Preparation and characterization of a porous scaffold based on poly(D,L-lactide) and N-hydroxyapatite by phase separation

In this study, a series of porous scaffolds were prepared from poly(D,L-lactide) (PLA) and nanohydroxyapatite (HA) using the phase separation method. HA/PLA composite membranes and PLA membranes with a microporous structure (pore size around 10-20 μm) were observed by scanning electron microscopy and these micropores were well distributed throughout the PLA membranes. The surface morphology of HA/PLA composite membranes was significantly improved compared to pure PLA membrane. Also, the mechanical property and contact angle of composite membranes were different from that of pure PLA films. The immortalized rat osteoblastic ROS 17/2.8 cell line was used in this research to study the cell adhesion and proliferation behavior, and the results indicated that composite membranes had great cell affinity and good biocompatibility.     

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.     

New concept of 3D printed bone clip (polylactic acid/hydroxyapatite/silk composite) for internal fixation of bone fractures

Abstract

Open reduction with internal fixation is commonly used for the treatment of bone fractures. However, postoperative infection associated with internal fixation devices (intramedullary nails, plates, and screws) remains a significant complication, and it is technically difficult to fix multiple fragmented bony fractures using internal fixation devices. In addition, drilling in the bone to install devices can lead to secondary fracture, bone necrosis associated with postoperative infection. In this study, we developed bone clip type internal fixation device using three- dimensional (3D) printing technology. Standard 3D model of the bone clip was generated based on computed tomography (CT) scan of the femur in the rat. Polylacticacid (PLA), hydroxyapatite (HA), and silk were used for bone clip material. The purpose of this study was to characterize 3D printed PLA, PLA/HA, and PLA/HA/Silk composite bone clip and evaluate the feasibility of these bone clips as an internal fixation device. Based on the results, PLA/HA/Silk composite bone clip showed similar mechanical property, and superior biocompatibility compared to other types of the bone clip. PLA/HA/Silk composite bone clip demonstrated excellent alignment of the bony segments across the femur fracture site with well-positioned bone clip in an animal study. Our 3D printed bone clips have several advantages: (1) relatively noninvasive (drilling in the bone is not necessary), (2) patient-specific design (3) mechanically stable device, and (4) it provides high biocompatibility. Therefore, we suggest that our 3D printed PLA/HA/Silk composite bone clip is a possible internal fixation device.     

HA/ZrO2 不同复合材料对MSCs贴壁、增殖及成骨分化影响的差异

目的: 观察间充质干细胞(MSCs)在不同生物材料表面的生长及成骨分化情况。方法: 采用干铺法制备氧化锆(ZrO₂)单层复合羟基磷灰石(HA)及ZrO₂梯度复合HA 两种复合材料,观察复合材料表面形貌特征。分离和培养兔MSCs,分别培养于HA/ZrO₂单层复合材料、HA/ZrO₂梯度复合材料、纯HA及ZrO₂材料薄片上,观察细胞贴壁、增殖情况和碱性磷酸酶活性,提取细胞总RNA并检测Ⅰ型胶原、骨钙蛋白和骨桥蛋白mRNA 表达情况。结果: 制备的HA/ZrO₂单层复合材料具有不连续的HA表面层,可以清晰地观察到部分ZrO₂基体。而HA/ZrO₂梯度复合材料表面较为粗糙,呈多孔状。X射线衍射分析显示,经高温烧结后,两种复合材料表面的ZrO₂相依旧存在,HA相转变为β-Ca₃(PO₄)₂(β-TCP)、α-Ca₃(PO₄)₂(α-TCP)和CaZrO₃相。细胞培养显示,HA/ZrO₂梯度复合材料更有利于细胞贴壁。细胞在纯HA上碱性磷酸酶活性较其它组显著升高;细胞在复合材料和纯HA上Ⅰ型胶原、骨钙蛋白和骨桥蛋白表达较对照组均有不同程度升高,其中Ⅰ型胶原表达升高更为明显。结论: HA/ZrO₂梯度复合材料可促进MSCs的增殖,并可促进MSCs的成骨分化。     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

An amphiphilic degradable polymer/hydroxyapatite composite with enhanced handling characteristics promotes osteogenic gene expression in bone marrow stromal cells

Electrospun polymer/hydroxyapatite (HA) composites combining biodegradability with osteoconductivity are attractive for skeletal tissue engineering applications. However, most biodegradable polymers such as poly(lactic acid) (PLA) are hydrophobic and do not blend with adequate interfacial adhesion with HA, compromising the structural homogeneity, mechanical integrity and biological performance of the composite. To overcome this challenge, we combined a hydrophilic polyethylene glycol (PEG) block with poly(d,l-lactic acid) to improve the adhesion of the degradable polymer with HA. The amphiphilic triblock copolymer PLA–PEG–PLA (PELA) improved the stability of HA–PELA suspension at 25 wt.% HA content, which was readily electrospun into HA–PELA composite scaffolds with uniform fiber dimensions. HA–PELA was highly extensible (failure strain >200% vs. <40% for HA–PLA), superhydrophilic (～0° water contact angle vs. >100° for HA–PLA), and exhibited an 8-fold storage modulus increase (unlike deterioration for HA–PLA) upon hydration, owing to the favorable interaction between HA and PEG. HA–PELA also better promoted osteochondral lineage commitment of bone marrow stromal cells in unstimulated culture and supported far more potent osteogenic gene expression upon induction than HA–PLA. We demonstrate that the chemical incorporation of PEG is an effective strategy to improve the performance of degradable polymer/HA composites for bone tissue engineering applications.     

Mechanical performance and osteoblast-like cell responses of fluorine-substituted hydroxyapatite and zirconia dense composite

A fluorine-substituted hydroxyapatite (FHA) and zirconia (ZrO(2)) dense composite (50:50 by volume) was fabricated, and its feasibility for hard tissue applications was investigated in terms of its mechanical properties and osteoblast-like cell (MG63) responses in vitro. The incorporation of fluorine into the hydroxyapatite (HA) structure was highly effective in producing a completely dense apatite-ZrO(2) composite through a pressureless sintering route, by preventing the thermal degradation of the apatite and ZrO(2). The resultant FHA-ZrO(2) dense composite had excellent mechanical properties, such as flexural strength (310 MPa), fracture toughness (3.4 MPam(1/2)), hardness (10 GPa), and elastic modulus (160 GPa). The flexural strength and fracture toughness of the composite showed a noticeable improvement by a factor of approximately 4 with respect to the pure apatites (HA and FHA). The MG63 cellular responses to the composite were assessed in terms of the cell proliferation (cell number and [(3)H]-thymidine incorporation) and differentiation (alkaline phosphatase activity, osteocalcin, and collagen production). The cells on the FHA-ZrO(2) composite spread and grew well, and proliferated actively during the culture period. The expression of alkaline phosphatase, osteocalcin, and collagen by the cells on the composite showed a similar trend to that on the pure apatites, although slight down-regulations were observed, implying that the FHA-ZrO(2) 50:50 composite retains the osteoblastic functionality and traits of the pure HA ceramics to a high degree. This finding, in conjunction with the considerable improvements in mechanical properties, supports the extended use of this composite for hard tissue applications.     

电沉积HA-Ti/HA复合涂层及其生物学性能

为了提高HA涂层的结合强度，采用两步电沉积法制备HA—Ti／HA复合涂层，对涂层的组分结构、表面形貌和结合强度进行了研究，并对涂层进行模拟体液和体外细胞实验，以考察涂层的生物学性能。实验结果表明：HA—Ti／HA复合涂层的结合强度明显高于HA涂层，当涂层中Ti的质量分数为51．2wt％时，涂层与基体的结合强度达到21．2MPa，约为纯HA涂层的3倍。涂层经模拟体液浸泡后，表面覆盖一层碳磷灰石（Carbonate—apatite），表明涂层具有良好的生物活性。体外细胞实验表明，骨髓基质细胞能在涂层表面黏附繁殖生长，表明涂层具有良好的生物相容性。     

基于丝素/胶原/羟基磷灰石仿生骨材料的多维结构及其性能

目的 体外构建丝素蛋白（silk fibroin,SF）、I型胶原(type I collagen,Col-I)和羟基磷灰石(hydroxyapatite, HA)共混体系制备二维复合膜和三维仿生支架,研究其理化性质和生物相容性,探讨其在组织工程支架材料中应用的可行性。方法 通过在细胞培养小室底部共混SF/Col-I/HA以及低温3D打印结合真空冷冻干燥法制备二维复合膜及三维支架。通过机械性能测试、电子显微镜和Micro-CT检测材料的理化性质,检测细胞的增殖评估其生物相容性。结果 通过共混和低温3D打印获得稳定的二维复合膜及三维多孔结构支架;力学性能具有较好的一致性,孔径、吸水率、孔隙率和弹性模量均符合构建组织工程骨的要求;支架为网格状的白色立方体,内部孔隙连通性较好; HA均匀分布在复合膜中,细胞黏附在复合膜上,呈扁平状;细胞分布在支架孔壁周围,呈梭形状,生长及增殖良好。结论 利用SF/Col-I/HA共混体系成功制备复合膜及三维支架,具有较好的孔连通性与孔结构,有利于细胞和组织的生长以及营养输送,其理化性能以及生物相容性符合骨组织工程生物材料的要求。     

Chitosan/nanohydroxyapatite composite membranes via dynamic filtration for guided bone regeneration

Chitosan/hydroxyapatite (HA) composite membranes were prepared by the coprecipitation method and a subsequent dynamic filtration and freeze-drying process. The influences of the HA content of the membranes on their phase and morphology, mechanical properties, and bioactivity were investigated. FTIR analysis revealed that chitosan and HA had good miscibility over a wide range of compositions. Needle-like HA nanocrystals with low crystallinity were uniformly embedded in the chitosan matrix. As the HA content was increased, the tensile strength of the membranes exhibited a steady decrease, while the elastic modulus increased by a factor of 2 when 20% HA was added. The results of the in vitro cell culture showed that the highest alkaline phosphatase level was achieved when 30% HA was contained in the composites.     

Fabrication and biocompatibility of an antimicrobial composite membrane with an asymmetric porous structure

A composite slurry from silver ion-substituted nano-hydroxyapatite, titania nano-particles and polyamide 66 (Ag-nHA/TiO(2)/PA66) was prepared and used to fabricate a novel antimicrobial membrane with a gradient porous structure for guided bone regeneration (GBR). Subsequently, assays were performed to determine the cytocompatibility, as well as the bone biocompatibility of the prepared membranes. To investigate the cytocompatibility of the Ag-nHA/TiO(2)/PA66 membrane, in vitro studies were done with osteoblast-like cells (MG63) and the viability, alkaline phosphatase activity (ALP) and morphology of cells cultured on the membrane were determined. The bone biocompatibility of the membranes was finally assessed in animal experiments, in which nano-hydroxyapatite/polyamide 66 (nHA/PA66) and pure polyamide 66 (PA66) membranes were compared. The in vitro cell-culture experiments showed that Ag-nHA/TiO(2)/PA66 antimicrobial membrane evoked good cell affinity and cytocompatibility. The in vivo study showed that Ag-nHA/TiO(2)/PA66 asymmetric porous barrier membrane resulted in complete closure of 5-mm bone defects as created in the skull of rats after 8 weeks of implantation. In conclusion, the Ag-nHA/TiO(2)/PA66 membrane has the potential to be applied in GBR, especially in infected tissue or areas with high bacteria concentration.     

Biocompatibility of bioresorbable poly(L-lactic acid) composite scaffolds obtained by supercritical gas foaming with human fetal bone cells

The aim of this investigation was to test the biocompatibility of three-dimensional bioresorbable foams made of poly(L-lactic acid) (PLA), alone or filled with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), with human primary osteoblasts, using a direct contact method. Porous constructs were processed by supercritical gas foaming, after a melt-extrusion of ceramic/polymer mixture. Three neat polymer foams, with pore sizes of 170, 310, and 600 microm, and two composite foams, PLA/5 wt% HA and PLA/5 wt% beta-TCP, were examined over a 4-week culture period. The targeted application is the bone tissue-engineering field. For this purpose, human fetal and adult bone cells were chosen because of their highly osteogenic potential. The association of fetal bone cells and composite scaffold should lead to in vitro bone formation. The polymer and composite foams supported adhesion and intense proliferation of seeded cells, as revealed by scanning electron microscopy. Cell differentiation toward osteoblasts was demonstrated by alkaline phosphatase (ALP) enzymatic activity, gamma-carboxylated Gla-osteocalcin production, and the onset of mineralization. The addition of HA or beta-TCP resulted in higher ALP enzymatic activity for fetal bone cells and a stronger production of Gla-osteocalcin for adult bone cells.     

多孔HA/PU复合支架材料生物相容性的评估

进行了三维多孔立体结构的纳米羟基磷灰石/聚氨酯(HA/PU)复合支架材料体外细胞培养和体内肌肉埋植实验研究,评估材料的生物相容性。实验选用SD大鼠的骨髓基质干细胞(BMSCs)和健康的SD雌性大鼠,进行细胞相容性、形态学观察和组织学切片分析。HA/PU支架材料的多孔性为细胞的生长提供了良好的微环境,细胞在内部贴壁爬行、增殖并分化,细胞毒性为零级,材料与周围组织有良好的结合,降解的空间有结缔组织纤维长入。实验表明,HA/PU复合支架材料具有良好的细胞亲和性和组织学相容性,可作为一类新型组织工程支架材料。     

PLA/液晶复合膜的制备及其性能研究

利用本实验室合成的两种新型的亲水性液晶化合物与聚乳酸在一定的条件下制备了具有抗凝血性能的复合材料。通过接触角的测试，动态凝血实验，溶血实验发现液晶化合物能改善聚乳酸的抗凝血性能，研究结果发现，随着液晶化合物在复合材料中的含量的增加，接触角明显减少；且发现液晶在30％－40％时抗凝血性能最佳。     

Fabrication of chitosan/hydroxylapatite composite rods with a layer-by-layer structure for fracture fixation

A composite rod for fracture fixation using chitosan (CHI)/hydroxylapatite (HA) was prepared by means of in situ precipitation, which had a layer-by-layer structure, good mechanical properties, and cell compatibilities. The CHI/HA composite rods were precipitated from the chitosan solution with calcium and phosphorus precursors, followed by treatment with a tripolyphosphate-trisodium phosphate solution (pH >13) to crosslink the CHI and to hydrolyze the calcium phosphates to nanocrystalline HA. The results of FTIR, XRD, and TEM measurements confirmed that HA had been formed within the CHI matrix. The effects of the CHI/HA ratios (20/0, 20/1, 20/2, 20/4, and 20/5, w/w) on the mechanical properties were investigated. At the CHI/HA ratio of 20/4 (w/w), the bending strength and modulus of the rods were 133 MPa and 6.8 GPa, respectively. Pre-osteoblast MC3T3-E1 cells were cultured in an extract of the CHI/HA rods (20/4, w/w) to study the cell compatibilities of the composite. The observations indicated that the CHI/HA composite could promote the growth of MC3T3-E1 cells better than the composite without HA (p < 0.05). Furthermore, the co-cultivation of the cells and the CHI/HA composite showed that cells fully spread on the surface of the composite with an obvious cytoskeleton organization, which also revealed that the CHI/HA composite had a good biocompatibility.     

Initial responses of human osteoblasts to sol-gel modified titanium with hydroxyapatite and titania composition

Sol-gel thin films of hydroxyapatite (HA) and titania (TiO(2)) have received a great deal of attention in the area of bioactive surface modification of titanium (Ti) implants. Sol-gel coatings were developed on Ti substrates of pure HA and TiO(2) and two composite forms, HA+10% TiO(2) and HA+20% TiO(2), and the biological properties of the coatings were evaluated. All the coating layers exhibited thin and homogeneous structures and phase-pure compositions (either HA or TiO(2)). Primary human osteoblast cells showed good attachment, spreading and proliferation on all the sol-gel coated surfaces, with enhanced cell numbers on all the coated surfaces relative to uncoated Ti control at day 1, as observed by MTT assay and scanning electron microscopy. Cell attachment rates were also enhanced on the pure HA coating relative to control Ti. The pure HA and HA+10% TiO(2) composite coating furthermore enhanced proliferation of osteoblasts at 4 days. Moreover, the gene expression level of several osteogenic markers including bone sialoprotein and osteopontin, as measured by RT-PCR at 24h, was shown to vary according to coating composition. These findings suggest that human primary bone cells show marked and rapid early functional changes in response to HA and TiO(2) sol-gel coatings on Ti.     

HA/ZrO2复合材料颗粒的体外基因毒性的初步研究

目的: 采用体外微核法评价HA/ZrO₂复合材料颗粒的基因毒性。方法: 按不同比例将羟基磷灰石与二氧化锆粉体混合在高温高压下烧结制备HA/ZrO₂复合材料颗粒,纯HA和纯ZrO₂颗粒作为对照。制备复合材料颗粒的悬液,分离和培养兔间充质干细胞。MTT法检测复合材料颗粒悬液对兔间充质干细胞增殖促进作用,体外微核试验(MNT) 检测复合材料颗粒悬液对兔间充质干细胞的基因毒性。结果: MTT实验显示:纯HA和含HA的复合材料颗粒均对间充质干细胞增殖有一定促进作用,而纯ZrO₂颗粒对间充质干细胞增殖没有促进作用,差异显著(P<0.05)。MNT实验显示:HA组与阴性对照组相比差异不显著(P>0.05),与阳性对照组比差异显著(P<0.05); ZrO₂组与阴性对照组比差异显著(P<0.01),与阳性对照组比差异不显著(P>0.05)。结论: HA/ZrO₂复合材料颗粒的基因毒性随着ZrO₂比例和复合材料浓度的增加而增加,30%wtHA/70%wtZrO₂在200 mg/L时具有显著基因毒性(P<0.01)。     

Hydroxyapatite/polylactide biphasic combination scaffold loaded with dexamethasone for bone regeneration

This study presents a novel design of a ceramic/polymer biphasic combination scaffold that mimics natural bone structures and is used as a bone graft substitute. To mimic the natural bone structures, the outside cortical-like shells were composed of porous hydroxyapatite (HA) with a hollow interior using a polymeric template-coating technique; the inner trabecular-like core consisted of porous poly(D,L-lactic acid) (PLA) that was loaded with dexamethasone (DEX) and was directly produced using a particle leaching/gas forming technique to create the inner diameter of the HA scaffold. It was observed that the HA and PLA parts of the fabricated HA/PLA biphasic scaffold contained open and interconnected pore structures, and the boundary between both parts was tightly connected without any gaps. It was found that the structure of the combination scaffold was analogous to that of natural bone based on micro-computed tomography analysis. Additionally, the dense, uniform apatite layer was formed on the surface of the HA/PLA biphasic scaffold through a biomimetic process, and DEX was successfully released from the PLA of the biphasic scaffold over a 1-month period. This release caused human embryonic palatal mesenchyme cells to proliferate, differentiate, produce ECM, and form tissue in vitro. Therefore, it was concluded that this functionally graded scaffold is similar to natural bone and represents a potential bone-substitute material.     

Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications

New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization.