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Int. j. morphol ; 38(4): 909-913, Aug. 2020. tab, graf
Article in English | LILACS | ID: biblio-1124875


Porous titanium alloy scaffold was widely used in treating bone defect caused by traumatic injury and osteomyelitis, which was incapable of self-healing. The implantation of scaffold produced stress shielding thereby forming osteolysis. The objective of this study was to analysis trabecular morphological features of osseointegrated bone. 14 New Zealand rabbits were divided into two groups, surgery group and healthy control group. 7 rabbits in surgery group were selected to perform 3D printed porous titanium alloy scaffold implantation surgery with preload at the defect of femoral condyle for osseointegration. The other 7 rabbits in control group were feed free. After 90 days healing, femoral condyles were extracted to perform micro-CT scanning with hydroxyapatite calibration phantom. Mean bone mineral density (BMD), bone volume fraction (BV/TV), BS/TV (bone surface area ratio), Tb.Th (thickness of trabeculae), Tb.N (number of trabeculae), Tb.Sp (trabecular separation) and DA (degree of anisotropy) were calculated from micro-CT images. The results revealed that osseointegration inside and at the surface of scaffolds worked well from grey values of micro-CT images. After 12 weeks healing, mean bone mineral densities (BMD) in surgery group and healthy control group were calculated as 800±20mg/cm3 and 980±90mg/cm3, respectively. This revealed that the strength of trabeculae in surgery group might lower than that in the healthy group. Trabecular morphological parameters test showed that trabecular morphological parameters at the surface of scaffolds in the surgery group deteriorated significantly. It was found from micro-CT images that ingrowth bone was filled with pores of scaffold. Overall, the effect of osseointegration was promoted through the change of mechanical micro-environment in the scaffold region. Overall, preload could improve osseointegration effect in the long-term after surgery. However, the trabecular morphology in the surgery group was deteriorated, which might bring secondary fracture risk again.

La malla de aleación de titanio poroso se usó ampliamente en el tratamiento de defectos óseos causados por lesiones traumáticas y osteomielitis. El implante de la malla generó una protección contra el estrés, formando así osteolisis. El objetivo de este estudio fue analizar las características morfológicas trabeculares del hueso osteointegrado. Se dividieron 14 conejos (Neozelandeses) en dos grupos, grupo cirugía y grupo control saludable. Se seleccionaron 7 conejos en el grupo de cirugía para realizar una implantación de mallas de aleación de titanio poroso, impresas en 3D con precarga en el defecto del cóndilo femoral para la osteointegración. Los 7 conejos restantes del grupo control se mantuvieron sin alimentación. Después de 90 días de curación, se extrajeron los cóndilos femorales para realizar una exploración por micro-CT con un espectro de calibración de hidroxiapatita. Se calcularon a partir de imágenes de micro-CTDensidad mineral ósea media (DMO), fracción de volumen óseo (BV / TV), BS / TV (relación de área de superficie ósea), Tb.Th (espesor de trabéculas), Tb.N (número de trabéculas), Tb.Sp (trabecular separación) y DA (grado de anisotropía). Los resultados revelaron que la osteointegración dentro y en la superficie de los andamios funcionó bien a partir de los valores grises de las imágenes de micro-CT. Después de 12 semanas de curación, las densidades medias de minerales óseos (DMO) en el grupo cirugía y en el grupo control sano se calcularon como 800 ± 20 mg/cm3 y 980 ± 90 mg/cm3, respectivamente. Esto reveló que la fuerza de las trabéculas en el grupo de cirugía podría ser menor que la del grupo sano. La prueba de parámetros morfológicos trabeculares mostró que en el grupo de cirugía, la superficie de las mallas, se deterioraron significativamente. Se descubrió a partir de imágenes de microCT que el hueso en crecimiento estaba lleno de poros de andamio. En general, el efecto de la osteointegración se promovió mediante el cambio del microambiente mecánico en la región de la malla. En general, la precarga podría mejorar el efecto de osteointegración a largo plazo después de la cirugía. Sin embargo, la morfología trabecular en el grupo de cirugía se deterioró, lo que podría traer un nuevo riesgo de fractura secundaria.

Animals , Rabbits , Bone Diseases/surgery , Osseointegration/physiology , Tissue Scaffolds/chemistry , Printing, Three-Dimensional , Prostheses and Implants , Titanium/chemistry , Porosity , Alloys , X-Ray Microtomography , Femur/surgery
Braz. j. med. biol. res ; 52(8): e8318, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011603


Currently, there is great clinical need for suitable synthetic grafts that can be used in vascular diseases. Synthetic grafts have been successfully used in medium and large arteries, however, their use in small diameter vessels is limited and presents a high failure rate. In this context, the aim of this study was to develop tissue engineering scaffolds, using poly(trimethylene carbonate-co-L-lactide) (PTMCLLA), for application as small diameter vascular grafts. For this, copolymers with varying trimethylene carbonate/lactide ratios - 20/80, 30/70, and 40/60 - were submitted to electrospinning and the resulting scaffolds were evaluated in terms of their physicochemical and biological properties. The scaffolds produced with PTMCLLA 20/80, 30/70, and 40/60 showed smooth fibers with an average diameter of 771±273, 606±242, and 697±232 nm, respectively. When the degradation ratio was evaluated, the three scaffold groups had a similar molecular weight (Mw) on the final day of analysis. PTMCLLA 30/70 and 40/60 scaffolds exhibited greater flexibility than the PTMCLLA 20/80. However, the PTMCLLA 40/60 scaffolds showed a large wrinkling and their biological properties were not evaluated. The PTMCLLA 30/70 scaffolds supported the adhesion and growth of mesenchymal stem cells (MSCs), endothelial progenitor cells, and smooth muscle cells (SMCs). In addition, they provided a spreading of MSCs and SMCs. Given the results, the electrospun scaffolds produced with PTMCLLA 30/70 copolymer can be considered promising candidates for future applications in vascular tissue engineering.

Humans , Polyesters/chemistry , Blood Vessel Prosthesis , Dioxanes/chemistry , Tissue Scaffolds/chemistry , Materials Testing , Cells, Cultured/cytology , Myocytes, Smooth Muscle/cytology , Cell Proliferation , Mesenchymal Stem Cells/cytology , Endothelial Progenitor Cells/cytology
J. appl. oral sci ; 24(1): 76-84, Jan.-Feb. 2016. graf
Article in English | LILACS, BBO | ID: lil-777354


ABSTRACT Objective The purpose of this study was to investigate the biological effects of epicatechin (ECN), a crosslinking agent, on human dental pulp cells (hDPCs) cultured in collagen scaffolds. Material and Method To evaluate the effects of ECN on the proliferation of hDPCs, cell counting was performed using optical and fluorescent microscopy. Measurements of alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reactions were performed to assess odontogenic differentiation. The compressive strength and setting time of collagen scaffolds containing ECN were measured. Differential scanning calorimetry was performed to analyze the thermal behavior of collagen in the presence of ECN. Results Epicatechin increased ALP activity, mineralized nodule formation, and the mRNA expression of dentin sialophosphoprotein (DSPP), a specific odontogenic-related marker. Furthermore, ECN upregulated the expression of DSPP in hDPCs cultured in collagen scaffolds. Epicatechin activated the extracellular signal-regulated kinase (ERK) and the treatment with an ERK inhibitor (U0126) blocked the expression of DSPP. The compressive strength was increased and the setting time was shortened in a dose-dependent manner. The number of cells cultured in the ECN-treated collagen scaffolds was significantly increased compared to the cells in the untreated control group. Conclusions Our results revealed that ECN promoted the proliferation and differentiation of hDPCs. Furthermore, the differentiation was regulated by the ERK signaling pathway. Changes in mechanical properties are related to cell fate, including proliferation and differentiation. Therefore, our study suggests the ECN treatment might be desirable for dentin-pulp complex regeneration.

Humans , Catechin/pharmacology , Collagen/pharmacology , Cross-Linking Reagents/pharmacology , Dental Pulp/cytology , Dental Pulp/drug effects , Tissue Scaffolds/chemistry , Time Factors , Calorimetry, Differential Scanning , Gene Expression , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Blotting, Western , Reproducibility of Results , Analysis of Variance , Extracellular Signal-Regulated MAP Kinases/analysis , Cell Proliferation/drug effects , Alkaline Phosphatase/analysis , Real-Time Polymerase Chain Reaction
Braz. oral res. (Online) ; 30(1): e120, 2016. tab, graf
Article in English | LILACS | ID: biblio-951977


Abstract This study was designed to determine the in vivo performance of three different materials as scaffolds for dental pulp stem cells (DPSC) undergoing induced odontogenic differentiation. An odontogenic medium modified by the addition of recombinant human bone morphogenetic protein 2 was used in the experimental groups to induce differentiation. Mesenchymal stem cell medium was used in the control groups. DPSC were transplanted onto the backs of mice via three scaffolds: copolymer of L-lactide and DL-lactide (PLDL), copolymer of DL-lactide (PDL) and hydroxyapatite tricalcium phosphate (HA/TCP). The expression levels of dentin sialo-phosphoprotein (DSPP), dentin matrix protein-1 (DMP1), enamelysin/matrix metalloproteinase 20 (MMP20) and phosphate-regulating gene with homologies to endopeptidases on X chromosome (PHEX) were analysed using RT-PCR. The expressions in the experimental groups were compared to those in the control groups. The transcript expressions at 6 and 12 weeks were significantly different for all scaffolds (p < 0.05), except for the expression of DSPP in the PLDL group with regard to the time variable. Although there was a decrease in the expression of enamelysin/MMP20 in PLDL and HA/TCP at 12 weeks, all other expressions increased and reached their highest level at 12 weeks. The highest DSPP expression was in the PDL group (p < 0.05). The highest expression of DMP1 was detected in the HA/TCP group (p < 0.05). The highest expression of PHEX was in the PLDL group (p < 0.05). Consequently, PLDL and PDL seemed to be promising scaffold candidates for odontogenic regeneration at least as HA-TCP, when they were applied with the DPSC induced for odontogenic differentiation.

Humans , Animals , Polymers/chemistry , Stem Cells/physiology , Cell Differentiation/physiology , Dental Pulp/cytology , Tissue Scaffolds/chemistry , Phosphoproteins/analysis , Sialoglycoproteins/analysis , Time Factors , Biocompatible Materials/chemistry , Calcium Phosphates/chemistry , Gene Expression , Reproducibility of Results , Extracellular Matrix Proteins/analysis , Durapatite/chemistry , Cell Culture Techniques , Dental Enamel/chemistry , Dentin/chemistry , Dioxanes/chemistry , Matrix Metalloproteinase 20/analysis , PHEX Phosphate Regulating Neutral Endopeptidase/analysis
Braz. dent. j ; 26(2): 93-98, Mar-Apr/2015. tab, graf
Article in English | LILACS | ID: lil-741212


The aim of this study was to evaluate the influence of the poly-L-lactic acid (PLLA)-based scaffold's pore size on the proliferation and differentiation of dental pulp stem cells (DPSCs). The scaffolds were prepared in pulp chambers of 1-mm-thick tooth slices from third molars using salt crystals (150-250 µm or 251-450 µm) as porogen. DPSC (1x105 cells) were seeded in the scaffolds with different pore sizes, and cultured in 24-well plates. The cell proliferation was evaluated using the WST-1 assay after 3-21 days. Furthermore, RT-PCR was used to assess the differentiation of the DPSCs into odontoblasts, using markers of odontoblastic differentiation (DSPP, DSP-1 and MEPE). RNA from human odontoblasts was used as control. Cell proliferation rate was similar in both scaffolds except at the 14th day period, in which the cells seeded in the scaffolds with larger pores showed higher proliferation (p<0.05). After 21 days DPSCs seeded in both evaluated scaffolds were able of expressing odontoblastic markers DMP-1, DSPP and MEPE. In summary, both scaffolds tested in this study allowed the proliferation and differentiation of DPSCs into odontoblast-like cells.

O objetivo desse estudo foi avaliar a influência do tamanho dos poros de um scaffold à base de poli ácido láctico (PLLA) sobre a proliferação e diferenciação de células tronco da polpa dental (dental pulp stem cells - DPSC). Os scaffolds foram preparados dentro da câmara pulpar de discos de terceiros molares (1 mm), utilizando sal como porógeno (150-250 µm ou 251-450 µm). DPSC (1x105 células) foram semeadas nos scaffolds com diferentes tamanhos de poros e cultivadas em placas de 24 poços. A proliferação celular foi avaliada utilizando WST-1 após 3-21 dias. Além disso, RT-PCR foi utilizado para avaliar a diferenciação odontoblástica das DPSC utilizando marcadores da diferenciação odontoblástica (DSPP, DMP-1 e MEPE). RNA obtido de odontoblastos humanos foi utilizado como controle. A taxa de proliferação celular foi semelhante nos dois scaffolds avaliados, exceto no 14° dia, no qual as células cultivadas nos scaffolds com os maiores poros apresentaram uma maior taxa de proliferação (p<0,05). Após 21 dias, as DSPC cultivadas em ambos scaffolds avaliados foram capazes de expressar os marcadores odontoblásticos DMP-1, DSPP e MEPE. Em resumo, ambos scaffolds avaliados nesse estudo permitiram a proliferação e diferenciação odontoblástica das DPSC. .

Dental Pulp/cytology , Polyesters/chemistry , Stem Cells/physiology , Tissue Scaffolds/chemistry , Biocompatible Materials/chemistry , Cell Differentiation , Cell Proliferation , Dental Pulp Cavity/anatomy & histology , Molar, Third , Real-Time Polymerase Chain Reaction , Surface Properties , Tissue Culture Techniques , Tissue Engineering
Article in English | WPRIM | ID: wpr-164160


We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 microL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 microL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 microL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 microL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 microL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.

Adult Stem Cells/cytology , Animals , Biocompatible Materials/chemistry , Cell Differentiation , Fibroblast Growth Factor 2/administration & dosage , Heparin/administration & dosage , Humans , Materials Testing , Models, Animal , Poloxamer , Polyesters , Rats , Reconstructive Surgical Procedures , Regeneration , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Urinary Bladder/anatomy & histology , Urine/cytology
Braz. j. med. biol. res ; 47(8): 715-720, 08/2014. tab, graf
Article in English | LILACS | ID: lil-716274


Our objective was to observe the biodegradable and osteogenic properties of magnesium scaffolding under in vivo conditions. Twelve 6-month-old male New Zealand white rabbits were randomly divided into two groups. The chosen operation site was the femoral condyle on the right side. The experimental group was implanted with porous magnesium scaffolds, while the control group was implanted with hydroxyapatite scaffolds. X-ray and blood tests, which included serum magnesium, alanine aminotransferase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) were performed serially at 1, 2, and 3 weeks, and 1, 2, and 3 months. All rabbits were killed 3 months postoperatively, and the heart, kidney, spleen, and liver were analyzed with hematoxylin and eosin (HE) staining. The bone samples were subjected to microcomputed tomography scanning (micro-CT) and hard tissue biopsy. SPSS 13.0 (USA) was used for data analysis, and values of P<0.05 were considered to be significant. Bubbles appeared in the X-ray of the experimental group after 2 weeks, whereas there was no gas in the control group. There were no statistical differences for the serum magnesium concentrations, ALT, BUN, and CREA between the two groups (P>0.05). All HE-stained slices were normal, which suggested good biocompatibility of the scaffold. Micro-CT showed that magnesium scaffolds degraded mainly from the outside to inside, and new bone was ingrown following the degradation of magnesium scaffolds. The hydroxyapatite scaffold was not degraded and had fewer osteoblasts scattered on its surface. There was a significant difference in the new bone formation and scaffold bioabsorption between the two groups (9.29±1.27 vs 1.40±0.49 and 7.80±0.50 vs 0.00±0.00 mm3, respectively; P<0.05). The magnesium scaffold performed well in degradation and osteogenesis, and is a promising material for orthopedics.

Animals , Male , Rabbits , Absorbable Implants , Bone Substitutes/therapeutic use , Implants, Experimental , Magnesium/therapeutic use , Osteogenesis/physiology , Tissue Scaffolds/chemistry , Alanine Transaminase/blood , Blood Urea Nitrogen , Biocompatible Materials/therapeutic use , Creatinine/blood , Durapatite/therapeutic use , Femur , Femur/surgery , Heart/anatomy & histology , Kidney/anatomy & histology , Liver/anatomy & histology , Magnesium/blood , Porosity , Spleen/anatomy & histology , X-Ray Microtomography
Braz. j. med. biol. res ; 47(7): 533-539, 07/2014. tab
Article in English | LILACS | ID: lil-712964


Development and selection of an ideal scaffold is of importance for tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible bioresorbable copolymer that belongs to the polyhydroxyalkanoate family. Because of its good biocompatibility, PHBHHx has been widely used as a cell scaffold for tissue engineering. This review focuses on the utilization of PHBHHx-based scaffolds in tissue engineering. Advances in the preparation, modification, and application of PHBHHx scaffolds are discussed.

Humans , /chemistry , Biocompatible Materials/chemistry , Caproates/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , /therapeutic use , Biocompatible Materials/therapeutic use , Bone and Bones/physiology , Caproates/therapeutic use , Cartilage/physiology , Freeze Drying , Muscle, Smooth/physiology , Regeneration , Surface Properties
Clinics ; 67(6): 629-638, 2012. ilus, graf
Article in English | LILACS | ID: lil-640214


OBJECTIVE: Dark poly(caprolactone) trifumarate is a successful candidate for use as a bone tissue engineering scaffold. Recently, a white polymeric scaffold was developed that shows a shorter synthesis time and is more convenient for tissue-staining work. This is an in vitro comparative study of both the white and dark scaffolds. METHODS: Both white and dark poly(caprolactone) trifumarate macromers were characterized via Fourier transform infrared spectroscopy before being chemically cross-linked and molded into disc-shaped scaffolds. Biodegradability was assessed by percentage weight loss on days 7, 14, 28, 42 and 56 (n = 5) after immersion in 10% serum-supplemented medium or distilled water. Static cell seeding was employed in which isolated and characterized rat bone marrow stromal cells were seeded directly onto the scaffold surface. Seeded scaffolds were subjected to a series of biochemical assays and scanning electron microscopy at specified time intervals for up to 28 days of incubation. RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at days 1 and 14 revealed cellular growth and attachment. CONCLUSIONS: There was no cell growth advantage between the two forms, but the white scaffold had a slower biodegradability rate, suggesting that the newly synthesized poly(caprolactone) trifumarate is more suitable for use as a bone tissue engineering scaffold.

Animals , Rats , Absorbable Implants , Mesenchymal Stem Cells/chemistry , Polyesters/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Cells, Cultured , Color , Confidence Intervals , Materials Testing , Microscopy, Electron, Scanning , Spectroscopy, Fourier Transform Infrared , Time Factors
Braz. dent. j ; 23(4): 315-321, 2012. ilus
Article in English | LILACS | ID: lil-658004


It is known that current trends on bone bioengineering seek ideal scaffolds and explore innovative methods to restore tissue function. In this way, the objective of this study was to evaluate the behavior of anorganic bovine bone as osteoblast carrier in critical-size calvarial defects. MC3T3-E1 osteoblast cells (1x10(5) cells/well) were cultured on granules of anorganic bovine bone in 24-well plates and after 24 h these granules were implanted into rat critical-size calvarial defects (group Biomaterial + Cells). In addition, other groups were established with different fillings of the defect: Blood Clot (negative control); Autogenous Bone (positive control); Biomaterial (only granules) and Cells (only MC3T3-E1 cells). After 30 days, the animals were euthanized and the calvaria were technically processed in order to allow histological and morphometric analysis. It was possible to detect blood vessels, connective tissue and newly formed bone in all groups. Particularly in the Biomaterial + Cells group, it was possible to observe a profile of biological events between the positive control group (autogenous bone) and the group in which only anorganic bovine granules were implanted. Altogether, the results of the present study showed that granules of anorganic bovine bone can be used as carrier to osteoblasts and that adding growth factors at the moment of implantation should maximize these results.

Sabe-se que uma das atuais tendências na bioengenharia óssea é procurar um carreador ideal e explorar métodos inovadores para restaurar a função do tecido. Desta forma, nosso objetivo foi avaliar o comportamento do osso bovino inorgânico como carreador de osteoblastos em defeitos ósseos de tamanho crítico em calvária de ratos. Osteoblastos da linhagem MC3T3-E1 (1x10(5) células/poço) foram cultivadas em grânulos de osso bovino inorgânico sob placas de 24 poços e após 24 h esses grânulos foram implantados em defeitos ósseos de tamanho crítico em calvária de ratos. Além deste grupo experimental (Biomaterial + Células), foram estabelecidos outros grupos com diferentes preenchimentos do defeito crítico: coágulo sanguíneo (controle negativo); osso autógeno (controle positivo); Biomaterial (apenas grânulos) e Células (apenas células MC3T3-E1). Após 30 dias, os animais foram eutanasiados e as calvárias foram processadas histotecnicamente, a fim de permitir a análise histológica e morfometria. Nossos resultados mostraram que em todos os grupos avaliados foi possível detectar vasos sanguíneos, tecido conjuntivo e osso neoformado. Em especial para o grupo tratado com Biomaterial + Células, foi possível observar um perfil de eventos biológicos intermediário ao grupo controle positivo (osso autógeno) e o grupo de biomaterial (apenas grânulos inorgânico bovino). Ao todo, nossos resultados mostraram que os grânulos de osso bovino inorgânico podem ser usados como carreador de osteoblastos e que a adição de fatores de crescimento no momento em que ocorre o implante deve maximizar os resultados.

Animals , Cattle , Mice , Rats , Biocompatible Materials , Bone and Bones , Bone Diseases/surgery , Osteoblasts/physiology , Tissue Scaffolds , Tissue Engineering/methods , Blood , Bone Transplantation , Biocompatible Materials/chemistry , Blood Vessels/pathology , Cell Culture Techniques , Collagen , Connective Tissue/pathology , Fibroblasts/pathology , Frontal Bone/pathology , Frontal Bone/surgery , Osteogenesis/physiology , Parietal Bone/pathology , Parietal Bone/surgery , Time Factors , Transplantation, Autologous , Tissue Scaffolds/chemistry
Acta cir. bras ; 26(1): 7-11, jan.-fev. 2011. ilus, tab
Article in English | LILACS | ID: lil-572227


Purpose: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. Methods: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN®. Results: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. Conclusion: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.

Objetivo: Avaliar a capacidade de suportes tridimensionais macroporosos de cimento de fosfato de cálcio (CFC), de permitir a adesão, proliferação e diferenciação de células-tronco mesenquimais derivadas da medula óssea humana. Métodos: células obtidas da crista ilíaca de um doador humano adulto foram processadas e cultivadas sobre suportes de CFC, macroporosos, que tiveram como corpo gerador de poros, microesferas de parafina, com tamanho entre 100 e 250µm. Os períodos de cultura estabelecidos foram de cinco, 10 e 15 dias. Após estes períodos, o comportamento e a morfologia das células junto ao biomaterial foram avaliados por meio de Microscopia Eletrônica de Varredura. Os níveis de expressão dos genes BGLA e SSP1 bem como a atividade da Fosfatase Alcalina (ALP) foram quantificados pela técnica de PCR em Tempo Real (QT-PCR) utilizando o fluoróforo SYBR Green®. Resultados: O QT-PCR detectou a expressão dos genes BGLA e SSP1 e a atividade da fosfatase alcalina nos períodos de 10 e 15 dias de cultura. No período de cinco dias, não foi observada a expressão de nenhum dos genes investigados. Conclusão: O CFC, macroporoso, com tamanho de poros entre 100 e 250µm, criados por meio da utilização de microesferas de parafina, permite a adesão, proliferação e diferenciação de células-tronco mesenquimais em células osteogênicas, podendo ser utilizado como arcabouço para engenharia de tecido ósseo.

Adult , Humans , Biocompatible Materials , Bone and Bones , Bone Cements , Calcium Phosphates , Mesenchymal Stem Cells , Tissue Scaffolds , Tissue Engineering/methods , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Biocompatible Materials/chemistry , Bone Cements/chemistry , Cell Differentiation , Cell Proliferation , Calcium Phosphates/chemistry , Extracellular Matrix/metabolism , Gene Expression , Mesenchymal Stem Cells , Microscopy, Electron, Scanning , Osteogenesis , Osteocalcin/genetics , Osteocalcin/metabolism , Osteocytes/cytology , Osteopontin/genetics , Osteopontin/metabolism , Polymerase Chain Reaction/methods , Time Factors , Tissue Scaffolds/chemistry
Article in English | WPRIM | ID: wpr-121322


We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation.

Animals , Bioreactors , Bone Regeneration , Cell Differentiation , Cell Line , Cell Proliferation , Lactic Acid/chemistry , Mechanical Phenomena , Mechanotransduction, Cellular/physiology , Mice , Osteoblasts/cytology , Polyesters/chemistry , Polymers/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry
Article in English | WPRIM | ID: wpr-202561


Fibroblast-collagen matrix culture has facilitated the analysis of cell physiology under conditions that more closely resemble an in vivo-like environment compared to conventional 2-dimensional (2D) cell culture. Furthermore, it has led to significant progress in understanding reciprocal and adaptive interactions between fibroblasts and the collagen matrix, which occur in tissue. Recent studies on fibroblasts in 3-dimensional (3D) collagen matrices have revealed the importance of biomechanical conditions in addition to biochemical cues for cell signaling and migration. Depending on the surrounding mechanical conditions, cells utilize specific cytoskeletal proteins to adapt to their environment. More specifically, cells utilize microtubule dependent dendritic extensions to provide mechanical structure for matrix contraction under a low cell-matrix tension state, whereas cells in a high cell-matrix tension state utilize conventional acto-myosin activity for matrix remodeling. Results of collagen matrix contraction and cell migration in a 3D collagen matrix revealed that the use of appropriate growth factors led to promigratory and procontractile activity for cultured fibroblasts. Finally, the relationship between cell migration and tractional force for matrix remodeling was discussed.

Animals , Cell Culture Techniques , Cell Movement , Collagen/chemistry , Fibroblasts/cytology , Humans , Tissue Scaffolds/chemistry