Mineralization of Collagen‐Coated Electrospun Poly(lactide‐co‐glycolide) Nanofibrous Mesh to Enhance Growth and Differentiation of Osteoblasts and Bone Marrow Mesenchymal Stem Cells

To obtain the biomimetic scaffolding materials for bone tissue engineering, poly(lactide‐co‐glycolide) (PLGA) nanofibrous mesh (NFM) was mineralized in a 5× simulated body fluid (SBF) for different time after it was treated by air plasma for 15 min and subsequent collagen coating. The apatite particles were nucleated on the surface of individual nanofibers, gradually grew up, and finally covered the whole NFM surface. The mineral aggregates were mainly composed of tiny hydroxyapatite (HA) nanoparticles, whose content reached a constant value of 54 µg · cm^?2 after 9 days. The collagen coating and apatite deposition enhanced the NFM strength pronouncedly too. In vitro cell culture demonstrated that the non‐ or less mineralized NFMs were more beneficial of cell spreading and proliferation than those highly mineralized NFMs, but the latter ones could strongly promote secretion of alkaline phosphatase (ALP) by osteoblasts after cultured for 14 days. Moreover, the highly mineralized NFMs also could significantly up‐regulated ALP activity and calcium synthesis of bone marrow mesenchymal stem cells (BMSCs), demonstrating that these NFMs are more favorable of the osteoblast phenotype expression and osteogenic induction. Therefore, the biomimetic apatite deposited PLGA/collagen NFM could be a promising candidate scaffold for bone tissue engineering.     

Porous calcium phosphate ceramics modified with PLGA–bioactive glass

Porous calcium phosphate ceramics (mainly hydroxyapatite) with interconnected macropores (∼1 mm) and micropores (∼5 μm) as well as high porosities (∼80%) were prepared by firing polyurethane foams that were coated with calcium phosphate cement at 1200 °C. In order to improve the mechanical properties such as compressive strength and compressive modulus and maintain the desirable bioactivity (i.e. the ability of apatite layer formation), the open micropores of the struts were infiltrated with poly(lactic-co-glycolic acid) (PLGA) to achieve an interpenetrating bioactive ceramic/biodegradable polymer composite structure. The PLGA filled struts were further coated with a 58S bioactive glass (33 wt.%)–PLGA composite coating. The PLGA–bioactive glass modified porous calcium phosphate ceramics proved to be bioactive and exhibited compressive strengths up to 7.7 MPa and compressive moduli up to 3 GPa, which were comparable to those of natural spongy bones. The obtained complex porous bioactive/biodegradable composites could be used as tissue engineering scaffolds for low-load bearing applications.     

Preparation and characterization of interpenetrating phased TCP/HA/PLGA composites

The purpose of this study was to fabricate composites consisting of three interpenetrating networks: tricalcium phosphate (TCP), hydroxyapatite (HA), and poly(dl-lactide-co-glycolide) (PLGA). The porous TCP network was first produced by coating a polyurethane (PU) foam with hydrolysable alpha-TCP slurry. The HA network was derived from a calcium phosphate cement (CPC) filled in the porous TCP network. The remaining open pore network in the HA/TCP composite was further infiltrated with a PLGA network. The three sets of spatially continuous networks would have different biodegradation rates and thus bone tissue would grow towards the fastest biodegrading network while the remaining networks still maintaining their geometrical shape and carrying the physiological load for the tissue ingrowth.     

聚乳酸组织工程支架表面涂覆钙磷盐的工艺研究

提出了以聚乳酸／钙磷盐／胶原的骨组织工程支架快速成形制造的材料系统，对比了两种在聚乳酸(PLA)表面涂覆钙磷盐的工艺．一种是将聚乳酸组织工程支架浸泡在模拟体液中采用平衡反应法沉积钙磷盐；另一种是在聚乳酸薄膜上采用非平衡反应法沉积钙磷盐；系统地研究了沉积时间和沉积量、钙磷摩尔比和相结构演变的关系；通过控制反应时间和调整反应物配比获得磷酸四钙(TetCP)-PLA和无定形磷酸钙(ACP)—PLA等材料组合；并对所获得的复合材料进行生物相容性试验．对比试验证明细胞在材料表面生长良好，采用两种方法涂覆钙磷盐均改进了聚乳酸的生物相容性．     

PLGA/TSF静电纺纳米纤维的仿生矿化及生物相容性

利用静电纺丝和模拟体液仿生矿化技术制备了聚乳酸-羟基乙酸共聚物/柞蚕丝素/羟基磷灰石((PLGA/TSF/HA)骨组织工程复合支架。通过扫描电子显微镜(SEM)、透射电子显微镜(TEM)、X射线衍射测试(XRD)和热重分析(TG)对复合纳米纤维的形貌结构进行了表征。此外,在复合纳米纤维支架材料上接种人骨髓间充质干细胞(hMSCs),通过四甲基偶氮噻唑蓝比色(Four methyl azo thiazole blue colorimetric,MTT)法,观察细胞在材料表面的生长情况评价纳米纤维的生物相容性。结果显示,PLGA/TSF纳米纤维毡具有精细的三维结构,纤维直径分布均匀,表面光滑。矿化后HA颗粒均匀地分布在PLGA/TSF纳米纤维表面,矿物含量约占63%。与PLGA/TSF纳米纤维支架相比,PLGA/TSF/HA纳米纤维支架的亲水性、生物相容性都得到显著提高。     

In vivo evaluation of composites of PLGA and apatite with two different levels of crystallinity

The cortical bone response towards poly(lactide-co-glycolide) (70/30) (PLGA) (70/30)/apatite complex scaffolds with different levels of crystallinity was investigated. Apatite with different levels of crystallinity, Ca-deficient hydroxyapatite (CDHA), which has a low crystallinity, and a mixture of carbonated hydroxyapatite (CHA) and CDHA, which has a higher crystallinity, were prepared from an aqueous mixture of Ca-EDTA complex, H₂O₂, H₃PO₄, and NH₄OH. Two porous PLGA(70/30)/apatite composite scaffolds, composite scaffold A (containing low crystallinity CDHA) and composite scaffold B (containing the higher crystallinity CHA/CDHA mixture), were prepared. Afterwards, pure porous PLGA and the two composite scaffolds were implanted into the cortical bone of rabbit tibiae for 12 weeks. High-resolution microfocus X-ray computed tomography and histological examinations revealed a better bone response for composite scaffold A compared with PLGA and composite scaffold B. For composite scaffold A, the original bone defect was almost filled with new bone. Quantitative analysis revealed that composite scaffold A produced a significantly greater amount of new bone. The present study demonstrated that the level of apatite crystallinity influences bone response. A PLGA/apatite porous composite with a low level of apatite crystallinity shows promise as a bone substitute or scaffold material for bone tissue engineering.     

Prolonged release from PLGA/HAp scaffolds containing drug-loaded PLGA/gelatin composite microspheres

Porous scaffolds that can prolong the release of bioactive factors are urgently required in bone tissue engineering. In this study, PLGA/gelatin composite microspheres (PGMs) were carefully designed and prepared by entrapping poly(l-lactide-co-glycolide) (PLGA) microspheres (PMs) in gelatin matrix. By mixing PGMs with PLGA solution directly, drug-loaded PLGA/carbonated hydroxyapatite (HAp)/PGMs composite scaffolds were successfully fabricated. In vitro release of fluorescein isothiocyanate-dextran (FD70S) as a model drug from the scaffolds as well as PMs and PGMs was studied by immersing samples in phosphate buffered saline (pH = 7.4) at 37°C for 32 days. Compared with PMs, PGMs and PLGA/HAp/PGMs scaffolds exhibited slow and steady release behavior with constant release rate and insignificantly original burst release. The swelling of PGMs, diffusion of drugs, and degradation of polymer dominated the release behaviors synergistically. The PLGA/HAp/PGMs scaffold offers a novel option for sequential or simultaneous release of several drugs in terms of bone regeneration.     

Sand dollar skeleton as templates for bacterial cellulose coating and apatite precipitation

In this work, the skeleton of Sand Dollar (Clypeaster subdepressus) was coated by bacterial cellulose (BC) produced by Gluconacetobacter hansenii and subsequently coated with calcium phosphate (apatite). The skeleton of sand dollar is composed of magnesium calcite ((Ca,Mg)CO₃) and exhibits an hierarchically porous structure with interconnected porosity. After coating, the small-sized pores were partially covered by cellulose microfibrils, where apatite particles were homogeneously deposited. The pore geometry of sand dollar is adequate for bone regeneration, allows cell migration through its large cells and permit vascularization through the small pores. Moreover, BC/apatite coating offers a bioactive surface for cell adhesion.     

Electrospinning of PLGA/gelatin randomly-oriented and aligned nanofibers as potential scaffold in tissue engineering

Electrospinning technique can be used to produce the three-dimensional nanofibrous scaffold similar to natural extracellular matrix, which satisfies particular requirements of tissue engineering scaffold. Randomly-oriented and aligned poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin biocomposite scaffolds were successfully produced by electrospinning in the present study. The resulting nanofibrous scaffolds exhibited smooth surface and high porous structure. Blending PLGA with gelatin enhanced the hydrophilicity but decreased the average fiber diameter and the mechanical properties of the scaffolds under the same electrospinning condition. The cell culture results showed that the elongation of the osteoblast on the aligned nanofibrous scaffold was parallel to the fiber arrangement and the cell number was similar to that of randomly-oriented scaffold, indicating that the aligned nanofibrous scaffold provide a beneficial approach for the bone regeneration.     

Differentiation of human adipose-derived stem cells seeded on mineralized electrospun co-axial poly(ε-caprolactone) (PCL)/gelatin nanofibers

Mineralized poly(ε-caprolactone)/gelatin core–shell nanofibers were prepared via co-axial electrospinning and subsequent incubation in biomimetic simulated body fluid containing ten times the calcium and phosphate ion concentrations found in human blood plasma. The deposition of calcium phosphate on the nanofiber surfaces was investigated through scanning electronic microscopy and X-ray diffraction. Energy dispersive spectroscopy results indicated that calcium-deficient hydroxyapatite had grown on the fibers. Fourier transform infrared spectroscopy analysis suggested the presence of hydroxyl-carbonate-apatite. The results of a viability assay (MTT) and alkaline phosphatase activity analysis suggested that these mineralized matrices promote osteogenic differentiation of human adipose-derived stem cells (hASCs) when cultured in an osteogenic medium and have the potential to be used as a scaffold in bone tissue engineering. hASCs cultured in the presence of nanofibers in endothelial differentiation medium showed lower rates of proliferation than cells cultured without the nanofibers. However, endothelial cell markers were detected in cells cultured in the presence of nanofibers in endothelial differentiation medium.     

The influences of poly(lactic-co-glycolic acid) (PLGA) coating on the biodegradability, bioactivity, and biocompatibility of calcium silicate bioceramics

Calcium silicate (CaSiO₃) bioceramics and polyesters have complementary qualities as potential bone substituted materials. In this study, sintered CaSiO₃ bioceramics were prepared and coated with poly(lactic-co-glycolic acid) (PLGA), and the influences of the PLGA coating on the degradation, hydrophilicity, bioactivity, and biocompatibility of CaSiO₃ ceramics were investigated. The results showed that the degradation rate was reduced, while hydrophilicity was decreased with the increase of the polymer coating. In addition, the polymer coating resulted in a decrease of the alkaline pH value during the degradation of the ceramics, which indicated an increase of the cell biocompatibility, confirmed by the attachment and proliferation of rMSCs on the surface of the polymer-coated ceramics. Furthermore, the apatite-forming ability of the PLGA-coated CaSiO₃ bioceramics was maintained. This study suggested that the coating with PLGA might be a useful method to improve the integrative properties of CaSiO₃ bioceramics for applications in bone regeneration and bone tissue engineering.     

Functionalization of poly(caprolactone) scaffolds by the surface mineralization for use in bone regeneration

The surface of a synthetic biopolymer scaffold was tailored by a mineralization with calcium phosphate for use as a functional bone tissue engineering matrix. Poly(ε-caprolactone) scaffold with a defined pore configuration constructed by a robocasting method was treated in a series of solutions involving steps of surface activation and calcium phosphate induction. The scaffold surface was completely covered with calcium phosphate nanocrystallites that had typical characteristics of bone mineral-like carbonate apatite. The scaffold with mineralized-surface demonstrated to support more favorable bone cell responses, including initial cell adhesion and proliferation and to allow higher loading of protein than the untreated-scaffold. The results suggest the developed scaffold has the potential for use as a bone regenerative matrix.     

The fabrication of biomineralized fiber-aligned PLGA scaffolds and their effect on enhancing osteogenic differentiation of UCMSC cells

The key factor of scaffold design for bone tissue engineering is to mimic the microenvironment of natural bone extracellular matrix (ECM) and guide cell osteogenic differentiation. The biomineralized fiber-aligned PLGA scaffolds (a-PLGA/CaPs) was developed in this study by mimicking the structure and composition of native bone ECM. The aligned PLGA fibers was prepared by wet spinning and then biomineralized via an alternate immersion method. Introduction of a bioceramic component CaP onto the PLGA fibers led to changes in surface roughness and hydrophilicity, which showed to modulate cell adhesion and cell morphology of umbilical cord mesenchymal stem cells (UCMSCs). It was found that organized actin filaments of UCMSCs cultured on both a-PLGA and a-PLGA/CaP scaffolds appeared to follow contact guidance along the aligned fibers, and those cells grown on a-PLGA/CaP scaffolds exhibited a more polarized cellular morphology. The a-PLGA/CaP scaffold with multicycles of mineralization facilitated the cell attachment on the fiber surfaces and then supported better cell adhesion and contact guidance, leading to enhancement in following proliferation and osteogenic differentiation of UCMSCs. Our results give some insights into the regulation of cell behaviors through design of ECM-mimicking structure and composition and provide an alternative wet-spun fiber-aligned scaffold with HA-mineralized layer for bone tissue engineering application.     

Surface modification of three-dimensional Ca-P/PHBV nanocomposite scaffolds by physical entrapment of gelatin and its in vitro biological evaluation

The properties of bone tissue engineering scaffolds such as architecture, porosity, mechanical properties and surface properties have significant effects on cellular response and play an important role in bone regeneration. In this study, three-dimensional nanocomposite scaffolds consisting of calcium phosphate (Ca-P) nanoparticles and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) copolymer with controlled external and internal architectures were successfully produced via selective laser sintering (SLS), one of the versatile rapid prototyping techniques. The Ca-P/PHBV nanocomposite scaffolds had a porosity of (61.75±1.24)%, compressive strength of (2.16±0.21) MPa and Young’s modulus of (26.98±2.29) MPa. The surface modification of scaffolds by gelatin was achieved through physical entrapment. The amount of entrapped gelatin could be controlled by varying the solvent composition and reaction time. The surface modification improved the hydrophilicity of scaffolds but did not significantly affect the surface morphology and mechanical properties. Osteoblast-like cells (SaOS-2) were cultured on scaffolds with and without gelatin surface modification. The majority of SaOS-2 cells were viable and proliferated in both types of scaffolds for up to 14 d in culture, as indicated by MTT assay and live and dead assay. Surface modification significantly increased cell proliferation for surface modified scaffolds, which could be due to the improvement in hydrophilicity of the scaffolds.     

PLGA/HA骨支架材料的降解特性

采用有机泡沫法获得了HA多孔骨架.运用溶胶浇铸法将PLGA溶胶填充入多孔骨架中,制备出骨组织工程用PLGA/HA复合支架材料,并考察了其在模拟体液中的降解性能,通过SEM观察了其表面组织形貌.结果表明,随浆料中HA含量的增加,材料降解后质量损失增大,且随降解时间的延长,PLGA/HA骨支架材料表面粘附的磷灰石相增多,表明该复合材料具有良好的生物活性.     

Synthesis and bioactivity of porous Ti alloy prepared by foaming with TiH2

A novel porous Ti–6Al–4V with an open cell structure was fabricated by powder metallurgy process with the addition of TiH₂ as the pore forming and active agent. Control of porosity of porous Ti alloy made it possible to obtain the porous Ti with the Young's modulus value of 5.8–9.5 GPa, which was similar to that of human cancellous bone. This kind of porous Ti alloy with good biomechanical properties is potential to alleviate the problem of mechanical mismatch between the bone and the Ti implant. The porous Ti alloy prepared by the addition of TiH₂ as foaming agent had a uniform distribution of pores with pore size of 90–190 μm and porosity of 43–59%. In order to improve the biological properties, the duplex titania/apatite coatings were applied onto the surface of porous Ti alloy. The titania coating was deposited by chemical treatment and the apatite coating was subsequently applied by immersing the samples in a simulated body fluid. Results showed that a homogeneous nanocrystallite titania coating with a thickness of 0.8 μm was formed on the surface of the Ti alloy after chemical treatment. The carbonate-containing apatite coating with a thickness of 1 μm was deposited on the surface of titania coating after immersion in simulated body fluid for 7 days. The nucleation of the carbonate-containing apatite can be induced from the electrostatic interaction between the OH-containing groups on the surface of titania coating and the calcium and phosphate ions in the metastable simulated body fluid on those specific superficial sites. The growth kinetics of the coatings was also discussed. Cell culture test showed the well stretched and proliferated cells on the surface of the sample, indicating the good biocompatibility of porous Ti alloy.     

冠脉支架表面PLGA涂层制备及其血液相容性研究

采用静电喷涂沉积(electrospray deposition ESD)法在冠脉支架表面制备了PLGA涂层.采用OLYMPUS体式显微镜、原子力显微镜(AFM)观察了涂层宏观表面形貌及三维形貌;通过对涂层支架进行球囊扩张考察了PLGA涂层与支架的结合力;通过血小板粘附实验和动态凝血时间测定研究PLGA涂层的血液相容性.结果表明:ESD法在冠脉支架表面制备PLGA涂层,支架筋拐角处无明显的聚合物胶体缠绕、粘连且涂层表面光滑;PLGA涂层将316L不锈钢基体的微坑覆盖,基体Ra=16.174nm,PLGA涂层Ra=0.149nm,涂层表面粗糙度小;涂层支架撑开后在最大塑性变形位置无涂层撕裂、翘起等缺陷,涂层与支架有良好结合力;PLGA涂层血小板粘附量少,变形小,未引起血小板激活,动态凝血时间长,直到50min未产生凝血,PLGA涂层具有较好的血液相容性.     

Morphological,mechanical, and biocompatibility characterization of macroporous alumina scaffolds coated with calcium phosphate/PVA

In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate cells and guide the tissue regeneration in three-dimension. Calcium phosphate (CaP) ceramics are widely used for bone substitution and repair due to their biocompatibility, bioactivity, and osteoconduction. However, compared to alumina ceramics, either in the dense or porous form, the mechanical strength achieved for calcium phosphates is generally lower. In the present work, the major goal was to develop a tri-dimensional macroporous alumina scaffold with a biocompatible PVA/calcium phosphate coating to be potentially used as bone tissue substitute. This approach aims to combine the high mechanical strength of the alumina scaffold with the biocompatibility of calcium phosphate based materials. Hence, the porous alumina scaffolds were produced by the polymer foam replication procedure. Then, these scaffolds were submitted to two different coating methods: the biomimetic and the immersion in a calcium phosphate/polyvinyl alcohol (CaP/PVA) slurry. The microstructure, morphology and crystallinity of the macroporous alumina scaffolds samples and coated with CaP/PVA were characterized by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDX) analysis. Also, specific surface area was assessed by BET nitrogen adsorption method and mechanical behavior was evaluated by axial compression tests. Finally, biocompatibility and cytotoxicity were evaluated by VERO cell spreading and attachment assays under SEM. The morphological analysis obtained from SEM photomicrograph results has indicated that 3D macroporous alumina scaffolds were successfully produced, with estimated porosity of over 65% in a highly interconnected network. In addition, the mechanical test results have indicated that porous alumina scaffolds with ultimate compressive strength of over 3.0 MPa were produced. Concerning to the calcium phosphate coatings, the results have showed that the biomimetic method was not efficient on producing a detectable layer onto the alumina scaffolds. On the other hand, a uniform and adherent inorganic–organic coating was effectively formed onto alumina macroporous scaffold by the immersion of the porous structure into the CaP/PVA suspension. Viable VERO cells were verified onto the surface of alumina porous scaffold samples coated with PVA–calcium phosphate. In conclusion, a new method was developed to produce alumina with tri-dimensional porous structure and uniformly covered with a biocompatible coating of calcium phosphate/PVA. Such system has high potential to be used in bone tissue engineering.     

Bioactivity of gelatin coated magnetic iron oxide nanoparticles: in vitro evaluation

Current research explores formation of bone like apatite on gelatin coated magnetic iron oxide nanoparticles (GIOPs) to evaluate the bioactivity of the material. The GIOPs were soaked in simulated body fluid (SBF) and the apatite formation on the surface was investigated in regular interval of time. Fourier transform-infrared (FT-IR) and x-ray diffraction spectroscopic (XRD) analyses were done to investigate the chemical changes and field emission-scanning electron microscopic (FE-SEM) analysis was done to investigate the morphological changes occurring on the surface of the GIOPs after soaking in different time intervals. The kinetic studies of the apatite growth in SBF suggest that initially calcium and phosphorous ions were deposited to the surface of the GIOPs from the SBF leading to formation of amorphous Ca/P particles. Later, after 9 days of the incubation the amorphous particles were fused to form needle and blade like crystalline structures of bone like apatite.     

Compositional and structural control in bone regenerative coatings

The development of a low-temperature method of producing bioactive coatings for medical implants has been shown to bypass the problems associated with high temperature processing routes, in particular the appearance of amorphous phases and non-stoichiometric hydroxyapatite (HA), and delamination of the coating from the substrate. An electric field/aqueous solution technique for producing adherent, crack-free calcium phosphate coatings on titanium and stainless steel substrates is described. The characteristics of the coating are a function of electrode spacing, supersaturation, temperature and current and voltage conditions. Scanning electron microscopy (SEM) characterized the surface morphology of the coatings, which were shown to be HA. The possibility of producing a coating of carbonate-substituted HA having the same chemical composition as bone apatite, and forming at physiological temperatures, has also been demonstrated. The size of the microstructure decreased and the morphology changed as the carbonate ion concentration in the calcium and phosphate ion solution increased. © 1999 Kluwer Academic Publishers