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1.
The influence of variant pore-size and porosity on the microstructure and the mechanical properties of poly-epsilon-caprolactone (PCL) and hydroxyapatite (HA) composite were examined for an optimal scaffold in bone tissue engineering. Three various amounts of sodium chloride (NaCl, as porogens) with two distinct particle size ranges (212-355 mum and 355-600 mum) were blended into PCL and HA mixture, followed by a leaching technique to generate PCL-HA scaffolds with various pores and porosity. The porosities of the scaffolds were correlated with the porogen size and concentration. The morphological properties of the resulting scaffolds were assessed by micro-computerized tomography (muCT), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). Extensive PCL-HA pore interconnections with thinner pore walls were present in scaffolds with higher concentration (4:1 w/w) and larger particulate of porogen used in the fabrication process. Embedding of HA particles in the scaffold resulted in rough surfaces on the composites. Instron actuator testing indicated that the tensile strengths and Young's moduli of scaffolds were influenced by both the porosities and pore sizes of the scaffold. It was apparent that increasing the concentration of porogen compromised the mechanical properties; and a larger porogen particle size led to increased tensile strength but a reduction in Young's modulus. Overall, the data indicated that modification of the concentration and particle size of porogen altered the porous features and mechanical strength of HA-PCL scaffolds. This provided means to manipulate the properties of biocompatible cell-supporting scaffolds for use as bone graft substitutes.  相似文献   

2.
A method of compression-molding fine-powder blends of polycaprolactone (PCL) and poly(ethylene oxide) (PEO) and subsequently dissolving the PEO phase was investigated to prepare porous PCL scaffolds. Different mixing ratios of the two polymers from 20 to 70% PCL were used to study the effect of the mixing ratio on the morphology formation of the scaffold. The mixing ratio was found to play an important role in affecting the porosity of the scaffold and the size of pores. Murine embryonic stem cell derived osteogenic cells were utilized to test the suitability of these scaffolds in tissue engineering applications. The seeded cells were able to colonize and grow in these scaffolds. Based on the overall consideration of morphology, mechanical performance, and ability for cell attachment and proliferation, the scaffolds with approximately 30-40% PCL appear to be an appropriate choice for tissue engineering. These findings suggest that sacrificial compression-molding of PCL-PEO powder blends can be used in the generation of biocompatible scaffolds with controllable porosity and pore size and may be used for in vitro tissue engineering applications.  相似文献   

3.
Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and an average pore size of 280-450 microm were fabricated using gelatin particles as porogen. The particles were bonded together by incubation in saturated water vapor at 70 degrees C for 3.5 h. After casting the PLLA/1,4-dioxane solution, freeze-drying and porogen leaching with 70 degrees C water, a porous scaffold with well-interconnected pores and some nano-fibers was obtained. The biological performance of the scaffold was evaluated by in vitro chondrocyte culture and in vivo implantation. In comparison with the control scaffold fabricated with NaCl particles as porogen under the same conditions, the experimental scaffold had better biological performance because the gelatin molecules were stably entrapped onto the pore surfaces. A larger number of cells in the experimental scaffold were observed by confocal laser scanning microscopy after the viable cells had been stained with fluorescein diacetate. The chondrocytes showed more spreading morphology. Higher cytoviability and secretion of glycosaminoglycan (GAG) were also determined in the experimental scaffold. After implantation of the chondrocytes/PLLA scaffold construct to the subcutaneous dorsum of nude mice for 30-120 days, cartilage-like specimens were harvested. Histological examination showed that the regenerated cartilages had a large quantity of collagen and GAG.  相似文献   

4.
Shor L  Güçeri S  Wen X  Gandhi M  Sun W 《Biomaterials》2007,28(35):5291-5297
Computer-aided tissue-engineering approach was used to develop a novel precision extrusion deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0 degrees /90 degrees pattern. Two groups of scaffolds having 60% and 70% porosity and with pore sizes of 450 and 750 microm, respectively, were evaluated for their morphology and compressive properties using scanning electron microscopy (SEM) and mechanical testing. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue-engineering application. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.  相似文献   

5.
Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.  相似文献   

6.
In this work, three-dimensional porous composite scaffolds, based on poly(epsilon-caprolactone) (PCL), were fabricated through the combination of a filament winding technique and a phase inversion/salt leaching process. Sodium chloride crystals were used as the porogen agent, and poly(lactic acid) (PLA) fibers and calcium phosphates as reinforcement. The aim of the current work is to assess the effective synergistic role of bioactive particles (i.e. alpha-tricalcium phosphates (alpha-TCP)) and PLA fibers on the morphology and mechanical response of the final scaffold. Morphological investigations performed on fiber-reinforced composite scaffolds with different PCL/alpha-TCP volume ratios (0%, 13%, 20% and 26%) show a high porosity degree (ca. 80%), pore interconnection and a homogeneous distribution of pores within the scaffold. More specifically, a bimodal pore size distribution was observed. This comprised microporosity (pores with radii ranging from 0.1 to 10 microm, which were strictly related to solvent extraction) and macroporosity (pores with radii from 10 to 300 microm, which were ascribable to the leaching of porogen elements). Static compressive tests showed that the effect of alpha-TCP on the mechanical response was to increase the elastic modulus up to a maximum value of 2.21+/-0.24 MPa, depending on the concentration of alpha-TCP added. This effect may be explained through the interaction of calcium-deficient hydroxyapatite crystals, formed as a consequence of a hydrolysis reaction of alpha-TCP, and the fiber-reinforced polymer matrix. The correct balance between chemical composition and spatial organization of reinforcement systems allows the attainment of an ideal compromise between mechanical response and bioactive potential, facilitating the development of composite scaffolds for bone tissue engineering applications.  相似文献   

7.
Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and an average pore size of 280–450 μm were fabricated using gelatin particles as porogen. The particles were bonded together by incubation in saturated water vapor at 70°C for 3.5 h. After casting the PLLA/1,4-dioxane solution, freeze-drying and porogen leaching with 70°C water, a porous scaffold with well-interconnected pores and some nano-fibers was obtained. The biological performance of the scaffold was evaluated by in vitro chondrocyte culture and in vivo implantation. In comparison with the control scaffold fabricated with NaCl particles as porogen under the same conditions, the experimental scaffold had better biological performance because the gelatin molecules were stably entrapped onto the pore surfaces. A larger number of cells in the experimental scaffold were observed by confocal laser scanning microscopy after the viable cells had been stained with fluorescein diacetate. The chondrocytes showed more spreading morphology. Higher cytoviability and secretion of glycosaminoglycan (GAG) were also determined in the experimental scaffold. After implantation of the chondrocytes/PLLA scaffold construct to the subcutaneous dorsum of nude mice for 30–120 days, cartilage-like specimens were harvested. Histological examination showed that the regenerated cartilages had a large quantity of collagen and GAG.  相似文献   

8.
聚乳酸多孔支架制备及细胞实验   总被引:4,自引:0,他引:4  
以冰粒子作为致孔剂,采用冷冻干燥-粒子滤出复合法制备了块状聚乳酸多孔支架。将聚乳酸溶于氯仿溶液后加入冰粒子,在液氮中冷冻后冷冻干燥获得多孔支架。对支架孔隙结构分析表明,该工艺制备的多孔支架无致孔剂残留,其孔隙大小由加入的冰粒子大小决定。细胞实验表明该多孔支架具有较好的生物相容性并且无细胞毒性。  相似文献   

9.
In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.  相似文献   

10.
Novel three-dimensional scaffolds consisting of nano- and microsized hydroxyapatite (HA)/poly(epsilon-caprolactone) (PCL) composite were fabricated using a modified rapid-prototyping (RP) technique for bone tissue engineering applications. The size of the nano-HA ranged from 20 to 90 nm, whereas that of the micro-HA ranged from 20 to 80 microm. The scaffold macropores were well interconnected, with a porosity of 72-73% and a pore size of 500 microm. The compressive modulus of the nano-HA/PCL and micro-HA/PCL scaffolds was 3.187 +/- 0.06 and 1.345 +/- 0.05 MPa, respectively. The higher modulus of the nano-HA/PCL composite (n-HPC) was to be likely caused by a dispersion strengthening effect. The attachment and proliferation of MG-63 cells on n-HPC were better than that on the micro-HA/PCL composite (m-HPC) scaffold. The n-HPC was more hydrophilic than the m-HPC because of the greater surface area of HA exposed to the scaffold surface. This may give rise to better cell attachment and proliferation. Bioactive n-HA/PCL composite scaffold prepared using a modified RP technique has a potential application in bone tissue engineering.  相似文献   

11.
Scaffolds for tissue engineering applications must incorporate porosity for optimal cell seeding, tissue ingrowth, and vascularization, but common fabrication methods for achieving porosity are incompatible with a variety of polymers, limiting widespread use. In this study, porous scaffolds consisting of poly(1,8-octanediol-co-citrate) (POC) containing hydroxyapatite nanocrystals (HA) were fabricated using low-pressure foaming (LPF). LPF is a novel method of fabricating an interconnected, porous scaffold with relative ease. LPF takes advantage of air bubbles that act as pore nucleation sites during a polymer mixing step. Vacuum is applied to expand the nucleation sites into interconnected pores that are stabilized through cross-linking. POC was combined with 20%, 40%, and 60% by weight HA, and the effect of increasing HA particle content on porosity, mechanical properties, and alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSC) was evaluated. The effect of the prepolymer viscosity on porosity and the mechanical properties of POC with 40% by weight HA (POC-40HA) were also assessed. POC-40HA scaffolds were also implanted in an osteochondral defect of a rabbit model, and the explants were assessed at 6 weeks using histology. With increasing HA content, the pore size of POC-HA scaffolds can be varied (85 to 1,003 μm) and controlled to mimic the pore size of native trabecular bone. The compression modulus increased with greater HA content under dry conditions and were retained to a greater extent than with porous scaffolds fabricated using salt-leaching under wet conditions. Furthermore, all POC-HA scaffolds prepared using LPF supported hMSC attachment, and an increase in ALP activity correlated with an increase in HA content. An increase in the prepolymer viscosity resulted in increased compression modulus, greater distance between pores, and less porosity. After 6 weeks in vivo, cell and tissue infiltration was present throughout the scaffold. This study describes a novel method of creating porous osteoconductive POC scaffolds without the need for porogen leaching and provides the groundwork for applying LPF to other elastomers and composites.  相似文献   

12.
Kim HW  Knowles JC  Kim HE 《Biomaterials》2004,25(7-8):1279-1287
Hydroxyapatite (HA) porous scaffold was coated with HA and polycaprolactone (PCL) composites, and antibiotic drug tetracycline hydrochloride was entrapped within the coating layer. The HA scaffold obtained by a polymeric reticulate method, possessed high porosity ( approximately 87%) and controlled pore size (150-200 microm). Such a well-developed porous structure facilitated usage in a drug delivery system due to its high surface area and blood circulation efficiency. The PCL polymer, as a coating component, was used to improve the brittleness and low strength of the HA scaffold, as well to effectively entrap the drug. To improve the osteoconductivity and bioactivity of the coating layer, HA powder was hybridized with PCL solution to make the HA-PCL composite coating. With alteration in the coating concentration and HA/PCL ratio, the morphology, mechanical properties, and biodegradation behavior were investigated. Increasing the concentration rendered the stems thicker and some pores to be clogged; as well increasing the HA/PCL ratio made the coating surface be rough due to the large amount of HA particles. However, for all concentrations and compositions, uniform coatings were formed, i.e., with the HA particles being dispersed homogeneously in the PCL sheet. With the composite coating, the mechanical properties, such as compressive strength and elastic modulus were improved by several orders of magnitude. These improvements were more significant with thicker coatings, while little difference was observed with the HA/PCL ratio. The in vitro biodegradation of the composite coatings in the phosphate buffered saline solution increased linearly with incubation time and the rate differed with the coating concentration and the HA/PCL ratio; the higher concentration and HA amount caused the increased biodegradation. At short period (<2 h), about 20-30% drug was released especially due to free drug at the coating surface. However, the release rate was sustained for prolonged periods and was highly dependent on the degree of coating dissolution, suggesting the possibility of a controlled drug release in the porous scaffold with HA+PCL coating.  相似文献   

13.
In this study, we fabricated polycaprolactone/hydroxyapatite (PCL/HA) scaffolds with a multi-head deposition system, a solid free-form fabrication technology that was developed in our previous study. The bone regeneration potential of the scaffolds was compared with that of PCL scaffolds fabricated with the same system. The fabricated scaffolds had a pore size of 400 μm and a porosity of 66.7%. The PCL/HA scaffolds had higher mechanical strength and modulus than the PCL scaffolds. To compare the osteogenic potential, the two types of scaffolds were seeded with rat osteoblasts and cultured in vitro or implanted subcutaneously into athymic mice. The cells cultured on PCL/HA scaffolds expressed higher levels of osteopontin and osteonectin, both of which are osteogenic proteins. The PCL/HA scaffolds resulted in larger bone area and calcium deposition in the implants compared to the PCL scaffolds.  相似文献   

14.
Preparation of poly(D,L-lactic acid) scaffolds using alginate particles   总被引:1,自引:0,他引:1  
A kind of novel natural polysaccharide (sodium alginate) porogen was developed to prepare a 3D biodegradable tissue-engineering scaffold. The sodium alginate particles were prepared by emulsification and subsequent ionic gelation. The size and morphology of the alginate particles was simply controlled by the stirring rate and the concentration of the cross-linking agent. ATR-FTIR spectroscopy demonstrated the existence of alginate molecules on the surface of the PLA scaffold. The water uptake of the scaffold made from alginate particles was obviously improved compared with the scaffold fabricated by KCl porogens. A MC3T3 osteoblast culture on the scaffolds showed that the alginate-modified PLA scaffolds significantly enhanced the osteoblast adhesion and proliferation. These results indicate that the alginate particle is a good porogen in the fabrication of 3D scaffolds for bone tissue engineering.  相似文献   

15.
Abstract

Biomaterial scaffolds have been increasingly used for tissue engineering applications as well as three dimensional (3D) cell culture models. Herein, we report a simple procedure combining compression molding, heating, and leaching methods for the fabrication of 3D micro-porous poly(ε-caprolactone) (PCL) biomaterial scaffolds. In this procedure, PCL micro particles are mixed with NaCl of defined sizes and compression molded, followed by heating and subsequent leaching of NaCl particles. This technique eliminates the gas foaming method, which is commonly used in the fabrication of PCL scaffolds. Process and scaffold parameters (i.e., heating time, NaCl concentration, and NaCl particle size) were varied and analyzed to determine their impact on the overall scaffold structural and mechanical properties. An increase in NaCl particle size led to an increase in pore area but did not significantly impact the mechanical properties of the scaffolds. Additionally, NaCl concentration did not show a significant effect on pore area, but considerably impacted the mechanical properties, water absorption capacity and porosity of the scaffolds. Variations in the heating time did not have an effect in the pore area, porosity, water absorption capacity or mechanical properties of the scaffolds. We also demonstrated the ability of these scaffolds to support the proliferation of breast cancer cells. Overall, these results elucidated structure-property relationships in the fabricated micro-porous PCL scaffolds. Further, this procedure could be potentially scaled up for the fabrication of micro-porous PCL scaffolds.  相似文献   

16.
For tissue-engineering applications, a 3D porous chitosan scaffold was simply prepared from a mixture of acidic chitosan solution and sodium acetate particles as the porogen by a salt-leaching method. Differences in the porous structure in terms of pore morphology and interconnectivity between the salt-leached chitosan scaffold and phase-separated scaffold as the control were examined by using scanning electron microscopy, protein release and enzymatic degradation tests. A fibroblast (NIH-3T3) cell culture was performed for cell affinity evaluation. The chitosan scaffold prepared by salt-leaching showed good interconnectivity and improved mechanical properties. Furthermore, the chitosan scaffolds showed a high initial cell adhesion after 4 h cell culture and increased cell proliferation than the control. Thus, salt-leached chitosan scaffolds can be used for various tissue-engineering applications.  相似文献   

17.
Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and pore size ranging from 150 to 700 microm were conveniently prepared with paraffin spheres used as porogen. PLLA/1,4-dioxane solution containing a given amount of paraffin spheres was frozen at -25 degrees C to obtain a solidified mixture, followed with freeze drying and subsequent leaching with hexane to remove the 1,4-dioxane and paraffin spheres, respectively. The fabricated PLLA scaffolds were highly porous with evenly distributed and interconnected pores. The microstructures of the PLLA scaffolds as a function of paraffin-sphere size, paraffin-sphere dosage, and PLLA concentration were characterized by confocal laser scanning microscopy (CLSM) and scanning-electronic microscopy (SEM). To improve the cytocompatibility of the bioinert material, a hybrid PLLA scaffold containing Type I collagen was prepared by pressing the collagen solution into the scaffold under reduced pressure. The amounts of the collagen introduced in the scaffolds were detected by ninhydrin method. The distribution of the collagen in the scaffolds was studied with CLSM. Finally, in vitro cell culture was performed by injecting a chondrocyte suspension into the scaffolds. The results showed that the chondrocytes were more evenly distributed and more spread out in the collagen-modified PLLA scaffolds than in the unmodified ones.  相似文献   

18.
In this study, highly porous collagen-HA scaffolds were prepared by solid-liquid phase separation method. Microstructure of the composites was characterized by SEM, TEM and XRD. The results show that collagen-HA scaffolds are porous with three-dimension interconnected fiber microstructure, pore sizes are 50-150 microm, and HA particles are dispersed evenly among collagen fiber. Compared with pure collagen, the mechanical property of collagen-HA composite improves significantly. To gain further insight into cell growth throughout 3D scaffolds, the cell proliferation and attachment on the scaffold in vitro was investigated. The collagen-HA composite has good biocompatibility, and adding HA does not affect the histocompatibility of the scaffold materials. The porous collagen-HA composite is suitable as scaffold used for bone tissue engineering.  相似文献   

19.
Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds   总被引:6,自引:0,他引:6  
A three-dimensional (3D) hyaluronic acid (HA) nanofibrous scaffold was successfully fabricated to mimic the architecture of natural extracellular matrix (ECM) based on electrospinning. Thiolated HA derivative, 3,3'-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), was synthesized and electrospun to form 3D nanofibrous scaffolds. In order to facilitate the fiber formation during electrospinning, Poly (ethylene oxide) (PEO) was added into the aqueous solution of HA-DTPH at an optimal weight ratio of 1:1. The electrospun HA-DTPH/PEO blend scaffold was subsequently cross-linked through poly (ethylene glycol)-diacrylate (PEGDA) mediated conjugate addition. PEO was then extracted in DI water to obtain an electrospun HA-DTPH nanofibrous scaffold. NIH 3T3 fibroblasts were seeded on fibronectin-adsorbed HA-DTPH nanofibrous scaffolds for 24h in vitro. Fluorescence microscopy and laser scanning confocal microscopy revealed that the 3T3 fibroblasts attached to the scaffold and spread, demonstrating an extended dendritic morphology within the scaffold, which suggests potential applications of HA-DTPH nanofibrous scaffolds in cell encapsulation and tissue regeneration.  相似文献   

20.
Biocompatible polycaprolactone (PCL) and hydroxyapatite (HA) were fabricated into nanofibrous scaffolds for the mineralization of osteoblasts in bone tissue engineering. PCL and PCL/HA nanofibrous surface were modified using oxygen plasma treatment and showing 0 degrees contact angle for the adhesion and mineralization of osteoblast cells. The fiber diameter, pore size and porosity of nanofibrous scaffolds were estimated to be 220-625 nm, 3-20 microm, and 87-92% respectively. The ultimate tensile strength of PCL was about 3.37 MPa and PCL/HA was 1.07 MPa to withstand the long term culture of osteoblasts on nanofibrous scaffolds. Human fetal osteoblast cells (hFOB) were cultured on PCL and PCL/HA surface modified and unmodified nanofibrous scaffolds. The osteoblast proliferation rate was significantly (p < 0.001) increased in surface-modified nanofibrous scaffolds. FESEM showed normal phenotypic cell morphology and mineralization occurred in PCL/HA nanofibrous scaffolds, HA acting as a chelating agent for the mineralization of osteoblast to form bone like apatite for bone tissue engineering. EDX and Alizarin Red-S staining indicated mineral Ca(2+) and phosphorous deposited on the surface of osteoblast cells. The mineralization was significantly increased in PCL/HA-modified nanofibrous scaffolds and appeared as a mineral nodule synthesized by osteoblasts similar to apatite of the natural bone. The present study indicated that the PCL/HA surface-modified nanofibrous scaffolds are potential for the mineralization of osteoblast for bone tissue engineering.  相似文献   

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