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

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

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.     

Fabrication and Characterization of Hydroxyapatite-Coated Polystyrene Disks for Use in Osteoprogenitor Cell Culture

A simple method is reported for fabricating polystyrene disk inserts coated with biomimetic carbonated hydroxyapatite (cHA) to be used for culturing osteoprogenitor cells or other stem cells. Roughened disks cut from tissue-culture polystyrene (TCPS) were coated in simulated body fluid with 5 × normal physiologic ionic concentrations (SBFx5) by a 2-step, 2-day method. The coatings were rigorously characterized by various methods and assessed in cell culture. An adherent, nearly 10 mm thick, relatively uniform layer of single-phase cHA was formed in two days. MC3T3-E1 and mouse calvaria-derived osteoprogenitor cells (pCOBs) were cultured on the cHA for various time points. Despite less initial attachment of both cell types to the cHA, proliferation rates on cHA were similar to that on TCPS. Two-fold greater cell attachment (P < 0.05) of the MC3T3-E1 cells was observed relative to the pCOBs, on both the TCPS and the cHA. Importantly, the coatings were relatively smooth, without the extensive agglomerates observed in other studies and remained adherent and morphologically unchanged after 21 days of culture. This technique can be used to rapidly produce high-quality cHA-coated TCPS disks for cell-culture studies.     

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-l-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.     

Exposed hydroxyapatite particles on the surface of photo-crosslinked nanocomposites for promoting MC3T3 cell proliferation and differentiation

We present a systematic study for investigating the role of exposed hydroxyapatite (HA) nanoparticles in influencing surface characteristics and mouse pre-osteoblastic MC3T3-E1 cell behavior using nanocomposites prepared by photo-crosslinking poly(ε-caprolactone) diacrylate (PCLDA) with HA. PCLDA530 and PCLDA2000 synthesized from poly(ε-caprolactone) diol precursors with nominal molecular weights of 530 and 2000 g mol(-1) were used as the polymer matrices. Crosslinked PCLDA530 was amorphous while crosslinked PCLDA2000 was semi-crystalline. Crosslinked PCLDA/HA composites with different compositions of HA (10%, 20% and 30%) as well as crosslinked PCLDAs were characterized in terms of their composition-dependent physicochemical properties. The tensile, compressive and shear moduli were greatly enhanced by incorporating HA nanoparticles with the polymer matrices. The disk surfaces of original crosslinked PCLDA/HA nanocomposites were removed by cutting using a blade to expose HA nanoparticles that were embedded in the polymer substrates. The composition of HA was much higher on the cut surface, particularly in semi-crystalline crosslinked PCLDA2000/HA nanocomposites. The surface characteristics of original and cut crosslinked PCLDA/HA nanocomposites were compared and correlated with cell behavior on these nanocomposites. MC3T3-E1 cell attachment, proliferation and differentiation were significantly enhanced when the HA composition was increased in original crosslinked PCLDA/HA nanocomposites due to more bioactive HA, higher surface stiffness and rougher topography. More exposed HA on the surface of cut semi-crystalline PCLDA2000/HA nanocomposites resulted in improved hydrophilicity and significantly better MC3T3 cell attachment, proliferation and differentiation compared with the original surfaces. This study suggests that HA nanoparticles may not be fully exploited in polymer/HA nanocomposites where the top polymer surface covers the particles. The removal of this polymer layer can generate more desirable surfaces and osteoconductivity for bone repair and regeneration.     

MC3T3-E1细胞与多组分纳米羟基磷灰石基三维复合支架材料的相容性

目的 观察MC3T3-E1细胞在复合支架材料上的黏附、增殖及形态,评价多组分纳米羟基磷灰石基三维复合支架材料的生物相容性.方法 采用仿生学方法,将壳聚糖、羟基磷灰石、明胶、果胶按照一定比例制作成多组分纳米羟基磷灰石基三维复合支架材料.在复合支架材料上接种MC3T3-E1细胞,通过倒置相差显微镜、HE染色、扫描电镜、四甲...     

Fabrication and characterization of modified nanofibrous poly(L-lactic acid) scaffolds by thermally induced phase separation technique and aminolysis for promoting cyctocompatibility

Modified nanofibrous Poly(L-lactic acid) (PLLA) scaffolds were fabricated by aminolysis combined with thermally induced phase separation technique using PLLA/1,4-dioxane/urea-NaOH-H₂O system at ?40 °C freeze temperature. Aminolysis led to the modification of scaffold resulting in enhancement in the bioactivity. The surface of the modified nanofibrous scaffold provided a good environment for attachment and proliferation of MC3T3-E1 subclone 14 cells, exhibiting significant potential for bone tissue regeneration and for promoting cytocompatibility.     

The precision structural regulation of PLLA porous scaffold and its influence on the proliferation and differentiation of MC3T3-E1 cells

A thermal-induced phase separation combined sugar template method was used to fabricate the Poly (L-lactide) acid (PLLA) scaffolds with precisely regulated porous structure. The effect of tuned porous structure of scaffolds on osteoblasts proliferation and differentiation was investigated. The results showed that the pore diameters (200–300, 300–400, 400–500 μm), porosity and interconnectivity of PLLA scaffolds can be accurately controlled indicated by scanning electron microscope. The results of cell experiments showed that the porous structure including the pore size and interconnectivity of scaffolds dramatically influence the cell proliferation and differentiation. The scaffold with pore diameter of 400–500 μm exhibited the highest cell viability and alkaline phosphatase activity among all the scaffolds for the MC3T3-E1 cells. The higher cell proliferation and biocompatibility observed in the 400–500 μm scaffold indicated the high selectivity for MC3T3-E1cells on the pore size of scaffold in tissue engineering. The precise control of the porous structure of scaffold may better guide the cell–matrix interaction in the future research.     

Electrospinning of aniline pentamer-graft-gelatin/PLLA nanofibers for bone tissue engineering

Blends of aniline pentamer-graft-gelatin (AP-g-GA) and poly(l-lactide) (PLLA) were electrospun to prepare uniform nanofibers as biomimetic scaffolds. The nanofibers exhibited good electroactivity, thermal stability and biodegradability. The biocompatibility of the nanofibers in vitro was evaluated by the adhesion and proliferation of mouse preosteoblastic MC3T3-E1 cells. The cellular elongation was significantly greater on electroactive AP-g-GA/PLLA nanofibers than on PLLA nanofibers. Moreover, the AP-g-GA/PLLA nanofibers stimulated by an electrical pulsed signal could promote the differentiation of MC3T3-E1 cells compared with pure PLLA nanofibers. Our results demonstrated that the biodegradable and electroactive AP-g-GA/PLLA nanofibers had potential application in vivo as bone repair scaffold materials in tissue engineering.     

低强度脉冲超声波作用下羟基磷灰石/磷酸三钙三维支架上成骨细胞的生物学行为的研究

目的评价低强度脉冲超声波对羟基磷灰石(HA)/磷酸三钙(TCP)三维多孔支架材料上的MC3T3-E1成骨细胞的生物学行为的影响。方法将鼠MC3T3-E1成骨细胞与HA/TCP支架材料复合培养4 d和7 d。超声组每天接受20 min的低强度脉冲(1 MHz)超声波辐照,对照组为不开功率源的假辐照。通过观察成骨细胞在支架材料上的附着和细胞活力,检测细胞长入支架深度、DNA含量等指标评价低强度脉冲超声波对成骨细胞生物学行为的影响。结果 14 d和7 d时超声组HA/TCP支架材料上成骨细胞生长密度高于对照组。24 d和7 d时超声组HA/TCP支架材料上成骨细胞长入深度较对照组分别增加了34.45%和23.45%,4 d时二者差异无统计学意义(P=0.057),而7 d差异有统计学意义(P=0.032)。34 d和7 d时超声组较对照组DNA含量分别增加了27.54%(P=0.000)和24.39%(P=0.000),差异均有显著统计学意义。结论加载低强度脉冲超声波有利于成骨细胞在HA/TCP支架材料上的生长与增殖。     

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

Abstract

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

Characterization of a novel composite scaffold consisting of acellular bladder submucosa matrix,polycaprolactone and Pluronic F127 as a substance for bladder reconstruction

The bladder is an organ susceptible to a variety of congenital anomalies, injuries and disorders. To address the clinical limitations of existing scaffolds, we fabricated a novel scaffold that can be applied to morphological and functional bladder reconstruction. As a first step to prove the benefit of the scaffold, intensive in vitro and in vivo analyses were conducted. The novel composite scaffold was fabricated using polycaprolactone/Pluronic F127 (PCL/F127) and variable proportions (1, 3, 5 and 10 wt.%) of porcine acellular bladder submucosa matrix (BSM). Physicochemical properties and biocompatibilities of the scaffolds were characterized. For cell-mediated analysis, upper-urinary-tract-derived urine stem cells were used. Observations of tensile strength, modulus, porosity, cell adhesion, viability and proliferation characteristics of scaffolds indicated that the optimum proportion of BSM in the composite scaffolds was 3 or 5 wt.%. Based on comparison of 3 and 5 wt.% BSM/PCL/F127 scaffolds with respect to degradability, hydrophilicity, surface properties and functional group presence, the 3 wt.% BSM was chosen for in vivo studies. 8 weeks after kidney-subcapsular implantation of the 3 wt.% BSM/PCL/F127 scaffold, cells remained attached to the surface and there was no evidence of teratomas. A BSM content of 3 wt.% was the optimum proportion for fabrication of the neo scaffold. We predict that the 3 wt.% BSM/PCL/F127 composite scaffold could act as an ideal matrix after cystectomy based on its favorable physicochemical properties and biocompatibilities.     

Preparation and characterization of nano-hydroxyapatite/chitosan composite scaffolds

A novel nano-hydroxyapatite (HA)/chitosan composite scaffold with high porosity was developed. The nano-HA particles were made in situ through a chemical method and dispersed well on the porous scaffold. They bound to the chitosan scaffolds very well. This method prevents the migration of nano-HA particles into surrounding tissues to a certain extent. The morphologies, components, and biocompatibility of the composite scaffolds were investigated. Scanning electron microscopy, porosity measurement, thermogravimetric analysis, X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier transformed infrared spectroscopy were used to analyze the physical and chemical properties of the composite scaffolds. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 cells seeded on the scaffolds. The composite scaffolds showed better biocompatibility than pure chitosan scaffolds. The results suggest that the newly developed nano-HA/chitosan composite scaffolds may serve as a good three-dimensional substrate for cell attachment and migration in bone tissue engineering.     

Fabrication and characterization of chitosan/OGP coated porous poly(ε-caprolactone) scaffold for bone tissue engineering

As one of the stimulators on bone formation, osteogenic growth peptide (OGP) improves both proliferation and differentiation of the bone cells in vitro and in vivo. The aim of this work was the preparation of three dimensional porous poly(ε-caprolactone) (PCL) scaffold with high porosity, well interpore connectivity, and then its surface was modified by using chitosan (CS)/OGP coating for application in bone regeneration. In present study, the properties of porous PCL and CS/OGP coated PCL scaffold, including the microstructure, water absorption, porosity, hydrophilicity, mechanical properties, and biocompatibility in vitro were investigated. Results showed that the PCL and CS/OGP-PCL scaffold with an interconnected network structure have a porosity of more than 91.5, 80.8%, respectively. The CS/OGP-PCL scaffold exhibited better hydrophilicity and mechanical properties than that of uncoated PCL scaffold. Moreover, the results of cell culture test showed that CS/OGP coating could stimulate the proliferation and growth of osteoblast cells on CS/OGP-PCL scaffold. These finding suggested that the surface modification could be a effective method on enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering application and the developed porous CS/OGP-PCL scaffold should be considered as alternative biomaterials for bone regeneration.     

Shell-core bi-layered scaffolds for engineering of vascularized osteon-like structures

Bottom-up assembly of osteon-like structures into large tissue constructs represents a promising and practical strategy toward the formation of hierarchical cortical bone. Here, a unique two-step approach, i.e., the combination of electrospinning and twin screw extrusion (TSE) techniques was used to fabricate a microfilament/nanofiber shell–core scaffold that could precisely control the spatial distribution of different types of cells to form vascularized osteon-like structures. The scaffold contained a helical outer shell consisting of porous microfilament coils of polycaprolactone (PCL) and biphasic calcium phosphates (BCP) that wound around a hollow electrospun PCL nanofibrous tube (the core). The porous helical shell supported the formation of bone-like tissues, while the luminal surface of nanofibrous core enabled endothelialization to mimic the function of Haversian canal. Culture of mouse pre-osteoblasts (POBs, MC 3T3-E1) onto the coil shells revealed that coils with pitch sizes greater than 135 μm, in the presence of BCP, favored the proliferation and osteogenic differentiation of POBs. The luminal surface of PCL nanofibrous core supported the adhesion and spreading of mouse endothelial cells (ECs, MS-1) to form a continuous endothelial lining with the function similar to blood vessels. Taken together, the shell–core bi-layered scaffolds with porous, coil-like shell and nanofibrous tubular cores represent a new scaffolding technology base for the creation of osteon analogs.     

Physical properties and cellular responses to crosslinkable poly(propylene fumarate)/hydroxyapatite nanocomposites

A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity.     

A mesoporous bioactive glass/polycaprolactone composite scaffold and its bioactivity behavior

Composite scaffolds of mesoporous bioactive glass (MBG)/polycaprolactone (PCL) and conventional bioactive glass (BG)/PCL were fabricated by a solvent casting-particulate leaching method, and the structure and properties of the composite scaffolds were characterized. The measurements of the water contact angles suggest that the incorporation of either MBG or BG into PCL can improve the hydrophilicity of the composites, and the former is more effective than the later. The bioactivity of the composite scaffold is evaluated by soaking the scaffolds in a simulated body fluid (SBF) and the results show that the MBG/PCL composite scaffolds can induce a dense and continuous layer of apatite after soaking in SBF for 3 weeks, as compared with the scattered and discrete apatite particles on the BG/PCL composite scaffolds. Such improvements (improvements of the hydrophilicity and apatite forming ability) should be helpful for the extensive applications of PCL scaffold in tissue engineering.     

Cellular Migration to Electrospun Poly(Lactic Acid) Fibermats

Abstract

Nonwoven fabrics prepared via an electrospinning method, so-called electrospun fibermats, are expected to be promising scaffold materials for bone tissue engineering. In the present work, poly(L-lactic acid) (PLLA) fibermats, consisting of fibers with diameters ranging from 1 to 10 μm, were prepared by electrospinning. Mouse osteoblast-like cells (MC3T3-E1) were seeded on the fibermats with various fiber diameters (10, 5 and 2 μm; they are denoted by samples A, B and C, respectively) and cultured in two different directions in order to compare the migration behaviours into the scaffold of the normal condition and the anti-gravity condition. The cells in/on the fibermats were observed by laser confocal microscopy to estimate the cellular migration ability into them. When the MC3T3-E1 cells were cultured in the normal direction, the thickness of their layer increased to approx. 90 μm in the sample A, consisting of 10-μm fibers after 13 days of culture, while that in the sample C, consisting of 2-μm fibers, did not increase. When the MC3T3-E1 cells were cultured in the anti-gravity condition, the thickness of the cell layer in the sample A increased to approx. 60 μm. These results mean that the MC3T3-E1 cells migrated into the inside of sample A in either the normal direction or the anti-gravity one. The cellular proliferation showed no significant difference among the fibermats with three different fiber diameters; MC3T3-E1 cells on the fibermat with 2 μm fiber diameter grew two-dimensionally, while they grew three-dimensionally in the fibermat with 10 μm fiber diameter.     

Porous alginate/poly(ε-caprolactone) scaffolds: preparation, characterization and in vitro biological activity

In bone tissue engineering, scaffolds with controlled porosity are required to allow cell ingrowth, nutrient diffusion and sufficient formation of vascular networks. The physical properties of synthetic scaffolds are known to be dependent on the biomaterial type and its processing technique. In this study, we demonstrate that the separation phase technique is a useful method to process poly(ε-caprolactone) (PCL) into a desired shape and size. Moreover, using poly(ethylene glycol), sucrose, fructose and Ca2+ alginate as porogen agents, we obtained PCL scaffolds with three-dimensional porous structures characterized by different pore size and geometry. Scanning electron microscopy and porosity analysis indicated that PCL scaffolds prepared with Ca2+ alginate threads resemble the porosity and the homogeneous pore size distribution of native bone. In parallel, MicroCT analysis confirmed the presence of interconnected void spaces suitable to guarantee a biological environment for cellular growth, as demonstrated by a biocompatibility test with MC3T3-E1 murine preosteoblastic cells. In particular, scaffolds prepared with Ca2+ alginate threads increased adhesion and proliferation of MC3T3-E1 cells under basal culture conditions, and upon stimulation with a specific differentiation culture medium they enhanced the early and later differentiated cell functions, including alkaline phosphatase activity and mineralized extracellular matrix production. These results suggest that PCL scaffolds, obtained by separation phase technique and prepared with alginate threads, could be considered as candidates for bone tissue engineering applications, possessing the required physical and biological properties.     

Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique

Aiming to develop a scaffold architecture mimicking morphological and mechanically that of a blood vessel, a sequential multi-layering electrospinning (ME) was performed on a rotating mandrel-type collector. A bi-layered tubular scaffold composed of a stiff and oriented PLA outside fibrous layer and a pliable and randomly oriented PCL fibrous inner layer (PLA/PCL) was fabricated. Control over the level of fibre orientation of the different layers was achieved through the rotation speed of the collector. The structural and mechanical properties of the scaffolds were examined using scanning electron microscopy (SEM) and tensile testing. To assess their capability to support cell attachment, proliferation and migration, 3T3 mouse fibroblasts and later human venous myofibroblasts (HVS) were cultured, expanded and seeded on the scaffolds. In both cases, the cell-polymer constructs were cultured under static conditions for up to 4 weeks. Environmental-scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), histological examination and biochemical assays for cell proliferation (DNA) and extracellular matrix production (collagen and glycosaminoglycans) were performed. The findings suggest the feasibility of ME to design scaffolds with a hierarchical organization through a layer-by-layer process and control over fibre orientation. The resulting scaffolds achieved the desirable levels of pliability (elastic up to 10% strain) and proved to be capable to promote cell growth and proliferation. The electrospun PLA/PCL bi-layered tube presents appropriate characteristics to be considered a candidate scaffold for blood vessel tissue engineering.