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‣ Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering

Sicchieri, Luciana Goncalves; Crippa, Grasiele Edilaine; de Oliveira, Paulo Tambasco; Beloti, Marcio Mateus; Rosa, Adalberto Luiz
Fonte: WILEY-BLACKWELL; MALDEN Publicador: WILEY-BLACKWELL; MALDEN
Tipo: Artigo de Revista Científica
Português
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A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)calcium phosphate (PLGACaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470590, 590850 and 8501200 mu m. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470590 mu m. These results show that PLGACaP is a promising biomaterial for bone engineering. However...

‣ Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects

Zambuzzi, Willian F.; Fernandes, Gustavo V. O.; Iano, Flávia G.; Fernandes, Mileni da S.; Granjeiro, José Mauro; Oliveira, Rodrigo Cardoso
Fonte: Fundação Odontológica de Ribeirão Preto Publicador: Fundação Odontológica de Ribeirão Preto
Tipo: Artigo de Revista Científica
Português
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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.

‣ Avaliação do uso de placas sinterizadas de titânio com e sem recobrimento de beta-fosfato tricálcico no reparo de falhas ósseas em calvária de ratos = : Evaluation of sintered titanium scaffolds with and without beta-tricalcium phosphate coating for calvarial defects repair in rats; Evaluation of sintered titanium scaffolds with and without beta-tricalcium phosphate coating for calvarial defects repair in rat

Davi Reis Calderoni
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 16/12/2014 Português
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Introdução: A reparação de um defeito craniano é um procedimento desafiador, que requer não somente o preenchimento da falha óssea mas também estabilidade a longo prazo do material empregado e restabelecimento do contorno e simetria. Diversos tipos de materiais com diferentes características bem como diferentes métodos de produção dos implantes vêm sendo testados, sem que ainda haja um substituto ósseo considerado ideal. No presente estudo foram investigadas as propriedades de osteointegração de implantes da liga Ti6Al4V construídos por prototipagem com poros tridimensionalmente conectados considerando a influência do recobrimento dos mesmos com uma camada delgada de ?-fosfato tricálcico nestas propriedades. Método: Trinta ratos foram submetidos à criação de defeitos ósseos bilaterais na calvária, que foram preenchidos com implantes, recobertos ou não com ?-fosfato tricálcico, de modo aleatório. Os animais foram distribuídos em grupos e sacrificados 15, 45 e 90 dias após o procedimento. A integração dos implantes foi inicialmente avaliada por ensaio de compressão. A interface osso-implante foi analisada por meio de microscopia eletrônica de varredura. Resultados: A força máxima para produzir o deslocamento inicial dos implantes foi aumentando durante o período estudado...

‣ Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects

Zambuzzi,Willian F.; Fernandes,Gustavo V. O.; Iano,Flávia G.; Fernandes,Mileni da S.; Granjeiro,José Mauro; Oliveira,Rodrigo Cardoso
Fonte: Fundação Odontológica de Ribeirão Preto Publicador: Fundação Odontológica de Ribeirão Preto
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2012 Português
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1702.3911%
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.

‣ Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering

Sefcik, Lauren S.; Petrie Aronin, Caren E.; Wieghaus, Kristen A.; Botchwey, Edward A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cells types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects and healing of bony defects was assessed by radiograph x-ray, microcomputed tomography (μCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together...

‣ Comparative effects of scaffold pore size, pore volume, and total void volume on cranial bone healing patterns using microsphere-based scaffolds

Petrie Aronin, Caren E.; Sadik, Karim W.; Lay, Ann L.; Rion, Dave B.; Tholpady, Sunil S.; Ogle, Roy C.; Botchwey, Edward A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/2009 Português
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Bony craniofacial deficits resulting from injury, disease, or birth defects remain a considerable clinical challenge. In this study, microsphere-based scaffold fabrication methods were use to study the respective effects of scaffold pore size, open pore volume, and total void volume fraction on osseous tissue infiltration and bone regeneration in a critical size rat cranial defect. To compare the healing effects of these parameters, three different scaffolds types were fabricated: solid 100 μm spheres, solid 500 μm spheres, and hollow 500 μm spheres. These constructs were implanted into surgically created rat calvarial defects. By 90-days post op, results of micro computed tomography (CT) analysis showed that all scaffolds generated similar amounts of new bone which was significantly greater than untreated controls. Interestingly, the spatial distribution of new bone within the defect area varied by scaffold group. MicroCT and histological analysis demonstrated healing restricted to the dural side in the hollow 500 μm group, whereas the solid 500 μm group demonstrated healing along the dural side and within the center of the defect. Solid 100 μm groups demonstrated healing along the dural layer, periosteal layer, and within the center of the defect. These results suggest that pore size and closed void volume may both play important roles in scaffold degradation patterns and associated bone healing.

‣ Bone Progenitors Produced by Direct Osteogenic Differentiation of the Unprocessed Bone Marrow Demonstrate High Osteogenic Potential In Vitro and In Vivo

Ginis, Irene; Weinreb, Miron; Abramov, Natalie; Shinar, Doron; Merchav, Shoshana; Schwartz, Aharon; Shirvan, Mitchell
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Publicado em /04/2012 Português
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Tissue-engineered bone grafts seeded with mesenchymal stem cells (MSCs) have been sought as a replacement for bone grafts currently used for bone repair. For production of osteogenic constructs, MSCs are isolated from bone marrow (BM) or other tissues, expanded in culture, then trypsinized, and seeded on a scaffold. Predifferentiation of seeded cells is often desired. We describe here bone progenitor cells (BPCs) obtained by direct osteogenic differentiation of unprocessed BM bypassing isolation of MSCs. Human BM aspirates were incubated for 2 weeks with a commonly used osteogenic medium (OM), except no fetal calf serum, serum substitutes, or growth factors were added, because responding stem and/or progenitor cells were present in the BM milieu. The adherent cells remaining after the culture medium and residual BM were washed out, expressed high levels of bone-specific alkaline phosphatase (ALP) on their surface, demonstrated high ALP activity, were capable of mineralization of the intercellular space, and expressed genes associated with osteogenesis. These parameters in BPCs were similar and even at higher levels compared to MSCs subjected to osteogenic differentiation for 2 weeks. The yield of BPCs per 1 mL BM was 0.71±0.39×106. In comparison...

‣ High Calcium Bioglass Enhances Differentiation and Survival of Endothelial Progenitor Cells, Inducing Early Vascularization in Critical Size Bone Defects

Eldesoqi, Karam; Seebach, Caroline; Nguyen Ngoc, Christina; Meier, Simon; Nau, Christoph; Schaible, Alexander; Marzi, Ingo; Henrich, Dirk
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 14/11/2013 Português
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Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40...

‣ Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration

Ansari, Sahar; Moshaverinia, Alireza; Pi, Sung Hee; Han, Alexander; Abdelhamid, Alaa I.; Zadeh, Homayoun H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro...

‣ Safety Evaluation of a Bioglass–Polylactic Acid Composite Scaffold Seeded with Progenitor Cells in a Rat Skull Critical-Size Bone Defect

Eldesoqi, Karam; Henrich, Dirk; El-Kady, Abeer M.; Arbid, Mahmoud S.; Abd El-Hady, Bothaina M.; Marzi, Ingo; Seebach, Caroline
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 03/02/2014 Português
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Treating large bone defects represents a major challenge in traumatic and orthopedic surgery. Bone tissue engineering provides a promising therapeutic option to improve the local bone healing response. In the present study tissue biocompatibility, systemic toxicity and tumorigenicity of a newly developed composite material consisting of polylactic acid (PLA) and 20% or 40% bioglass (BG20 and BG40), respectively, were analyzed. These materials were seeded with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) and tested in a rat calvarial critical size defect model for 3 months and compared to a scaffold consisting only of PLA. Serum was analyzed for organ damage markers such as GOT and creatinine. Leukocyte count, temperature and free radical indicators were measured to determine the degree of systemic inflammation. Possible tumor occurrence was assessed macroscopically and histologically in slides of liver, kidney and spleen. Furthermore, the concentrations of serum malondialdehyde (MDA) and sodium oxide dismutase (SOD) were assessed as indicators of tumor progression. Qualitative tissue response towards the implants and new bone mass formation was histologically investigated. BG20 and BG40, with or without progenitor cells...

‣ Low-Dose Bone Morphogenetic Protein-2/Stromal Cell-Derived Factor-1β Cotherapy Induces Bone Regeneration in Critical-Size Rat Calvarial Defects

Herberg, Samuel; Susin, Cristiano; Pelaez, Manuel; Howie, R. Nicole; Moreno de Freitas, Rubens; Lee, Jaebum; Cray, James J.; Johnson, Maribeth H.; Elsalanty, Mohammed E.; Hamrick, Mark W.; Isales, Carlos M.; Wikesjö, Ulf M.E.; Hill, William D.
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Português
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Increasing evidence suggests that stromal cell-derived factor-1 (SDF-1/CXCL12) is involved in bone formation, though underlying molecular mechanisms remain to be fully elucidated. Also, contributions of SDF-1β, the second most abundant splice variant, as an osteogenic mediator remain obscure. We have shown that SDF-1β enhances osteogenesis by regulating bone morphogenetic protein-2 (BMP-2) signaling in vitro. Here we investigate the dose-dependent contribution of SDF-1β to suboptimal BMP-2-induced local bone formation; that is, a dose that alone would be too low to significantly induce bone formation. We utilized a critical-size rat calvarial defect model and tested the hypotheses that SDF-1β potentiates BMP-2 osteoinduction and that blocking SDF-1 signaling reduces the osteogenic potential of BMP-2 in vivo. In preliminary studies, radiographic analysis at 4 weeks postsurgery revealed a dose-dependent relationship in BMP-2-induced new bone formation. We then found that codelivery of SDF-1β potentiates suboptimal BMP-2 (0.5 μg) osteoinduction in a dose-dependent order, reaching comparable levels to the optimal BMP-2 dose (5.0 μg) without apparent adverse effects. Blocking the CXC chemokine receptor 4 (CXCR4)/SDF-1 signaling axis using AMD3100 attenuated the osteoinductive potential of the optimal BMP-2 dose...

‣ Application of AMOR in Craniofacial Rabbit Bone Bioengineering

Freire, Marcelo; Choi, Jeong-Ho; Nguyen, Anthony; Chee, Young Deok; Kook, Joong-Ki; You, Hyung-Keun; Zadeh, Homayoun H.
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
Português
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Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2, BMP-4, and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end, we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks, de novo bone formation was demonstrated by micro-CT imaging, histology, and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration, suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects, suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans...

‣ A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue

Ferroni, Letizia; Gardin, Chiara; Sivolella, Stefano; Brunello, Giulia; Berengo, Mario; Piattelli, Adriano; Bressan, Eriberto; Zavan, Barbara
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 02/03/2015 Português
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321.05332%
Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue.

‣ New LLLT protocol to speed up the bone healing process-histometric and immunohistochemical analysis in rat calvarial bone defect

Marques, Leonardo; Holgado, Leandro A.; Francischone, Leda A.; Ximenez, Joao P. B.; Okamoto, Roberta; Kinoshita, Angela
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica Formato: 1225-1230
Português
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); A new low-level laser therapy (LLLT) protocol is proposed and compared to another previously studied, in animal models, aiming to establish a more practical LLLT protocol. Protocol 1, the same used in other works and based on the clinical LLLT protocol for bone regeneration, consists of punctual transcutaneous applications in the defect region with fluence of 16 J/cm(2) every 48 h for 15 days. Protocol 2, proposed in this work, consists of three sessions: the first application directly on the defect site with fluency of 3.7 J/cm(2), during the surgical procedure, followed by two transcutaneous applications, 48 and 120 h postoperatively. The Thera LaseA (R) (lambda = 830 nm) was used, and the dosimetry of the first application of protocol 2 was calculated based on in vitro studies. Forty-five male rats were used, in which critical-size bone defects with 8 mm of diameter were surgically created in calvaria. The animals were randomly divided into three groups of 15 animals, named group 1 (protocol 1), group 2 (protocol 2), and control, which was not submitted to laser treatment. After 7, 15, and 45 days, five animals of each group were euthanized, and the pieces of calvarial bone were collected for microscopic and immunohistochemistry for vascular endothelial growth factor (VEGF)...

‣ Comparative Evaluation of Nanofibrous Scaffolding for Bone Regeneration in Critical-Size Calvarial Defects

Woo, Kyung Mi; Chen, Victor J.; Jung, Hong-Moon; Kim, Tae-Il; Shin, Hong-In; Baek, Jeong-Hwa; Ryoo, Hyun-Mo; Ma, Peter X.
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Português
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In a previous study we found that nanofibrous poly(l-lactic acid) (PLLA) scaffolds mimicking collagen fibers in size were superior to solid-walled scaffolds in promoting osteoblast differentiation and bone formation in vitro. In this study we used an in vivo model to confirm the biological properties of nanofibrous PLLA scaffolds and to evaluate how effectively they support bone regeneration against solid-walled scaffolds. The scaffolds were implanted in critical-size defects made on rat calvarial bones. Compared with solid-walled scaffolds, nanofibrous scaffolds supported substantially more new bone tissue formation, which was confirmed by micro-computed tomography measurement and von Kossa staining. Goldner's trichrome staining showed abundant collagen deposition in nanofibrous scaffolds but not in the control solid-walled scaffolds. The cells in these scaffolds were immuno-stained strongly for Runx2 and bone sialoprotein (BSP). In contrast, solid-walled scaffolds implanted in the defects were stained weakly with trichrome, Runx2, and BSP. These in vivo results demonstrate that nanofibrous architecture enhances osteoblast differentiation and bone formation.