Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering

Electrospun fiber matrices composed of scaffolds of varying fiber diameters were investigated for potential application of severe skin loss. Few systematic studies have been performed to examine the effect of varying fiber diameter electrospun fiber matrices for skin regeneration. The present study reports the fabrication of poly[lactic acid-co-glycolic acid] (PLAGA) matrices with fiber diameters of 150-225, 200-300, 250-467, 500-900, 600-1200, 2500-3000 and 3250-6000nm via electrospinning. All fiber matrices found to have a tensile modulus from 39.23+/-8.15 to 79.21+/-13.71MPa which falls in the range for normal human skin. Further, the porous fiber matrices have porosity between 38 to 60% and average pore diameters between 10 to 14mum. We evaluated the efficacy of these biodegradable fiber matrices as skin substitutes by seeding them with human skin fibroblasts (hSF). Human skin fibroblasts acquired a well spread morphology and showed significant progressive growth on fiber matrices in the 350-1100nm diameter range. Collagen type III gene expression was significantly up-regulated in hSF seeded on matrices with fiber diameters in the range of 350-1100nm. Based on the need, the proposed fiber skin substitutes can be successfully fabricated and optimized for skin fibroblast attachment and growth.     

Apatite nano-crystalline surface modification of poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering: implications for protein adsorption

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.     

Apatite nano-crystalline surface modification of poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering: implications for protein adsorption

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.     

The sintered microsphere matrix for bone tissue engineering: in vitro osteoconductivity studies

A tissue engineering approach has been used to design three-dimensional synthetic matrices for bone repair. The osteoconductivity and degradation profile of a novel polymeric bone-graft substitute was evaluated in an in vitro setting. Using the copolymer poly(lactide-co-glycolide) [PLAGA], a sintering technique based on microsphere technology was used to fabricate three-dimensional porous scaffolds for bone regeneration. Osteoblasts and fibroblasts were seeded onto a 50:50 PLAGA scaffold. Morphologic evaluation through scanning electron microscopy demonstrated that both cell types attached and spread over the scaffold. Cells migrated through the matrix using cytoplasmic extensions to bridge the structure. Cross-sectional images indicated that cellular proliferation had penetrated into the matrix approximately 700 microm from the surface. Examination of the surfaces of cell/matrix constructs demonstrated that cellular proliferation had encompassed the pores of the matrix by 14 days of cell culture. With the aim of optimizing polymer composition and polymer molecular weight, a degradation study was conducted utilizing the matrix. The results demonstrate that degradation of the sintered matrix is dependent on molecular weight, copolymer ratio, and pore volume. From this data, it was determined that 75:25 PLAGA with an initial molecular weight of 100,000 has an optimal degradation profile. These studies show that the sintered microsphere matrix has an osteoconductive structure capable of functioning as a cellular scaffold with a degradation profile suitable for bone regeneration.     

Synthesis, characterization of chitosans and fabrication of sintered chitosan microsphere matrices for bone tissue engineering

The objective of the present study was to synthesize and characterize chitosans with different degrees of deacetylation (DDA%), prepare chitosan microspheres with controlled chemistry and geometry, and fabricate three-dimensional (3-D) chitosan matrices based on microspheres with appropriate pore size, porosity and mechanical properties suitable for bone tissue engineering applications. Chitosans with three DDA% of 69%, 79% and 97% were obtained using a thermomechanochemical technique by varying the applied pressure and NaOH solution concentration. The prepared chitosans were comprehensively characterized by proton nuclear magnetic resonance, elemental analysis, viscosity measurements, thermal analyses and X-ray diffraction. In addition, chitosan microspheres were prepared using an ionotropic gelation method. Three-dimensional chitosan matrices were fabricated via a sintered microsphere technique. Scanning electron microscopy revealed rough surfaces of the prepared chitosan microspheres. Mercury intrusion porosimetry revealed a porosity of 19.2% and a median pore diameter of 199.62microm of the fabricated 3-D matrix. The compressive modulus of the sintered microsphere matrix (662.26+/-54.53MPa) was in the range of human cancellous bone (10-2000MPa), making it suitable for bone tissue engineering applications.     

聚乳酸-羟基乙酸纳米粒的制备*

背景：聚乳酸-羟基乙酸是一种生物相容性良好的可降解材料,确定其最佳制备工艺条件,有利于聚乳酸-羟基乙酸后续药物载体研究与工业化生产条件的确立。 目的：以聚乳酸-羟基乙酸为包裹材料,探索纳米粒的制备条件对粒径、表面形态等的影响,确定最佳制备工艺条件。 方法：采用乳化-溶剂挥发法制备聚乳酸-羟基乙酸纳米粒,以粒径为观察指标,探讨乳化剂种类、乳化剂含量、油相种类、超声时间、挥发时间、油相与水相体积比(W∶O)以及聚合物质量浓度等制备条件对纳米粒粒径的影响,确定制备聚乳酸-羟基乙酸纳米粒的最佳工艺条件。 结果与结论：优化后的制备工艺条件是在室温下,以一定的搅拌速度和滴加速度,选择常用无毒的乳化剂,浓度在0.3%~1.0%,丙酮为有机相,超声时间8~15 min、挥发时间6~10 h、水油相比(W∶O)>25∶1,聚合物质量浓度<60 g/L。提示该制备工艺简单、稳定,优化制备条件,可制备出表面形态规整、粒径适宜的聚乳酸-羟基乙酸纳米粒。     

In vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering

In vitro degradation behaviors of three-dimensional tissue engineering porous scaffolds made from amorphous poly(D,L-lactide-co-glycolide) with three different formulations have been systematically investigated up to 26 weeks in phosphate buffer saline solution at 37 degrees C. The following properties of the scaffolds were measured as a function of degradation time: dimensions, weight, compressive strength and modulus, polymer molecular weight and its distribution, and pore morphology. Of special interest was the determination of mechanical properties in wet environment. The pH of the PBS media was also detected. According to the characteristic changes of the various properties of porous scaffolds, the degradation process is suggested to be roughly divided into three stages tentatively named as quasi-stable stage, decrease-of-strength stage, loss-of-weight and disruption-of-scaffold stage.     

A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering

A new method to prepare PLA/CMP (poly-L-lactide/calcium metaphosphate) composite scaffolds was developed for effective bone tissue engineering. This novel sintering method is composed of pressing the mixture of PLA, CMP, and salt particles at 150 MPa for 3 min followed by heat treatment at 210 degrees C for 30 min. The scaffolds had a homogeneously interconnected porous structure without a skin layer, and they exhibited a narrower pore size distribution and higher mechanical strength in comparison with scaffolds made by a solvent casting method. The scaffolds were seeded by osteoblasts and cultured in vitro or implanted into nude mice subcutaneously for up to 5 weeks. The number of cells attached to and proliferated on the scaffolds at both in vitro and in vivo was in the order of; PLA by novel sintering < PLA/CMP by solvent casting < PLA/CMP by novel sintering. In addition, the alkaline phosphatase activity of and calcium deposition in the scaffolds explanted from mice were enhanced significantly for the scaffolds by novel sintering compared to them by solvent casting. The in vitro results agreed well with the in vivo data. Such a superior characteristic of the novel sintering method should have resulted from the fact that the CMP particles could contact directly with cells/tissues to stimulate the cell proliferation and osteogenic differentiation, while the CMP particles would be coated by polymers and hindered to interact with cells/tissues in the case of a solvent casting method. As the novel sintering method does not use any solvents it offers another advantage to avoid problems associated with solvent residue.     

Physicomechanical properties of sintered scaffolds formed from porous and protein-loaded poly(DL-lactic-co-glycolic acid) microspheres for potential use in bone tissue engineering

An injectable poly(DL-lactic-co-glycolic acid) (PLGA) system comprising both porous and protein-loaded microspheres capable of forming porous scaffolds at body temperature was developed for tissue regeneration purposes. Porous and non-porous (lysozyme loaded) PLGA microspheres were formulated to represent ‘low molecular weight’ 22–34 kDa, ‘intermediate molecular weight’ (IMW) 53 kDa and ‘high molecular weight’ 84–109 kDa PLGA microspheres. The respective average size of the microspheres was directly related to the polymer molecular weight. An initial burst release of lysozyme was observed from both microspheres and scaffolds on day 1. In the case of the lysozyme-loaded microspheres, this burst release was inversely related to the polymer molecular weight. Similarly, scaffolds loaded with 1 mg lysozyme/g of scaffold exhibited an inverse release relationship with polymer molecular weight. The burst release was highest amongst IMW scaffolds loaded with 2 and 3 mg/g. Sustained lysozyme release was observed after day 1 over 50 days (microspheres) and 30 days (scaffolds). The compressive strengths of the scaffolds were found to be inversely proportional to PLGA molecular weight at each lysozyme loading. Surface analysis indicated that some of the loaded lysozyme was distributed on the surfaces of the microspheres and thus responsible for the burst release observed. Overall the data demonstrates the potential of the scaffolds for use in tissue regeneration.     

In vitro evaluation of potential calcium phosphate scaffolds for tissue engineering

Nanocrystalline calcium phosphates are very interesting candidates as scaffolds for bone tissue engineering. These materials show excellent in vivo biocompatibility, cell proliferation, and resorption. In this work we have studied the osteoblast-like cell behavior seeded onto HA and BCP synthesized by controlled crystallization method and treated at different temperatures. In vitro cell attachment, proliferation, differentiation, spreading, and cytotoxicity tests have been carried out. The results can be explained as a function of the phase composition and microstructure. Under in vitro closed conditions, nanocrystalline HA depletes the calcium of the medium avoiding cell proliferation, whereas well-crystallized HA enhances high cell proliferation. On the other hand, nanocrystalline BCPs supply Ca(2+) to the medium due to the higher solubility of the beta-TCP component, allowing an excellent in vitro cellular response when osteoblast-like cells are seeded on it. These features make BCPs excellent candidates as scaffolds for bone tissue engineering.     

In vivo tissue engineering of bone using poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) and collagen scaffolds

Porous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and calcium phosphate-loaded collagen (CaP-Gelfix) foams were seeded with rat bone marrow stromal cells and implanted into defects created in rat femurs to study in vivo bone formation and to test their suitability for use in bone tissue engineering. At 3 and 6 weeks, new bone formation was evaluated by macroscopy, radiography, dual-energy X-ray absorptiometry (DEXA), and quantitative computerized tomography (QCT). Atomic contents of the implants were further assessed by QCT. Some initial inflammation that significantly decreased with time was observed in the CaP-Gelfix group. PHBV inflammation was minimal at all stages. Fibrous tissue formation in the CaP-Gelfix group was more than in the PHBV group. Both cell-loaded and cell-free PHBV matrices elicited minimal fibrous tissue formation during the 6-week implantation duration. Macroscopic and radiological studies demonstrated better healing with PHBV matrices than with CaP-Gelfix in 3 weeks. Histologically, fibrous connective tissue establishment and inflammation scores were significantly higher in the CaP-Gelfix group when compared with the PHBV group at both time intervals. At 6 weeks, however, the extent of healing was almost the same with both implants. DEXA and QCT results indicated that there was an increase in bone mineral density in both PHBV and CaP-Gelfix implants at the end of 6 weeks. This study suggests that even though PHBV and CaP-Gelfix have different bulk and surface chemistries they both are promising cell carriers that may be suitable for use in bone tissue engineering.     

Porous poly (DL-lactic acid) modified chitosan-gelatin scaffolds for tissue engineering

Chitosan-gelatin (Cs-Gel) scaffolds are modified with poly (DL-lactic acid) (PDLLA) dichloromethane solution of different concentrations (0.1, 0.5, and 1.0%) and immersed in water after the evaporation of the solvent. The swollen scaffolds are freeze dried. The concentration of PDLLA has significant effects on both the physicomechanical properties and the cytocompatibility. Data reveal that only the 0.1% concentration could increase the tensile strength fourfold in comparison with the pristine Cs-Gel scaffold, while maintaining the human fibroblast adhesion, migration, and proliferation just like the Cs-Gel scaffold.     

In vitro bioactivity of poly(epsilon-caprolactone)-apatite (PCL-AP) scaffolds for bone tissue engineering: the influence of the PCL/AP ratio

Porous poly(epsilon-caprolactone) (PCL) is used as long-term bioresorbable scaffold for bone tissue engineering. The bone regeneration process can be enhanced by addition of carbonated apatites (AP). This study was aimed at evaluating the influence of the PCL/AP ratio on the in vitro degradation and bioactivity of PCL-AP composites. To this purpose, PCL-AP samples were synthesised with the following PCL/AP weight/weight ratios: 50/50, 60/40 and 75/25. Vibrational IR and Raman spectroscopies coupled to thermogravimetry (TG) and differential scanning calorimetry (DSC) were used to investigate the in vitro degradation mechanism in different media: 0.01 M NaOH solution (pH=12), saline phosphate buffer at pH 7.5 (SPB), esterase in SPB and simulated body fluid (SBF) at pH 7.5. The latter medium was used to evaluate the bioactivity of the composites. A control PCL sample was analysed before the addition of the AP component. As regards the untreated samples, the method of synthesis utilised for preparing the composite was found to enhance the crystallinity degree. The AP component revealed to be constituted of a B-type carbonated hydroxyapatite with a 3% carbonate content. After 28 days of treatment, the samples showed different degradation patterns and extents depending on the degradation medium, the starting PCL crystallinity and composite composition. Weight measurements, Raman and TG analyses revealed deposition of an apatitic phase on all the composites immersed in SBF. Therefore, all the samples displayed a good bioactivity; the sample which showed the most pronounced apatitic deposition was 50/50, i.e. that containing the highest amount of AP.     

丝素/壳聚糖/纳米羟基磷灰石构建的骨组织工程支架

背景：丝素蛋白、壳聚糖及纳米羟基磷灰石均是天然材料,具有良好的生物活性和理化特性,作为人体组织工程材料已取得了一定的成果,但3种材料在单独应用的研究中还存在一定的缺陷。 目的：制作丝素蛋白/壳聚糖/纳米羟基磷灰石三维支架材料,分析其特性。 方法：将丝素蛋白、壳聚糖、纳米羟基磷灰石分别配制成2%的溶液后,分别按照 1∶1∶0.5,1∶1∶1, 1∶1∶1.5 的体积比混合,采用冷冻干燥与化学交联技术制备成三维复合支架材料。检测三维复合支架的孔隙率、吸水膨胀率及热水溶失率,采用材料力学测验机测试干燥三维复合支架材料的拉伸和压缩弹性模量,采用扫描电镜检测三维复合支架的孔径。 结果与结论：丝素蛋白/壳聚糖/纳米羟基磷灰石三维复合支架在干燥状态下呈白色,无特殊气味,为稳定固态的圆柱体,触之有明显的抗压能力和弹性。随着复合支架材料中纳米羟基磷灰石含量的增高,支架材料的孔隙率、吸水膨胀率、平均孔径呈逐渐减小趋势,热水溶失率及抗压能力表现出相反的趋势,结果显示以1∶1∶1体积比制作的支架更符合骨替代材料要求,其平均孔径为85.67 µm、吸水膨胀率的为(135.65±4.56)%、热水溶失率为(22.84±1.06)%,支架材料内部孔隙均匀,呈现网状结构,孔隙之间交通发达,网状结构本身约10 µm。     

Porous polylactide/chitosan scaffolds for tissue engineering

Novel porous scaffolds were fabricated using biodegradable polylactide/chitosan blends. A combinational technique involving solvent-extracting, liquid-solid separation, and freeze-drying paths were employed. The processing parameters were optimized in order to produce desired porous scaffolds and thus obtained scaffolds showed well distributed and interconnected porous structures with controllable porosities varying from around 50-85% and regulative pore sizes being distributed within a region between 2 and 190 microm. These scaffolds exhibited remarkably improved hydrophilicity based on the measurements for their swelling index. The results obtained from in vitro incubation of scaffolds in phosphate buffered saline solutions at 37 degrees C during various periods up to 10 weeks indicated that chitosan component inside the scaffolds, on the one hand, effectively buffered the acidic degradation products of polylactide/chitosan scaffolds and on the other hand, the degradation of the scaffolds was also conspicuously delayed. These porous scaffolds maintained well-defined compressive mechanical properties and by well-blending polylactide with chitosan component, improved toughness on the resultant porous scaffolds was also observed.     

Nanofibrous poly(epsilon-caprolactone)/poly(vinyl alcohol)/chitosan hybrid scaffolds for bone tissue engineering using mesenchymal stem cells

In the present study, based on a biomimetic approach, novel 3D nanofibrous hybrid scaffolds consisting of poly(epsilon-caprolactone), poly(vinyl alcohol), and chitosan were developed via a multi-jet electrospinning method. The influence of chemical, physical, and structural properties of the scaffolds on the differentiation of mesenchymal stem cells into osteoblasts, and the proliferation of the differentiated cells were investigated. Osteogenically induced cultures revealed that cells were well-attached, penetrated into the construct and were uniformly distributed. The expression of early and late phenotypic markers of osteoblastic differentiation was upregulated in the constructs cultured in osteogenic medium.     

Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering

High porosity of scaffold is always accompanied by poor mechanical property; the aim of this study was to enhance the strength and modulus of the highly porous scaffold of nanohydroxyapatite/polyamide66 (n-HA/PA66) by coating chitosan (CS) and to investigate the effect of CS content on the scaffold physical properties and cytological properties. The results show that CS coating can reinforce the scaffold effectively. The compress modulus and strength of the CS coated n-HA/PA66 scaffolds are improved to 32.71 and 2.38 MPa, respectively, being about six times and five times of those of the uncoated scaffolds. Meanwhile, the scaffolds still exhibit a highly interconnected porous structure and the porosity is approximate about 78%, slightly lower than the value (84%) of uncoated scaffold. The cytological properties of scaffolds were also studied in vitro by cocultured with osteoblast-like MG63 cells. The cytological experiments demonstrate that the reinforced scaffolds display favorable cytocompatibility and have no significant difference with the uncoated n-HA/PA66 scaffolds. The CS reinforced n-HA/PA66 scaffolds can meet the basic mechanical requirement of bone tissue engineering scaffold, presenting a potential for biomedical application in bone reconstruction and repair.     

纳米羟基磷灰石/聚羟基丁酸酯骨组织工程支架的制备及性能表征

背景：高分子聚合物聚羟基丁酸酯具有优良的生物相容性、生物降解性及压电性,但也存在脆性大、亲水性较差等不足。 目的：制备不同组成比例的电纺纳米羟基磷灰石/聚羟基丁酸酯纤维支架材料,分析其结构及性能。 方法：通过气流-高压静电纺丝技术制备纳米羟基磷灰石质量百分比分别为0、10%、20%、30%的电纺纳米羟基磷灰石/聚羟基丁酸酯纤维支架,检测支架材料的微观结构、基团组成、晶相分布、热学性能及表面润湿性。 结果与结论：扫描电镜观察显示,随着纳米羟基磷灰石含量的增大,越来越多的纳米羟基磷灰石颗粒分布于复合纤维表面且分布趋于均匀,到含量达到30%时,纳米羟基磷灰石已基本布满纤维表面,纤维表面的粗糙度也随之增加;差示扫描量热法及X射线衍射结果表明,纳米羟基磷灰石的加入可以降低复合纤维中聚羟基丁酸酯的结晶度及结晶规整程度,且纳米羟基磷灰石含量越大效果越明显;随着纳米羟基磷灰石含量的增大,复合支架表面的接触角逐渐降低,亲水性有所提高。表明将纳米羟基磷灰石与聚羟基丁酸酯复合进行电纺可以有效提高材料的表面润湿性及结晶度,改善材料的亲水性及脆性,且纳米羟基磷灰石含量越高作用越明显。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

β-磷酸三钙/聚乳酸叠层生物材料的理化性能

本文研究了β—磷酸三钙／聚乳酸叠层复合支架在体外37℃生理盐水中的降解过程，内容包括材料的重量、力学强度、聚乳酸分子量、材料周围环境的pH值、Ca^2 浓度等参数随时间的变化，并证实材料理化特性适合于骨组织工程支架材料的要求。支架材料的生物相容性评价实验结果表明骨髓基质细胞与支架间有良好的亲和性。     

In vitro evaluation of novel bioactive composites based on Bioglass-filled polylactide foams for bone tissue engineering scaffolds

Highly porous poly(DL-lactic acid) (PDLLA) foams and Bioglass-filled PDLLA composite foams were characterized and evaluated in vitro as bone tissue engineering scaffolds. The hypothesis was that the combination of PDLLA with Bioglass in a porous structure would result in a bioresorbable and bioactive composite, capable of supporting osteoblast adhesion, spreading and viability. Composite and unfilled foams were incubated in simulated body fluid (SBF) at 37 degrees C to study the in vitro degradation of the polymer and to detect hydroxyapatite (HA) formation, which is a measure of the materials' in vitro bioactivity. HA was detected on all the composite samples after incubation in SBF for just 3 days. After 28 days immersion the foams filled with 40 wt % Bioglass developed a continuous layer of HA. The formation of HA for the 5 wt % Bioglass-filled foams was localized to the Bioglass particles. Cell culture studies using a commercially available (ECACC) human osteosarcoma cell line (MG-63) were conducted to assess the biocompatibility of the foams and cell attachment to the porous substrates. The osteoblast cell infiltration study showed that the cells were able to migrate through the porous network and colonize the deeper regions within the foam, indicating that the composition of the foams and the pore structures are able to support osteoblast attachment, spreading, and viability. Rapid formation of HA on the composites and the attachment of MG-63 cells within the porous network of the composite foams confirms the high in vitro bioactivity and biocompatibility of these materials and their potential to be used as scaffolds in bone tissue engineering and repair.