RESUMO
PURPOSE: Bone marrow-derived mesenchymal stem cells (MSC) can differentiate osteogenic lineages, but their tissue regeneration ability is inconsistent. The bone marrow mononuclear cell (BMMC) fraction of adult bone marrow contains a variety of progenitor cells that may potentiate tissue regeneration. This study examined the utility of BMMC, both alone and in combination with purified MSC, as a cell source for bone regeneration. METHODS: Fresh BMMC, culture-expanded MSC, and a combination of BMMC and MSC were encapsulated in collagen-chitosan hydrogel microbeads for pre-culture and minimally invasive delivery. Microbeads were cultured in growth medium for 3 days, and then in either growth or osteogenic medium for 17 days prior to subcutaneous injection in the rat dorsum. RESULTS: MSC remained viable in microbeads over 17 days in pre-culture, while some of the BMMC fraction were nonviable. After 5 weeks of implantation, microCT and histology showed that supplementation of BMMC with MSC produced a strong synergistic effect on the volume of ectopic bone formation, compared to either cell source alone. Microbeads containing only fresh BMMC or only cultured MSC maintained in osteogenic medium resulted in more bone formation than their counterparts cultured in growth medium. Histological staining showed evidence of residual microbead matrix in undifferentiated samples and indications of more advanced tissue remodeling in differentiated samples. CONCLUSIONS: These data suggest that components of the BMMC fraction can act synergistically with predifferentiated MSC to potentiate ectopic bone formation. The microbead system may have utility in delivering desired cell populations in bone regeneration applications.
Assuntos
Células da Medula Óssea/citologia , Separação Celular/métodos , Quitosana/farmacologia , Coristoma/patologia , Colágeno/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Células-Tronco Mesenquimais/citologia , Osteogênese , Animais , Densidade Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Coristoma/diagnóstico por imagem , Implantes Experimentais , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microesferas , Tamanho do Órgão/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos Endogâmicos F344 , Microtomografia por Raio-XRESUMO
Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 × 10(6) cells/mL, containing an estimated 5 × 10(4) MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 × 10(5) cells/mL) were added to a 65-35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.
Assuntos
Células da Medula Óssea/citologia , Osso e Ossos/fisiologia , Quitosana/farmacologia , Colágeno/farmacologia , Leucócitos Mononucleares/citologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , Bovinos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , DNA/metabolismo , Glicosaminoglicanos/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microesferas , Ortopedia , Osteocalcina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (µCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the µCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Quimiocina CXCL12/farmacologia , Osteogênese/efeitos dos fármacos , Receptores CXCR4/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Benzilaminas , Ciclamos , Compostos Heterocíclicos/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/antagonistas & inibidores , Proteínas Recombinantes/farmacologiaRESUMO
Enhanced understanding of differential gene expression and biological pathways associated with distinct phases of intramembranous bone regeneration following femoral marrow ablation surgery will improve future advancements regarding osseointegration of joint replacement implants, biomaterials design, and bone tissue engineering. A rat femoral marrow ablation model was performed and genome-wide microarray data were obtained from samples at 1, 3, 5, 7, 10, 14, 28, and 56 days post-ablation, with intact bones serving as controls at Day 0. Bayesian model-based clustering produced eight distinct groups amongst 9,062 significant gene probe sets based on similar temporal expression profiles, which were further categorized into three major temporal classes of increased, variable, and decreased expression. Osteoblastic- and osteoclastic-associated genes were found to be significantly expressed within the increased expression groups. Chondrogenesis was not detected histologically. Adipogenic marker genes were found within variable/decreased expression groups, emphasizing that adipogenesis was inhibited during osteogenesis. Differential biological processes and pathways associated with each major temporal group were identified, and significantly expressed genes involved were visually represented by heat maps. It was determined that the increased expression group exclusively contains genes involved in pathways for matrix metalloproteinases (MMPs), Wnt signaling, TGF-ß signaling, and inflammatory pathways. Only the variable expression group contains genes associated with glycolysis and gluconeogenesis, the notch signaling pathway, natural killer cell mediated cytotoxicity, and the B cell receptor signaling pathway. The decreased group exclusively consists of genes involved in heme biosynthesis, the p53 signaling pathway, and the hematopoietic cell lineage. Significant biological pathways and transcription factors expressed at each time point post-ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.
Assuntos
Medula Óssea , Regeneração Óssea , Fêmur , Perfilação da Expressão Gênica , Animais , Teorema de Bayes , Masculino , Metaloproteinases da Matriz/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMO
Cell differentiation, adhesion, and orientation are known to influence the functionality of both natural and engineered tissues, such as articular cartilage. Several attempts have been devised to regulate these important cellular behaviors, including application of inexpensive but efficient electrospinning that can produce patterned extracellular matrix (ECM) features. Electrospun and oriented polycaprolactone (PCL) scaffolds (500 or 3000 nm fiber diameter) were created, and human mesenchymal stem cells (hMSCs) were cultured on these scaffolds. Cell viability, morphology, and orientation on the fibrous scaffolds were quantitatively determined as a function of time. While the fiber-guided initial cell orientation was maintained even after 5 weeks, cells cultured in the chondrogenic media proliferated and differentiated into the chondrogenic lineage, suggesting that cell orientation is controlled by the physical cues and minimally influenced by the soluble factors. Based on assessment by the chondrogenic markers, use of the nanofibrous scaffold (500 nm) appears to enhance the chondrogenic differentiation. These findings indicate that hMSCs seeded on a controllable PCL scaffold may lead to an alternate methodology to mimic the cell and ECM organization that is found, for example, in the superficial zone of articular cartilage.