Stability and cellular responses to fluorapatite-collagen composites

Fluorapatite (FA)-collagen composites were synthesized via a biomimetic coprecipitation method in order to improve the structural stability and cellular responses. Different amounts of ammonium fluoride (NH4F), acting as a fluorine source for FA, were added to the precipitation of the composites. The precipitated composites were freeze-dried and isostatically pressed in a dense body. The added fluorine was incorporated nearly fully into the apatite structure (fluoridation), and a near stoichiometric FA-collagen composite was obtained with complete fluoridation. The freeze-dried composites had a typical biomimetic network, consisting of collagen fibers and precipitates of nano-sized apatite crystals. The human osteoblast-like cells on the FA-collagen composites exhibited significantly higher proliferation and differentiation (according to alkaline phosphatase activity) than those on the hydroxyapatite-collagen composite. These enhanced osteoblastic cell responses were attributed to the fluorine release and the reduced dissolution rate.     

Poly-epsilon-caprolactone/hydroxyapatite composites for bone regeneration: in vitro characterization and human osteoblast response

Polycaprolactone (PCL), a semicrystalline linear resorbable aliphatic polyester, is a good candidate as a scaffold for bone tissue engineering, due to its biocompatibility and biodegradability. However, the poor mechanical properties of PCL impair its use as scaffold for hard tissue regeneration, unless mechanical reinforcement is provided. To enhance mechanical properties and promote osteoconductivity, hydroxyapatite (HA) particles were added to the PCL matrix: three PCL-based composites with different volume ratio of HA (13%, 20%, and 32%) were studied. Mechanical properties and structure were analysed, along with biocompatibility and osteoconductivity. The addition of HA particles (in particular in the range of 20% and 32%) led to a significant improvement in mechanical performance (e.g., elastic modulus) of scaffold. Saos-2 cells and osteoblasts from human trabecular bone (hOB) retrieved during total hip replacement surgery were seeded onto 3D PCL samples for 1-4 weeks. Following the assessment of cell viability, proliferation, morphology, and ALP release, HA-loaded PCL was found to improve osteoconduction compared to the PCL alone. The results indicated that PCL represents a potential candidate as an efficient substrate for bone substitution through an accurate balance between structural/ mechanical properties of polymer and biological activities.     

Evaluation of biomimetic scaffold of gelatin-hydroxyapatite crosslink as a novel scaffold for tissue engineering: biocompatibility evaluation with human PDL fibroblasts, human mesenchymal stromal cells, and primary bone cells

Biomimetic gelatin (gel)-hydroxyapatite (HA) composites have been prepared for studying hard tissue engineering scaffolds. However, the biocompatibility test of this form of material using these three cell types, which are periodontal ligament (PDL) fibroblast cells, human mesenchymal stromal cells (HMSc) and primary cells from human hip bone (HBc) has never been evaluated. The objective of this article is to prepare and evaluate the biocompatibility of gel-HA crosslinked scaffold for tissue engineering. Two different scaffolds were prepared: preparation (1), 2.5% gel/2.5% HA; preparation (2), 2.5% gel/5% HA. Three cell types including PDL, HMSc, and HBc were used. Assessment of biocompatibility and osteoblastic cellular responses was evaluated using a three-dimensional cell culture method and scanning electron microscopy (SEM). From SEM, it was observed that scaffold (1) exhibits stable porous formation with well-blended and dispersed HA powder. All three cell types were able to proliferate in both scaffolds. The HMSc and HBc got attached to the scaffolds to a significantly higher degree and subsequently proliferated more than PDL. The alkaline phosphatase (ALP) activities of HMSc and HBc were stronger when cultured in scaffold (S1) than (S2). It was seen that the two scaffold preparations show good biocompatibility with all three cell types tested. The better cellular responses with scaffold (S1) than (S2) might be due to the different structural and morphological characteristics, that is, scaffold (S1) retained more small-sized apatite crystals and a better developed pore configuration than scaffold (S2). Based on these findings, the biomimetically synthesized composite scaffolds have the potential to be used in hard tissue regeneration and tissue engineering fields.     

Electrosprayed Hydroxyapatite on Polymer Nanofibers to Differentiate Mesenchymal Stem Cells to Osteogenesis

Abstract

Electrospraying of hydroxyapatite (HA) nanoparticles onto the surface of polymer nanofibers provides a potentially novel substrate for the adhesion, proliferation and differentiation of mesenchymal stem cells (MSCs) into bone tissue regeneration. HA nanoparticles (4%) were electrosprayed on the surface of electrospun polycaprolactone (PCL) nanofibers (420 ± 15 nm) for bone tissue engineering. PCL/HA nanofibers were comparatively characterized with PCL/Collagen (275 ± 56 nm) nanofibers by FT-IR analysis to confirm the presence of HA. Fabricated PCL/HA and PCL/Collagen nanofibers and TCP (control) were used for the differentiation of equine MSC into osteogenic lineages in the presence of DMEM/F12 medium supplemented with β-glycerophosphate, ascorbic acid and dexamethasone. Cell proliferation and differentiation into an osteogenic lineage was evaluated by MTS assay, SEM observation, ALP activity, ARS staining, quantification of mineral deposition and expression of osteocalcin. Proliferation of MSCs increased significantly (P ? 0.05) up to 12% in PCL/Collagen (day 15) compared to PCL/HA nanofibrous substrate. ALP activity was increased 20% in PCL/HA by day 10 confirming the direction of osteogenic lineage from MSCs differentiation. PCL/HA stimulated an increased mineral secretion up to 26% by day 15 on ARS staining compared to PCL/Collagen nanofibers and showing cuboidal morphology by expressing osteocalcin. These results confirmed that the specifically fabricated PCL/HA composite nanofibrous substrate enhanced the differentiation of MSCs into osteogenesis.     

Hydroxyapatite and fluor-hydroxyapatite layered film on titanium processed by a sol-gel route for hard-tissue implants

A double-layered coating, consisting of a hydroxyapatite (HA) outer film and a fluor-hydroxyapatite (FHA) inner film, was produced on a Ti substrate by a sol-gel route to improve the biocompatibility and functionality of the system. Dissolution behavior of and in vitro cellular responses to the layered film were investigated. Calcium nitrate and triethyl phosphite were used for calcium and phosphate precursors, respectively, and ammonium fluoride was added as a fluorine-ion source for FHA. The FHA layer was deposited on Ti by spin coating and subsequent heat treatment at 550 degrees C for 30 min in air, and then the HA layer was laid down over the FHA-coated Ti under the same conditions. After heat treatment, characteristic apatite structures and phases were developed on both FHA and HA films. The cross-section view of the HA/FHA film clearly showed a double-layered structure on Ti with each layer approximately 0.6-0.8-microm thickness. The coating layer was highly uniform and dense, and adhered to Ti substrate strongly with an adhesion strength of about 40 MPa. The in vitro solubility of the HA/FHA layered film in a physiological solution was between that of HA and FHA pure film, and the dissolution profile was quite biphasic, that is, an initial rapid period and a slowdown with increasing time, reflecting the gradient solubility of the fast HA outer structure/slow FHA inner structure. The human osteoblast-like HOS TE85 cells cultured on the HA/FHA layered film attached, spread, and grew favorably. The proliferation rate of the cells on the layered film was significantly higher (considered at p < 0.05 for n = 6) than that on Ti substrate and was similar to that on pure HA film. The alkaline phosphatase (ALP) activity and osteocalcin (OC) produced by the cells on the layered film were significantly higher (considered at p < 0.05 for n = 6) than those on Ti substrate. Moreover, the ALP and OC levels of cells on the layered film showed the trends of HA outer/FHA inner structure with respect to culture period, that is, HA initially and FHA later. These observations suggest that the HA/FHA layered film on Ti obtained by a sol-gel route possesses gradient functionality in terms of solubility and cellular responses, and find that those parameters can be tailored for specific use in hard-tissue implants.     

Preparation,Characterization and Osteoblastic Activity of Chitosan/Polycaprolactone/In Situ Hydroxyapatite Scaffolds

Abstract

Calcium-deficient hydroxyapatite (cd-HA) crystals with a rod-like shape, 10-30 nm in diameter and 60150 nm in length, were prepared via a hydrothermal method in the presence of poly(acrylic acid) (PAA) (in situ HA). Scaffolds composed of chitosan (CS), polycaprolactone (PCL) and in situ HA were prepared by freeze-drying, using a formic acid/acetone mixture as a shared solvent. The mass fraction of in situ HA in the scaffolds ranged from 0 to 40%. FT-IR and XRD studies indicated that hydrogen bonding interactions existed among CS, PCL and in situ HA, which suppressed the crystallization of PCL. The mechanical results demonstrated that the CS/PCL composites had the maximum flexural stress (308.14 ± 8.86 MPa), which was significantly higher than 2.92 ± 0.02 MPa for the CS/in situ HA control. The effects of scaffolds on MC3T3-E1 cells were studied by measuring the viability, proliferation, adhesion, alkaline phosphatase activity, as well as mineralization assay. The WST-1 assay showed that in situ HA-loaded scaffolds had higher cell viability than CS/PCL scaffolds. SEM images of the cell-seeded scaffolds revealed a significant promotion of cell adhesion in in situ HA-loaded scaffolds. Moreover, ALP and mineralization were found to be enhanced in in situ HA-loaded scaffolds. All these results indicate that in situ HA-loaded scaffolds support cellular functions of osteoblastic cells and may serve as promising bone scaffolds.     

Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2766-2774, 2012.     

Fluoridated apatite coatings on titanium obtained by electron-beam deposition

In this report, a series of fluoridated apatite coatings were obtained by the electron-beam deposition method. The fluoridation of the apatite was aimed to improve the stability of the coating and elicit the fluorine effect, which is useful in the dental restoration area. Apatites fluoridated at different levels were used as initial evaporants for the coatings. The as-deposited coatings were amorphous, but after heat treatment at 500 degrees C for 1 h, the coatings crystallized well to an apatite phase without forming any cracks. The adhesion strengths of the as-deposited coatings were about 40 MPa. After heat treatment at 500 degrees C, the strengths of the pure HA and FA coatings decreased to about 20 MPa, however, the partially fluoridated coatings maintained their initial strength. The dissolution rate of the fluoridated coatings was lower than that of the pure HA coating, and the rate was the lowest in the coatings with 25% and 50% fluorine substitutions. The osteoblast-like cells responded to the coatings in a similar manner to the dissolution behavior. The cells on the fluoridated coatings showed a lower (p < 0.05) proliferation level compared to those on the pure HA coating. The alkaline phosphatase activity of the cells was slightly lower than that on the pure HA coating, but this difference was not statistically significant.     

Hydroxyapatite and titania sol-gel composite coatings on titanium for hard tissue implants; mechanical and in vitro biological performance

Hydroxyapatite (HA) composites with titania (TiO2) up to 30 mol % were coated on a titanium (Ti) substrate by a sol-gel route, and the mechanical and biological properties of the coating systems were evaluated. Using polymeric precursors, highly stable HA and TiO2 sols were prepared prior to making composite sols and coatings. Coatings were produced under a controlled spinning and heat treatment process. Pure phases of HA and TiO2 were well developed on the composites after heat treatment above 450 degrees C. The HA-TiO2 composite coating layers were homogeneous and highly dense with a thickness of about 800-900 nm. The adhesion strength of the coating layers with respect to Ti substrate increased with increasing the TiO2 addition. The highest strength obtained was as high as 56 MPa, with an improvement of about 50% when compared to pure HA (37 MPa). The osteoblast-like cells grew and spread actively on all the composite coatings. The proliferation and alkaline phosphatase (ALP) activity of the cells grown on the composite coatings were much higher than those on bare Ti, and even comparable to those on pure HA coating. Notably, the HA-20% TiO2 composite coating showed a significantly higher proliferation and ALP expression compared to bare Ti (p < 0.05). These findings suggest that the sol-gel-derived HA-TiO2 composite coatings possess excellent properties for hard tissue applications from the mechanical and biological perspective.     

Biodegradable polymer/hydroxyapatite composites: surface analysis and initial attachment of human osteoblasts

Biodegradable polymer/hydroxyapatite (HA) composites have potential application as bone graft substitutes. Thin films of polymer/HA composites were produced, and the initial attachment of primary human osteoblasts (HOBs) was assessed to investigate the biocompatibility of the materials. Poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLA) were used as matrix materials for two types of HA particles, 50-microm sintered and submicron nonsintered. Using ESEM, cell morphology on the surfaces of samples was investigated after 90 min, 4 h, and 24 h of cell culture. Cell activity and viability were assessed after 24 h of cell culture using Alamar blue and DNA assays. Surface morphology of the polymer/HA composites and HA exposure were investigated using ESEM and EDXA, respectively. ESEM enabled investigation of both cell and material surface morphology in the hydrated condition. Combined with EDXA it permitted chemical and visual examination of the composite. Differences in HA exposure were observed on the different composite surfaces that affected the morphology of attached cells. In the first 4 h of cell culture, the cells were spread to a higher degree on exposed HA regions of the composites and on PLA than they were on PCL. After 24 h the cells were spread equally on all the samples. The cell activity after 24 h was significantly higher on the polymer/HA composites than on the polymer films. There was no significant difference in the activity of the cells on the various composite materials. However, cells on PCL showed higher activity compared to those on PLA. A polymer surface exhibiting "point exposure" of HA appeared to provide a novel and favorable substrate for primary cell attachment. The cell morphology and activity results indicate a favorable cell/material interaction and suggest that PLA and PCL and their composites with HA may be candidate materials for the reconstruction of bony tissue. Further investigations regarding long-term biomaterial/cell interactions and the effects of acidic degradation products from the biodegradable polymers are required to confirm their utility.     

Human osteoblast response to silicon-substituted hydroxyapatite

Human osteoblasts were cultured on hydroxyapatite (HA), 0.8 wt % silicon substituted hydroxyapatite (Si-HA) and 1.5 wt % Si-HA discs. The influence of these substrates on cell behaviour in vitro was assessed by measuring total protein in the cell lysate and the production of several phenotypic markers: collagen type I (COL I), alkaline phosphatase (ALP), osteocalcin (OC), and the formation of bone mineral. After 7 days, beta-glycerophosphate and physiological levels of hydrocortisone were added to the culture medium to stimulate cell differentiation and mineral production. There was a significantly higher production of ALP on 1.5 wt % Si-HA at day 7 following which, the addition of hydrocortisone promoted the differentiation of cells on the other two substrates. Hydrocortisone addition also decreased the production of OC. During the period, when hydrocortisone was present, no significant difference in behavior was seen between cells on Si-HA and HA; however, following removal of hydrocortisone, cells responded to 0.8 wt % Si-HA with a significant increase in protein production. Using fluorescence microscopy, nodular structures labeled with tetracycline were observed on the surface of all substrates after 21 days. These structures were deposited on areas of high cell density but were not related to the presence or level of silicon in the substrate. These results indicate that human osteoblasts are affected by the presence of silicon in the HA substrate and that the timing of these effects may be dependent upon the level of silicon substitution.     

Improvement of dual-leached polycaprolactone porous scaffolds by incorporating with hydroxyapatite for bone tissue regeneration

Polycaprolactone (PCL)/hydroxyapatite (HA) composite scaffolds were prepared by combining solvent casting and salt particulate leaching with a polymer leaching technique. The hydrophilicity of the dual-leached scaffold was improved by alkaline (NaOH) treatment. Well-defined interconnected pores were detected by scanning electron microscopy. The water absorption capacity of the NaOH-treated PCL/HA dual-leached scaffold increased greatly, confirming that the hydrophilicity of the scaffold was improved by NaOH treatment. The compressive modulus of the PCL/HA dual-leached scaffold was greatly increased by the addition of HA particles. An indirect evaluation of the cytotoxicity of all PCL dual-leached scaffolds with mouse fibroblastic cells (L929) and mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) indicated that the PCL dual-leached scaffolds are non-toxic to cells. The ability of the scaffolds to support mouse calvaria-derived pre-osteoblastic cell (MC3T3-E1) attachment, proliferation, differentiation, and mineralization was also evaluated. Although the viability of cells was lower on the PCL/HA dual-leached scaffold than on the tissue-culture polystyrene plates (TCPS) and on the other substrates at early time points, both the PCL and NaOH-treated PCL/HA dual-leached scaffolds supported the attachment of MC3T3-E1 at significantly higher levels than TCPS. During the proliferation period (days 1–3), all of the PCL dual-leached scaffolds were able to support the proliferation of MC3T3-E1 at higher levels than the TCPS; in addition, the cells grown on NaOH-treated PCL/HA dual-leached scaffolds proliferated more rapidly. The cells cultured on the surfaces of NaOH-treated PCL/HA dual-leached scaffolds had the highest rate of mineral deposition.     

Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin-hydroxyapatite for tissue engineering scaffolds

Collagen-derived gelatin/hydroxyapatite (HA) nanocomposites were biomimetically synthesized for hard tissue engineering scaffold. In vitro osteoblastic cellular responses to the nanocomposites were assessed in comparison with those conventionally mixed gelatin-HA composites. A three-dimensional culture method involving floating cells in a culture medium was introduced to assist in the initial attachment of the cells to the scaffolds, and the proliferation and differentiation behaviors of the cells were examined. The osteoblastic MG63 cells attached to the nanocomposites to a significantly higher degree and subsequently proliferated more. The alkaline phosphatase (ALP) activity and osteocalcin produced by the cells were significantly higher on the nanocomposite scaffolds than on the conventional composite scaffolds. These improved cellular responses on the nanocomposites are considered to result from the increased ionic release and serum protein adsorption on the nanocomposites, which was derived from the different structural and morphological characteristics, i.e., the nanocomposite scaffolds retained less-crystallized and smaller-sized apatite crystals and a more well-developed pore configuration than the conventional ones. Based on these findings, the biomimetically synthesized nanocomposite scaffolds are believed to be potentially useful in hard tissue regeneration and tissue engineering fields.     

Hydroxyapatite and gelatin composite foams processed via novel freeze-drying and crosslinking for use as temporary hard tissue scaffolds

Hydroxyapatite (HA) and gelatin composites were fabricated in a foam type via a novel freeze-drying and crosslinking technique. The morphological and mechanical properties of and in vitro cellular responses to the foams were investigated. The HA powder was added at up to 30 wt % into the gelatin solution, and the mixtures were freeze-dried and further crosslinked. The pure gelatin foam had a well-developed pore configuration with porosity and pore size of approximately 90% and 400-500 microm, respectively. With HA addition, the porosity decreased and pore shape became more irregular. The HA particulates, in sizes of about 2-5 microm, were distributed within the gelatin network homogeneously and made the framework surface rougher. All the foams had high water absorption capacities, showing typical hydrogel characteristics, even though the HA addition decreased the degree of water absorption. The HA addition made the foam much stronger and stiffer (i.e., with increasing HA amount the foams sustained higher compressive stress and had higher elastic modulus in both dry and wet states). The osteoblast-like human osteosarcoma cells spread and grew actively on all the foams. The cell proliferation rate, quantified indirectly on the cells cultured on Ti discs coated with gelatin and gelatin-HA composites using MTT assay, exhibited an up-regulation with gelatin coating compared with bare Ti substrate, but a slight decrease on the composite coatings. However, the alkaline phosphatase activities expressed by the cells cultured on composites foams as well as their coatings on Ti discs were significantly enhanced compared with those on pure gelatin foam and coating. These findings suggest that the gelatin-HA composite foams have great potential for use as hard tissue regeneration scaffolds.     

Solvent-free polymer/bioceramic scaffolds for bone tissue engineering: fabrication,analysis, and cell growth

This study examines the potential use of porous polycaprolactone (PCL) and polycaprolocatone/hydroxyapatite (PCL/HA) scaffolds fabricated through melt molding and porogen leaching for bone tissue engineering. While eliminating organic solvents is desirable, the process steps proposed in this study for uniformly dispersing HA particles (~5?μm in size) within the scaffold can also contribute to homogeneous properties for these porous composites. Poly(ethylene oxide) (PEO) was chosen as a porogen due to its similar density and melting point as PCL. Pore size of the scaffold was controlled by limiting the size of PCL and PEO particles used in fabrication. The percent of HA in the fabricated scaffolds was quantified by thermogravimetric analysis (TGA). Mechanical testing was used to compare the modulus of the scaffolds to that of bone, and the pore size distribution was examined with microcomputed tomography (μCT). Scanning electron microscopy (SEM) was used to examine the effect on scaffold morphology caused by the addition of HA particles. Both μCT and SEM results showed that HA could be incorporated into PCL scaffolds without negatively affecting scaffold morphology or pore formation. Energy-dispersive X-ray spectroscopy (EDS) and elemental mapping demonstrated a uniform distribution of HA within PCL/HA scaffolds. Murine calvaria-derived MC3T3-E1 cells were used to determine whether cells could attach on scaffolds and grow for up to 21 days. SEM images revealed an increase in cell attachment with the incorporation of HA into the scaffolds. Similarly, DNA content analysis showed a higher cell adhesion to PCL/HA scaffolds.     

Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent

Macroporous composites made of coralline:chitosan with new microstructural features were studied for their scaffolding potential in in vitro bone regeneration. By using different ratios of natural coralline powder, as in situ gas forming agent and reinforcing phase, followed by freeze-drying, scaffolds with controlled porosity and pore structure were prepared and cultured with mesenchymal stem cells (MSCs). Their supportive activity of cellular attachment, proliferation and differentiation were assessed through cell morphology studies, DNA content, alkaline phosphatase (ALP) activity and osteocalcin (OC) release. The coralline scaffolds showed by far the highest evaluation of cell number and ALP activity over all the other chitosan-based scaffolds. They were the only material on which the OC protein was released throughout the study. When used as a component of the chitosan composite scaffolds, these coralline's favourable properties seemed to improve the overall performance of the chitosan. Distinct cell morphology and osteoblastic phenotype expression were observed depending on the coralline-to-chitosan ratios composing the scaffolds. The coralline-chitosan composite scaffolds containing high coralline ratios generally showed higher total cell number, ALP activity and OC protein expression comparing to chitosan scaffolds. The results of this study strongly suggest that coralline:chitosan composite, especially those having a high coralline content, may enhance adhesion, proliferation and osteogenic differentiation of MSCs in comparison with pure chitosan. Coralline:chitosan composites could therefore be used as attractive scaffolds for developing new strategies for in vitro tissue engineering.     

Biomedical nanocomposites of hydroxyapatite/polycaprolactone obtained by surfactant mediation

The composite approach to combining bioactive ceramic and degradable polymer is a promising strategy in the development of bone regenerative matrices. Moreover, in the fabrication of composites, the nanoscale organization of each component should improve the level of structural integration as well as the resultant mechanical and biological properties. The aim of this study was to develop a novel nanocomposite system consisting of hydroxyapatite (HA) and poly(epsilon-caprolactone) (PCL), wherein the HA nanoparticles were uniformly dispersed within the PCL matrix. The strategy was based on applying an amphiphilic surfactant, oleic acid in this case, between the HA and PCL. Oleic acid, which belongs to the fatty acid family and is generally noncytotoxic at the levels used in this study, is believed to mediate the interaction between the hydrophilic HA and hydrophobic PCL. With the mediation of oleic acid, the HA nanoparticles were distributed uniformly within the PCL matrix on the nanoscale (distributed particle size of less than 1 microm), which is in marked contrast to the conventionally mixed HA-PCL composite, in which the HA particles were severely agglomerated. The developed nanocomposite had significantly higher mechanical strength than did the conventional composite and the pure PCL. Moreover, the osteoblastic cells showed a better proliferation behavior on the nanocomposite than on the conventional composite. This HA-PCL nanocomposite mediated by oleic acid is expected to be useful in the bone regeneration field. Moreover, this methodology is applicable to the nanocomposite processing of other biomedical materials.     

Compressive properties and degradability of poly(epsilon-caprolatone)/hydroxyapatite composites under accelerated hydrolytic degradation

Hydroxyapatite (HA) was incorporated as filler into polycaprolactone (PCL) matrix to improve the bioactivity as well as the compressive properties of the polymer composites that can be typically used in tissue engineering scaffolds. The compressive properties of five PCL/HA composites of different compositions were investigated in conjunction with the study of their rate of degradation. As PCL has a slow degradation rate, the experiment was conducted in a concentrated 5M sodium hydroxide medium to accelerate the degradation process. The compressive strength and modulus of all PCL/HA compositions were observed to decrease as the degradation experiment progressed, with samples having high HA content degraded most significantly as compared with samples with lower HA content. Pure PCL samples, however, were found to retain their mechanical properties comparatively well in the same degradation experiments. Although the addition of HA as filler into the PCL matrix was shown to have improved mechanical properties and bioactivity initially, these results do raise concerns of material properties being compromise during hydrolytic degradation.     

新型聚己内酯/磷酸钙支架的制备及生物相容性研究

探讨新型聚己内酯(PCL)/磷酸钙(CPC)复合材料支架的制备方法及对骨髓基质细胞(BMSCs)的生物相容性。采用溶液共混法,利用可溶盐晶体做造孔剂,制备PCL/CPC复合材料支架,以单纯PCL和CPC支架为对照组,Q800型动态力学分析仪进行动态力学性能试验(DMA),采用排水法测量孔隙率;灭菌后通过与犬BMSCs体外共同培养后细胞形态、生长曲线、碱性磷酸酶(ALP)染色和半定量及骨钙素(OC)半定量等方法检测细胞在支架材料上的黏附、增殖及成骨分化情况,动物体内异位成骨检测其成骨情况。结果显示,复合材料的储能模量在PCL/CPC比例为7:3时达到最大,制得的材料孔径为250～350μm,多孔支架的孔隙率为70%～80%;BMSCs在新型PCL/CPC组、CPC组支架表面分布均匀,生长增殖明显较PCL组活跃(P<0.05);PCL/CPC组、CPC组BMSCs成骨行为与PCL组之间有显著差异(P<0.05)。动物体内异位成骨检测提示,4周时PCL/CPC组为13.78%±1.60%、CPC组BMSCs为15.29%±1.20%,成骨显著强于PCL组BMSCs的7.56%±2.20%(P<0.05),表明PCL和CPC的复合明显改善了两种材料的缺陷,获得的PCL/CPC支架具有良好的生物相容性,可与BMSCs共同构建具有成骨能力的三维立体组织工程化骨。     

Evaluation of human mesenchymal stem cells response to biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine composite scaffolds for bone tissue engineering

The aim of this study was to examine in vitro the response of human mesenchymal stem cells (hMSCs) on the novel biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composite scaffold for potential use in bone tissue engineering. The initial attachment, the proliferation, migration and differentiation behavior of the cells on the BG-COL-HYA-PS composites were assessed in comparison with those on pure 58sBG, BG-COL, and BG-COL-HYA composites in either growth medium (L-DMEM supplemented with 10% fetal bovine serum) or osteogenic medium (growth medium supplemented with 0.1 microM dexamethasone, 10 mM beta-glycerophosphate, and 50 microM ascorbic acid). HMSCs attached, and subsequently proliferated and migrated on the BG-COL-HYA-PS composites to a significantly higher degree. The alkaline phosphatase (ALP) staining, ALP activity and the expression of the bone associated gene ALP, osteocalcin (OC), and osteopontin (OPN) was also significantly higher in the hMSCs on the BG-COL-HYA-PS scaffolds than those on the BG-COL, BG-COL-HYA composites and the pure 58sBG. These findings suggest that the BG-COL-HYA-PS composite porous scaffolds have high potential for use as scaffolds in bone tissue engineering and repair.