , Alicerces Teciduais/química , Animais , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , Células Cultivadas , Elastômeros/química , Masculino , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Poliésteres/química , Poliuretanos/química , Porosidade , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodosRESUMO
Bone is the second most transplanted tissue after blood and engineered bone substitutes represent an attractive alternative to autologous bone grafts. Therefore, strategies are needed to facilitate the development of engineered bone graft substitutes in vitro. Perfusion bioreactors have been investigated as a component of engineered bone strategies because they supply oxygen and nutrients to cells seeded within a scaffold while applying a mechanical stimulus to the cells. Evidence with planar cell cultures has shown that dynamic flow regimens elicit an enhanced cellular response over steady flow regimens but dynamic perfusion strategies have not yet been translated to 3D scaffold architectures. The objective of this research is to show that pulsatile flow patterns will enhance osteogenic differentiation of bone marrow stromal cells seeded in porous scaffolds over a continuous flow pattern as indicated by levels of alkaline phosphatase activity and the bone extracellular matrix protein, osteopontin. Our results indicate that all flow conditions enhanced alkaline phosphatase activity and osteopontin expression. Additionally, these markers are preferentially enhanced by pulsatile flow over continuous flow. A trend of increasing alkaline phosphatase activity with decreasing pulse frequency suggests that cells are sensitive to frequency of pulsatile flow. These results indicate that dynamic perfusion may be a useful component of the engineered bone tissue strategy.