Electrospinning of Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) nanofibers with feature surface microstructure

The development of surface microstructure with specific features in electrospun nanofibers has attracted more and more attention in recent years. In this article, a common biological polyester, poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) was electrospinning into nanofibers with “coral‐like” surface microstructure by a conventional‐electrospinning setup. The effect of the process parameters on the microstructure in electrospun nanofibers were investigated via a series of experiments. The formation mechanism of this feature structure and cytotoxicity assays of PHBV membrane were also discussed. The water contact angle of the electrospun PHBV membrane was higher than that of the PHBV cast film due to a very‐rough fiber surface including porous beads when PHBV was electrospun from the concentration of 4 wt %. Because of special hole shape and size distribution, the physical structure of surface of PHBV electrospun fibers offered it special properties, such as specific‐surface area, hydrophilic–hydrophobic properties, adhesion properties of cells and biological substances, etc. The demonstrated method of form coral structure would contribute to the areas such as filtration, sensor, tissue engineering scaffolds, and carriers of drugs or catalysis. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Fabrication and evaluation of nanofibrous polyhydroxybutyrate valerate scaffolds containing hydroxyapatite particles for bone tissue engineering

Tissue engineering is a new approach for regeneration of damaged tissues. The current clinical methods such as autograft and allograft transplantation are not effective for repairing bone damages, mainly due to the limited available sources and the donor-site side effects. In this research, the nanocomposite poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/nano hydroxyapatite (nHA) scaffolds with different nHA ratios for bone regeneration were utilized. The diameter and porosity of scaffolds were approximately 200?nm and 74%, respectively. The degradability test of the scaffolds suggests a low degradation rate with total degradation of 30% after 3 months. Cytotoxicity result showed that cultured osteoblast cells (MC3T3) on nanocomposite scaffolds had superiority in terms of higher proliferation and attachment in comparison with PHBV scaffold. The protein expression of alkaline phosphatase illustrated that nanofibrous scaffold containing hydroxyapatite had the highest alkaline phosphatase activities as a result of better proliferation. These results recommend that PHBV/nHA scaffolds are suitable candidates for bone tissue engineering.     

Fabrication and Evaluation of Polycaprolactone–Poly(hydroxybutyrate) or Poly(3‐Hydroxybutyrate‐co‐3‐Hydroxyvalerate) Dual‐Leached Porous Scaffolds for Bone Tissue Engineering Applications

Polycaprolactone (PCL) blend with poly(hydroxybutyrate) (PHB) or poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) dual‐leached scaffolds are prepared by using the solvent casting and salt–polymer‐leaching technique. The blending of the PHB and PHBV in PCL scaffolds results in decreased porosities of the scaffolds, and the water absorption capacities of the scaffolds also decrease. The compressive modulus of the PCL–PHB and PCL–PHBV dual‐leached scaffolds is greatly increased by the blending of PHB or PHBV matrix. An indirect cytotoxicity evaluation of all scaffolds with mouse fibroblastic cells (L929) and mouse calvaria‐derived preosteoblastic cell (MC3T3‐E1) indicates that all dual‐leached scaffolds are posed as nontoxic to cells. Both PCL–PHB and PCL–PHBV dual‐leached scaffolds are supported by the attachment of MC3T3‐E1 at significantly higher levels to tissue culture polystyrene plate (TCPS) and are able to support the proliferation of MC3T3‐E1 at higher levels to that cells on TCPS and PCL scaffolds. For mineralization, cells cultured on surfaces of PCL–PHB and PCL–PHBV dual‐leached scaffolds show higher mineral deposition than on TCPS and PCL scaffold.

     

An Overview of Poly(lactic-co-glycolic) Acid (PLGA)-Based Biomaterials for Bone Tissue Engineering

Poly(lactic-co-glycolic) acid (PLGA) has attracted considerable interest as a base material for biomedical applications due to its: (i) biocompatibility; (ii) tailored biodegradation rate (depending on the molecular weight and copolymer ratio); (iii) approval for clinical use in humans by the U.S. Food and Drug Administration (FDA); (iv) potential to modify surface properties to provide better interaction with biological materials; and (v) suitability for export to countries and cultures where implantation of animal-derived products is unpopular. This paper critically reviews the scientific challenge of manufacturing PLGA-based materials with suitable properties and shapes for specific biomedical applications, with special emphasis on bone tissue engineering. The analysis of the state of the art in the field reveals the presence of current innovative techniques for scaffolds and material manufacturing that are currently opening the way to prepare biomimetic PLGA substrates able to modulate cell interaction for improved substitution, restoration, or enhancement of bone tissue function.     

Compressive properties and creep resistance of a novel,porous, semidegradable poly(vinyl alcohol)/poly(lactic‐co‐glycolic acid) scaffold for articular cartilage repair

Tissue engineering for articular cartilage repair has shown success in ensuring the integration of neocartilage with surrounding natural tissue, but the rapid restoration of biomechanical functions remains a significant challenge. The poly(vinyl alcohol) (PVA) hydrogel is regarded as a potential articular cartilage replacement for its fair mechanical strength, whereas its lack of bioactivity limits its utility. To obtain a scaffold possessing expected bioactivity and initial mechanical properties, we herein report a novel salt‐leaching technique to fabricate a porous PVA hydrogel simultaneously embedded with poly(lactic‐co‐glycolic acid) (PLGA) microspheres. Through the investigation of environmental scanning electron microscopy, we found that the porous PVA/PLGA scaffold was successfully manufactured. The compression and creep properties were also comprehensively studied before and after cell culturing. The relationship between the compressive modulus and strain ratio of the porous PVA/PLGA scaffold showed significant nonlinear behavior. The elastic compressive modulus was influenced a little by the porogen content, whereas it went higher with a higher PLGA microsphere content. The cell‐cultured scaffolds presented higher compressive moduli than the initial ones. The creep resistance of the cell‐cultured scaffolds was much better than that of the initial ones. In all, this new scaffold is a promising material for articular cartilage repair. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40311.     

Preparation and characterization of polyhydroxybutyrate‐co‐hydroxyvalerate/silk fibroin nanofibrous scaffolds for skin tissue engineering

Nanofibrous scaffolds were obtained by co‐electrospinning poly (3‐hydroxybuty‐rate‐co‐3‐hydroxyvalerate) (PHBV) and fibroin regenerated from silk in different proportions using 1,1,1,3,3,3‐hexafluoro‐2‐isopropanol (HFIP) as solvent. Field emission scanning electron microscope (FESEM) investigation showed that the fiber diameters of the nanofibrous scaffolds ranged from 190 to 460 nm. X‐ray diffraction (XRD) and Fourier transform infrared spectroscopy analysis (FT‐IR) showed that the main structure of silk fibroin (SF) in the nanofibrous scaffold was β‐sheet. Compared to the PHBV nanofibrous scaffold, the surface hydrophilicity and water‐uptake capability of the PHBV/SF nanofibrous scaffold with 50/50 were improved. The results of cell adhesion experiment showed that the fibroblasts adhered more to the PHBV/SF nanofibrous scaffold with 50/50 than the pure PHBV nanofibrous scaffold. The proliferation of fibroblast on the PHBV/SF nanofibrous scaffold with 50/50 was higher than that on the pure PHBV nanofibrous scaffold. Our results indicated that the PHBV/SF nanofibrous scaffold with 50/50 may be a better candidate for biomedical applications such as skin tissue engineering and wound dressing. POLYM. ENG. SCI., 55:907–916, 2015. © 2014 Society of Plastics Engineers     

Fabrication and characterization of 3-dimensional PLGA nanofiber/microfiber composite scaffolds

Novel three-dimensional (3-D) nano-/microfibrous poly(lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by hybrid electrospinning, involving a combination of solution electrospinning and melt electrospinning. The scaffolds consisted of a randomly oriented structure of PLGA microfibers (average fiber diameter = 28 μm) and PLGA nanofibers (average fiber diameter = 530 nm). From mercury porosimetry, the PLGA nano-/microfiber (10/90) scaffolds were found to have similar pore parameters to the PLGA microfiber scaffolds. PLGA nano-/microfibrous scaffolds were examined and compared with the PLGA microfiber scaffolds in terms of the attachment, spreading and infiltration of normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF). The cell attachment and spreading of both cell types were several times higher in the nano-/microfiber composite scaffolds than in the microfibrous scaffolds without nanofibers. This shows that the presence of nanofibers enhanced the attachment and spreading of the cells on the nano-/microfiber composite scaffolds. Moreover, the nanofibers helped the cells infiltrate easily into the scaffolds. Overall, this novel nano-/microfiber structures has great potential for the 3-D organization and guidance of cells provided for tissue engineering.     

Fabrication and characterization of electrospun fibrous nanocomposite scaffolds based on poly(lactide‐co‐glycolide)/poly(vinyl alcohol) blends

Tissue engineering involves the fabrication of three‐dimensional scaffolds to support cellular in‐growth and proliferation. Ideally, the scaffolds should be similar to the native extracellular matrix (ECM). Electrospun polymer nanofibrous scaffolds are appropriate candidates for ECM mimetic materials since they mimic the nanoscale properties of ECM. Electrospun polymer nanocomposites based on poly(lactide‐co‐glycolide) (PLGA)/poly(vinyl alcohol) (PVA) and organically modified montmorillonite (OMMT) were prepared by a solution intercalation technique followed by electrospinning. The morphology of fibrous scaffolds based on these nanocomposites was investigated using scanning electron microscopy. The scaffolds showed highly porous structure within the nanofibres of diameters ranging from 400 to 700 nm. X‐ray diffractometry gave evidence of good dispersion of the OMMT in the blends with exfoliated morphology. Measurements of the water uptake and water contact angle of the fibrous scaffolds indicated significant improvement in the hydrophilicity of the scaffolds. Evaluations of the mechanical properties and unrestricted somatic stem cell culture of the electrospun fibrous nanocomposite scaffolds revealed that the PLGA90/PVA10/1.5% OMMT and PLGA90/PVA10/3% OMMT samples are the most useful from the tissue engineering application viewpoint. Copyright © 2010 Society of Chemical Industry     

碳纤维增强聚合物组织工程支架的制备及表征

以碳纤维（CF）作为增强材料,将CF有序排列于聚乳酸羟基乙酸（PLGA）多孔结构中,制备性能优良的CF/PLGA复合支架,并对其力学性能及细胞生物学性能进行表征.对增强体CF进行有序排列以提高支架的力学性能,扫描电子显微镜（SEM）观察CF/PLGA复合支架的微观形貌,可以看出CF在聚合物基体内部是呈有序结构并且二者结合情况良好.为了提高CF的生物相容性,利用对氨基苯甲酸对CF进行表面修饰,细胞生长在支架上的SEM照片反映了成纤维细胞对PLGA及CF/PLGA复合支架的黏附性能良好;通过细胞毒性测试,发现表面修饰的CF对细胞的生长没有负面作用,且在一定程度上促进了细胞的生长.研究结果表明,制备的CF/PLGA支架具有良好的力学性能和生物相容性,在骨组织工程支架的应用中具有一定的潜力.     

Solid-state NMR study on the structure and mobility of the noncrystalline region of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

The noncrystalline structures of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymers were studied by variable temperature solid-state wide-line ¹H NMR and solid-state high-resolution ¹³C NMR spectroscopy. It is found that at room temperature there exists a rich and rigid component in the noncrystalline region of PHB and PHBV. The content of this component decreases with the increase in 3-hydroxyvalerate content in PHBV and with the increase in temperature. The brittleness of PHB may be partly attributed to the rigidness of the noncrystalline region at room temperature, while the improvement of the properties of PHBV may come from the enhanced mobility of the noncrystalline region.     

Self-adhesive plasticized regenerated silk on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) for bio-piezoelectric force sensor and microwave circuit design

This study shows that regenerated silk (RS), a natural biodegradable and biocompatible polymer, can behave as a self-adhesive thermoplastic material with multifunctional properties. In particular, Ca ions-plasticized RS hybrids with gold nanorods have been produced. It has been found that at mild conditions of temperature and pressure, RS hybrids undergo to the loss of the β-sheet content and forms a tough self-adhesive material on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) substrate. The structure-dependent piezoelectricity of such RS adhesives on PHBV films was investigated and it was demonstrated that this forms a RS/PHBV piezoelectric sensor that can be used for the monitoring of force. The constitutive parameters (i.e., permittivity and loss tangent) of both PHBV and RS/PHBV were measured in view of their use as dielectric substrates in microwave circuit design. Being fully made of biodegradable and biocompatible materials, this self-adhesive material can be used in tissue engineering for different applications.     

PLGA/SF blend scaffolds modified with plasmid complexes for enhancing proliferation of endothelial cells

Biomimetic scaffolds have been investigated for vascular tissue engineering for many years. However, the design of an ideal biodegradable vascular scaffold is still in progress. The optimization of poly(lactide-co-glycolide)/silk fibroin (PLGA/SF) blend composition was performed to provide the designed scaffolds with adequate mechanical properties and favorable biocompatibility for the intended application. By systematically varying the weight ratio of PLGA and SF, we could control fiber diameter and hydrophilicity as well as mechanical properties of the fibrous scaffolds. These scaffolds with a weight ratio of PLGA/SF at 70/30 exhibited excellent performance, such as tensile strength of 1.5 ± 0.1 MPa, and elongation at break of 77.4 ± 6.4%. Therefore, PLGA/SF scaffold with a weight ratio of 70/30 was chose as the matrix because it matches at best the mechanical demands for application in vascular tissue engineering. In order to promote the endothelialization of electrospun scaffolds, we used pEGFP-ZNF580 plasmid (pZNF580) complexes to modify the electrospun scaffolds by electrospraying technique. pZNF580 complexes were prepared from pZNF580 and microparticles (MPs) of amphiphilic copolymer methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-2,5-morpholinedione-co-glycolide)-graft-polyethyleneimine. Negatively charged PLGA/SF fibers adsorbed the positively charged MPs via physical deposition and electrostatic force. Scanning electron microscope image indicated the forming of composite scaffold and MPs did not change fiber’s shape and 3-D structure. Cell culture experiments demonstrated that the scaffolds modified with MPs/pZNF580 complexes could promote human umbilical vein endothelial cell growth and inhibit human umbilical artery smooth muscle cell proliferation. Our results indicated that the composite scaffolds with MPs/pZNF580 complexes could be used as a potential scaffold for vascular tissue engineering.     

Formation of porous PLGA scaffolds by a combining method of thermally induced phase separation and porogen leaching

A novel method for the fabrication of porous poly(L -lactide-co-glycolide) (PLGA) scaffolds by combining thermally induced phase separation and porogen leaching is presented in this article. Big pores with about 75–400 μm diameters in the obtained scaffolds were generated by the porogen, sucrose particles, while small pores with diameters less than 20 μm induced via phase separation. Extraction of the solvent, chloroform by ethanol at cool temperatures could reduce the scaffold toxicity. Effects of PLGA concentration, freezing temperature, volume fraction of porogen, and introduction of β-tricalcium phosphate (β-TCP) on morphology, porosity, and compressive properties of the scaffolds were systematically discussed. Results showed that the size of small pores decreased by decreasing the polymer concentration and reducing the freezing temperature, whereas the interconnectivity of the scaffolds was improved by increasing the porogen fraction. The compressive modulus and strength were significantly lowered by increasing the scaffold porosity, that is, by increasing porogen fraction, or decreasing the polymer concentration, or reducing the freezing temperature. Addition of β-TCP into the scaffolds did not influence the compressive modulus significantly but tended to decrease the compressive strength. The obtained scaffolds with diverse pore sizes would be potentially used in bone tissue engineering. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Fabrication and characterization of aligned nanofibrous PLGA/Collagen blends as bone tissue scaffolds

Aligned nanofibrous blends of poly (d, l-lactide-co-glycolide) (PLGA) and collagen with various PLGA/collagen compositions (80/20, 65/35 and 50/50) were fabricated by electrospinning and characterized for bone tissue engineering. Morphological characterization showed that the addition of collagen to PLGA resulted in narrowing of the diameter distribution and a reduction in average diameter. Differential scanning calorimetric (DSC) studies showed that the triple helix structure of the native collagen was not destroyed during the fabrication process. However, the blending had a marked effect on the overall enthalpy of the blends, whereby the total enthalpy decreased as the collagen content decreased. Thermogravimetric analysis showed the addition of collagen increased the hydrophilicity of the scaffolds. The crosslinking of collagen to increase the biostability was done using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and an overall ∼25% degree of crosslinking was achieved. The EDC crosslinking had little effect on the nanofibrous morphology of the 80/20 blend system; however, the nanofibrous features were compromised to some extent at higher collagen concentrations. The mechanical characterization under dry and wet conditions showed that increasing collagen content resulted in a tremendous decrease in the mechanical properties. However, crosslinking resulted in the increase in elastic modulus from 47 MPa to 83 MPa for the wet PLGA/Collagen 80/20 blend system, with little effect on the tensile strength. In conclusion, the aligned nanofibrous scaffold used in this study constitutes a promising material for bone tissue engineering.     

Strontium Peroxide-Loaded Composite Scaffolds Capable of Generating Oxygen and Modulating Behaviors of Osteoblasts and Osteoclasts

The reconstruction of bone defects remains challenging. The utilization of bone autografts, although quite promising, is limited by several drawbacks, especially substantial donor site complications. Recently, strontium (Sr), a bioactive trace element with excellent osteoinductive, osteoconductive, and pro-angiogenic properties, has emerged as a potential therapeutic agent for bone repair. Herein, a strontium peroxide (SrO₂)-loaded poly(lactic-co-glycolic acid) (PLGA)-gelatin scaffold system was developed as an implantable bone substitute. Gelatin sponges serve as porous osteoconductive scaffolds, while PLGA not only reinforces the mechanical strength of the gelatin but also controls the rate of water infiltration. The encapsulated SrO₂ can release Sr²⁺ in a sustained manner upon exposure to water, thus effectively stimulating the proliferation of osteoblasts and suppressing the formation of osteoclasts. Moreover, SrO₂ can generate hydrogen peroxide and subsequent oxygen molecules to increase local oxygen tension, an essential niche factor for osteogenesis. Collectively, the developed SrO₂-loaded composite scaffold shows promise as a multifunctional bioactive bone graft for bone tissue engineering.     

Thermal and mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/cellulose nanowhiskers composites

Bacterial polyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was reinforced with cellulose nanowhiskers (CNW) in 1-5 wt.% concentrations using a solvent casting method. The CNW was prepared from microcrystalline cellulose (MCC) using sulfuric acid hydrolysis. The influence of CNW on the PHBV crystallization, thermal, dynamic mechanical and mechanical properties were evaluated using polarized optical microscope (POM), differential scanning calorimeter (DSC), dynamic mechanical analysis (DMA), tensile and bulge tests, respectively. POM test results demonstrated that CNW was an effective PHBV nucleation agent. Tensile strength, Young’s modulus and toughness of PHBV increased with the increasing concentration of CNW. DMA results showed an increased tan δ peak temperature and broadened transition peak, indicating restrained PHBV molecular mobility in the vicinity of the CNW surface. Storage modulus of the PHBV also increased with the addition of CNW, especially at the temperatures higher than the PHBV glass transition temperature. These results indicated that the CNW could substantially increase the mechanical properties of PHBV and this increase could be attributed to the strong interactions between these two phases.     

Miscibility and crystallization behavior of biodegradable blends of two aliphatic polyesters. Poly(3-hydroxybutyrate-co-hydroxyvalerate) and poly(ε-caprolactone)

Biodegradable polymer blends of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) blends were prepared with the ratio of PHBV/PCL ranging from 80/20-20/80 by co-dissolving the two polyesters in chloroform and casting the mixture. Differential scanning calorimetry (DSC) and optical microscopy (OM) were used to investigate the miscibility and crystallization of PHBV/PCL blends. Experimental results indicated that PHBV showed no miscibility with PCL for PHBV/PCL blends as evidenced by the existence of unchanged composition independent glass transition temperature and the biphasic melt. Crystallization of PHBV and PCL was studied with DSC and analyzed by the Avrami equation by using two-step crystallization in the PHBV/PCL blends. The crystallization rate of PHBV at 70 °C decreased with the increase of PCL in the blends, while the crystallization mechanism did not change. In the case of the isothermal crystallization of PCL at 42 °C, the crystallization rate increased with the addition of PHBV, and the crystallization mechanism changed, too, indicating that the crystallization of PHBV at 70 °C had an apparent influence on the crystallization of PCL at 42 °C.     

骨组织工程用PLGA多孔支架的制备及细胞毒性研究

制备能在骨组织工程研究中应用,并具有良好孔隙结构的块状聚（D,L-乳酸-CO-乙醇酸）（PLGA）多孔支架,探索出以冰粒子作为致孔剂,采用粒子滤出方法结合冷冻干燥工艺制备多孔支架的方法.首先将冰颗粒加入预冻的PLGA氯仿溶液中混合均匀,然后把混合物置于液氮中深度冷冻后冷冻干燥,制得多孔支架.对支架孔隙结构分析表明,该工艺制备的多孔支架无致孔剂残留、三维结构良好、孔径与孔隙可通过改变冰粒子的粒径和质量分数来控制;细胞毒性实验表明该多孔支架毒性在0～1级,可作为骨组织工程研究用多孔支架.     

Miscibility and crystallization behaviour of biodegradable blends of two aliphatic polyesters. Poly(3-hydroxybutyrate-co-hydroxyvalerate) and poly(butylene succinate) blends

Blends of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(butylene succinate) (PBSU), both biodegradable semicrystalline polyesters, were prepared with the ratio of PHBV/PBSU ranging from 80/20 to 20/80 by co-dissolving the two polyesters in chloroform and casting the mixture. Differential scanning calorimetry (DSC) and optical microscopy (OM) were used to study the miscibility and crystallization behaviour of PHBV/PBSU blends. Experimental results indicate that PHBV is immiscible with PBSU as shown by the almost unchanged glass transition temperature and the biphasic melt. Crystallization of PHBV/PBSU blends was studied by DSC using two-step crystallization and analyzed by the Avrami equation. The crystallization rate of PHBV decreases with the increase of PBSU in the blends while the crystallization mechanism does not change. In the case of the isothermal crystallization of PBSU, the crystallization mechanism does not change. The crystallization rate of PBSU in the blends is lower than that of neat PBSU; however, the change in the crystallization rate of PBSU was not so big in the blends. The different content of the PHBV in the blends does not make a significant difference in the crystallization rate of PBSU.     

Cellular Response of Limbal Stem Cells on Poly (Hydroxybuthyrate-co-Hydroxyvalerate) Porous Scaffolds for Ocular Surface Bioengineering

The aim of this study was to develop a modified-porous poly (3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffold for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human limbal stem cell was used to evaluate the biocompatibility of substrates (porous scaffold, human amniotic membrane and thermoresponsive substrate) based on their viability, proliferation, and attachment ability. Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the scaffold surface. Microscopic figures showed that the human limbal stem cell was firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the porous scaffold and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Gene expression results revealed no change in the expression profile of LECs grown on scaffold when compared to those grown on human amniotic membrane or thermo responsive substrate. In addition, porous PHBV substrate provides not only a milieu supporting LSCs expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.