Evaluation of type II collagen scaffolds reinforced by poly(epsilon-caprolactone) as tissue-engineered trachea

A novel composite scaffold comprising a poly(epsilon-caprolactone) (PCL) stent and a type II collagen sponge for tissue-engineered trachea was developed. The PCL stent with surface grooves was fabricated by casting and freeze drying the PCL solution in a mold container. The grooves on the stent were filled by the type II collagen with crosslinking treatment (ring-shaped collagen sponge). The rabbit chondrocytes (3 x 10(6) cells for each ring) were seeded onto the collagen sponge of the scaffold. The cell-scaffold constructs were implanted subcutaneously in the dorsum of nude mice. After 4 and 8 weeks, constructs were harvested and dedicated for measurement of mechanical properties, histology, and biochemical assays. It was found that the constructs were strong enough to retain their tubular shape against extrinsic forces in the dorsum of nude mice. The gross appearance of the constructs revealed cartilage-like tissue at 8 weeks, with modulus higher than that of native trachea. Histological and biochemical analyses of the tissue-engineered tracheal cartilage revealed evenly spaced lacunae embedded in the matrix, with abundant proteoglycans and type II collagen. The stent-sponge composite facilitated the proliferation of chondrocytes and was expected to provide adequate mechanical strength, and therefore was a promising material for use in trachea tissue engineering.     

Permeation of protein from porous poly(epsilon-caprolactone) films

The objective of this study was designed to extend the application of poly(epsilon-caprolactone) (PCL) in delivery of macromolecular proteins. The strategy applied here is to create a porous structure in PCL films in order to control the diffusion rate of protein. Various amounts of both high-molecular-weight and low-molecular-weight poly(ethylene glycol) (PEG) were used as pore-forming agents. The porous films were prepared by a solvent-casting-leaching method. The thicknesses of the prepared films were controlled to be in the range of 75.3 +/- 0.6 similar 81.7 +/- 0.6 mum. The pore fraction of films was determined to be 27.7 +/- 1.0% similar 52.5 +/- 0.8% for PEG(10000) and 26.6 +/- 1.8% similar 48.8 +/- 1.4% for PEG(4000). The pore fraction initially increased with increasing amounts of PEG, independent of the molecular weight of PEG. In the permeation study, lysozyme was used as a model diffuser. The permeation rate of protein increased as the pore fraction of films increased, especially when 30 similar 40% of PEG was added initially, and this phenomenon was more prominent when low-molecular-weight PEG was used. This result was probably due to the highly porous structure creating interconnected channels in the films, further enhancing protein diffusion. In addition, the size of micropores formed by PEG(4000) was observed to be larger than by PEG(10000), which also accounted for faster permeation rate of lysozyme through PCL-PEG(4000) porous films.     

Development of perforated microthin poly(epsilon-caprolactone) films as matrices for membrane tissue engineering

The design and fabrication of thin films based on bioresorbable polymers such as poly(epsilon-caprolactone) (PCL) has been the focus of a part of current biomedical research, especially as matrices for membrane tissue engineering. We have successfully developed perforated microthin PCL membrane for this purpose. Two critical issues are the control of moisture permeability and understanding the degradation of PCL microthin film. In order to increase the moisture permeability. PCL films were biaxially stretched to a thickness of 10 +/- 3 microm and perforated with uniform array of holes (180-275 microm) using a Sony Robotic system. After perforation, the water vapour transmission rate was increased by 50% to a value of 47.6 +/- 2.7 g/h per m2. Accelerated hydrolytic degradations were performed in 5 M NaOH. The degraded samples were characterised for changes in weight, surface morphology, mechanical properties, crystallinity and molecular weight. Hydrolytic degradation commenced with random chain scission of backbone ester bonds on the film surface and followed by loss of material due to surface erosion. In general, the perforated films degraded faster than the unperforated microthin films. Scanning electron microscopic images showed that surface erosion led to extensive formation of micropores, microcracks and increased in surface roughness.     

Transitory oxidative stress in L929 fibroblasts cultured on poly(epsilon-caprolactone) films

Poly(epsilon-caprolactone) (PCL) is considered as a potential substrate for wide medical applications. In previous studies we carried out the in vitro biocompatibility assessment of PCL films using L929 mouse fibroblasts, obtaining good cell behaviour but a transitory stimulation of mitochondrial activity and cell retraction. Reactive oxygen species (ROS), mainly formed in mitochondria, can impair the function of several cellular components and produce cell oxidative stress by changing the normal red-ox status of the major cell antioxidants as glutathione. The aim of this study was to measure intracellular ROS production and glutathione content of L929 fibroblasts cultured on PCL films. Cell size, internal complexity, cell cycle and lactate dehydrogenase release were also evaluated. The films were treated with NaOH before culture to improve the cell-polymer interaction. PCL induces a transitory but significant oxidative stress in L929 fibroblasts. The treatment of PCL films with NaOH reduces this effect. PCL also induces transitory changes on cell size and complexity. Nevertheless, after 7 days in culture, cells reach control levels for all the studied parameters. Neither cell cycle nor membrane integrity appears affected by this oxidative stress respect to control cells at any culture time. These results underline the cytocompatibility of PCL films and, therefore, its potential utility as a suitable scaffold in tissue engineering.     

The intracellular degradation of poly(epsilon-caprolactone)

Poly(epsilon-caprolactone) [PEC], a biodegradable aliphatic polyester, undergoes a two-stage degradation process: The first lengthy phase involves nonenzymatic hydrolytic cleavage of ester groups, the second phase beginning when the polymer is more highly crystalline, and of low molecular weight. The cellular events of the second phase were examined by implanting gelatin capsules containing 25 mg of low molecular weight (Mn 3000) PEC powders, 106 to 500 micron, in rats. PEC fragments ultimately were degraded in phagosomes of macrophages and giant cells, the process requiring less than 13 days for completion at some sites. PEC was also identified within fibroblasts. These studies support the intracellular degradation of PEC as the principal pathway of degradation once the molecular weight of the aged polymer is reduced to 3000 or less.     

One-step preparation of poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) nanoparticles for plasmid DNA delivery

In this article, a kind of biodegradable poly(epsilon-caprolactone)-Poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL, PCEC) copolymer was synthesized by ring-opening polymerization method. The PCEC nanoparticles were prepared at one-step by modified emulsion solvent evaporation method using CTAB as stabilizer. With increase in PCEC concentration, the particle size increased obviously, but zeta potential only increased slightly. The obtained cationic PCEC nanoparticle was employed to condense and adsorb DNA onto its surface. Plasmid GFP (pGFP) was used as model plasmid to evaluate the loading capacity of cationic PCEC nanoparticles in this work. The DNA/nanoparticles weight ratio at 1:16 induced almost neutral zeta potential of DNA-nanoparticles complex. At this time, the size of complex became abnormally large which implied aggregates formed. So DNA-nanoparticles weight ratio should be chosen carefully. The cationic PCEC nanoparticles had the capacity of condensing plasmid DNA into complex when the DNA/nanoparticles weight ratio was lower than 1:8, which was evidenced by gel retardation assay. In vitro release behavior of DNA/nanoparticle complexes was also studied here. The obtained cationic PCEC nanoparticles might have great potential application in DNA delivery.     

Alkaline-treated poly(epsilon-caprolactone) films: degradation in the presence or absence of fibroblasts

In the first stage, we observed the study of the degradation behavior of alkaline-treated poly(epsilon-caprolactone) (PCL) in two biologically-related media: phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) for 18 months, finding a much accelerated degradation in the last one. As expected, the degradation in the presence of cells is much pronounced even considering that the study is limited to 6 months. The characterization of the degraded substrates by chemiluminescence (CL) allows to explain the modifications of the substrate and their relations with transitory oxidative stress phenomena described in the fibroblasts seeded onto the PCL membranes.     

In vitro biocompatibility assessment of poly(epsilon-caprolactone) films using L929 mouse fibroblasts

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing vascular grafts, the in vitro biocompatibility of poly(epsilon-caprolactone) (PCL), recently suggested for several clinical applications, was evaluated in this study using L929 mouse fibroblasts. Different cellular aspects were analyzed in order to know the cell viability during cell culture on PCL films: adhesion, proliferation, morphology, LDH release and mitochondrial function. Since topography and other surface characteristics of materials play an essential part in cell adhesion, PCL membranes with either smooth or rough surface were prepared, characterized and used to carry out cell cultures. During short culture times, PCL produced a significant stimulation of mitochondrial activity evaluated by reduction of the MTT reagent. The results provide evidences of good adhesion, growth, viability, morphology and mitochondrial activity of cells on PCL films. Therefore, it can be concluded that PCL is a suitable and biocompatible material as a scaffold for vascular graft development.     

Surface modification of ultra thin poly (epsilon-caprolactone) films using acrylic acid and collagen

Poly (epsilon-caprolactone) (PCL) has been used as a bioresorbable polymer in numerous medical devices as well as for tissue engineering applications. Its main advantage is its biocompatibility and slow degradation rate. PCL surface, however, is hydrophobic and cell-biomaterial interaction is not the best. We attempt for the first time to modify an ultra thin PCL surface with collagen. The PCL film was prepared using solvent casting and biaxial stretching technique developed in our laboratory. This biaxial stretching produced an ultra thin PCL 3-7 microm thick, ideal for membrane tissue engineering applications. The PCL film was pretreated using Argon plasma, and then UV polymerized with acrylic acid (AAc). Collagen immobilization was then carried out. The modified film surface was characterized by Fourier Transform Infrared (FT-IR) and X-ray Photoelectron Spectroscopy (XPS). Water contact angles were also measured to evaluate the hydrophilicity of the modified surface. Results showed that the hydrophilicity of the surface has improved significantly after surface modification. The water contact angle dropped from 66 degrees to 32 degrees. Atomic Force Microscopy (AFM) showed an increase in roughness of the film. A change from 46 to 60 nm in the surface morphology was also observed. The effect of cells attachment on the PCL film was studied. Human dermal fibroblasts and myoblasts attachment and proliferation were improved remarkably on the modified surface. The films showed excellent cell attachment and proliferation rate.     

BHK cell attachment and growth on EDA-plasma-modified poly(L-lactide/epsilon-caprolactone) biodegradable films

In this study, attachment and growth of Baby Hamster Kidney (BHK) cells on ethylene diamine (EDA)-plasma-treated poly(L-lactide/epsilon-caprolactone) biodegradable copolymer films were investigated. The co-polymer (Mw: 58000; Mn: 35000 and PI 1.60) was synthesised by ring-opening polymerization of the respective dimers with using stannous octoate as the catalyst. The final ratio of L-lactide to epsilon-caprolactone obtained by 1H-NMR was 87:13. The co-polymer films were treated with the EDA-plasma in a glow-discharge apparatus. The BHK-30 cell line was cultured on plain and EDA-plasma-treated films and their pre-wetted forms (with ethanol and/or cell culture medium before use). Cell attachment and growth were followed. Alkaline phosphatase (ALP) activity and glucose uptake in cell culture medium were also investigated. There was no attachment in the first 12 h. Glow-discharge treatment increased significantly the attachment and growth. Pre-wetting with ethanol and cell culture medium was also increase significantly both the attachment and growth.     

Evaluation of cell affinity on poly(L-lactide) and poly(epsilon-caprolactone) blends and on PLLA-b-PCL diblock copolymer surfaces

An evaluation of cell proliferation and adhesion on biocompatible film supports was performed. A series of films were compression molded from commercially available poly (L-lactide), PLLA, and poly(epsilon-caprolactone), PCL, and from their melt mixed blends (PLLA/PCL blends). These were compared with compression molded films of PLLA-b-PCL model diblock copolymers. The samples were analyzed by differential scanning calorimetry (DSC), contact angle measurements, and scanning force microscopy (SFM). Cell adhesion and proliferation were performed with monkey derived fibroblasts (VERO) and with osteoblastic cells obtained either enzymatically or from explants cultures of Sprague-Dawley rat calvaria. Migration studies were performed with bone explants of the same origin. The results obtained indicate that although all materials tested were suitable for the support of cellular growth, a PLLA-b-PCL diblock copolymer sample with 93% PLLA was significantly more efficient. This sample exhibited a unique surface morphology with long range ordered domains (of the order of 2-3 mum) of edge-on PLLA lamellae that can promote "cell contact guidance." The influence of other factors such as chemical composition, degree of crystallinity, and surface roughness did not play a major role in determining cell preference toward a specific surface for the materials employed in this work.     

Phase transfer and characterization of poly(epsilon-caprolactone) and poly(L-lactide) microspheres

A method suitable for transfer of poly(epsilon-caprolactone) and poly(L-lactide) microspheres (synthesized by pseudoanionic dispersion polymerization of epsilon-caprolactone and L-lactide in heptane-1,4-dioxane mixed solvent) from heptane to water was developed. This method consists of treating the microspheres with KOH-ethanol in the presence of surfactants (nonionic Triton X-405, anionic sodium dodecyl sulfate (SDS), and zwitterionic ammonium sulfobetaine-2 (ASB)). Partial hydrolysis of polyesters results in the formation of hydroxyl and carboxyl groups in the surface layer of microspheres and enhances their stability in water-based media. Minimal concentrations of surfactants, needed to obtain stable suspensions of particles, were equal to 3 x 10(-2) and 6 x 10(-2), and 3 x 10(-2) mol l(-1) for Triton X-405. SDS, and ASB, respectively. In the case of poly(epsilon-caprolactone) microspheres, suspensions in water were stable for all three surfactants for pH values ranging from 3 to 11. Suspensions of poly(L-lactide) were stable in the same range of pH values only for ASB. Surface charge density determined by electrophoretic mobility varied for poly(epsilon-caprolactone) microspheres from 2.6 x 10(-7) to 8.9 x 10(-7) mol m(-2), for particles stabilized with Triton X-405 and ASB. respectively. In the case of poly(L-lactide) microspheres, surface charge density varied from 3.9 x 10(-7) (stabilizer: Triton X-405) to 7.4 x 10(-7) mol m(-2) (stabilizer: ASB). Carboxyl groups located in the surface layer of poly(L-lactide) microspheres were used for covalent immobilization of 6-aminoquinoline, a fluorophore with an amino group. Maximum surface concentration of immobilized 6-aminoquinoline was equal to 1.9 x 10(-6) mol m(-2). Poly(epsilon-caprolactone) microspheres transferred into water were loaded with ethyl salicylate. Loading up to 38% (w/w) was obtained.     

Chitosan/poly(epsilon-caprolactone) blend scaffolds for cartilage repair

Chitosan (CHT)/poly(?-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. μCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation.     

Biodegradable poly(ethylene oxide)/poly(epsilon-caprolactone) multiblock copolymers.

A series of poly(ethylene oxide) (PEO)/poly(epsilon-caprolactone) (PCL) containing biodegradable poly(ether ester urethane)s, covering a wide range of compositions, were synthesized and characterized. The synthesis consisted of a two-step process. During the first step, the ring-opening reaction of epsilon-caprolactone was carried out, initiated by the hydroxyl terminal groups of the PEO chain. The second step involved the chain extension of these PCL-PEO-PCL trimers with hexamethylene diisocyanate. By varying either the ethylene oxide/epsilon-caprolactone ratio or the length of both segments, we obtained a series of polymers having different morphologies and displaying a broad range of properties.     

Bone-like apatite-forming ability and mechanical properties of poly(epsilon-caprolactone)/silica hybrid as a function of poly(epsilon-caprolactone) content

Effect of poly(epsilon-caprolactone) (PCL) content on the bioactivity and mechanical properties of PCL/silica hybrid was investigated. The PCL/silica hybrids with different PCL contents were prepared through co-condensation reaction with triethoxysilane end capped PCL and tetraethyl orthosilicate. The higher the PCL content in the hybrid, the lower the apatite-forming rate and showed polymer-like ductile-tough fracture behavior. On the contrary, the lower the PCL content in the hybrid, the higher the apatite-forming rate and showed ceramic-like hard-brittle fracture behavior. At the intermediate PCL content, the apatite-forming rate and its mechanical properties showed also intermediate behaviors. The highest tensile strength and Young's modulus could be obtained at intermediate PCL content and they were around 20 and 600 MPa, respectively, while the strain at failure was around 50%. This new kind of hybrid material is likely to have the potential to be used as a bone repairing material because of its apatite-forming ability and the mechanical properties comparable to human cancellous bone.     

Laser surface modification of poly(epsilon-caprolactone) (PCL) membrane for tissue engineering applications

Ultra-thin polycaprolactone (PCL) produced by bi-axial stretching was previously shown to have significant advantage for membrane tissue engineering. However, the permeability of the membrane needs to be enhanced. In this study, ablation experiments using femtosecond laser and excimer laser were carried out to modify the PCL surface. The use of the femtosecond laser produces neat drilled-through holes while the excimer laser is employed to produce blind-holes on the membrane. The modified surface of the membrane was studied and analyzed for different laser parameters (such as pulse energy and pulse repetition rate and characterized using several techniques that include optical microscopy, scanning electron microscopy and water contact angle measurements). Results showed that the morphological surface changes with different laser parameters, and the water contact angle decreases as the surface of the membrane is modified. The decrease in water contact angle suggests that surface of the membrane had become more hydrophilic than the non-laser treated membrane. The present study demonstrated that laser surface modification on the PCL can be achieved with high degree of success and precision. This paved the way for further enhancement in membrane tissue engineering.     

Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) ultra-thin films

An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and—in general—in the field of bio-hybrid devices.     

Controlled release of heparin from poly(epsilon-caprolactone) electrospun fibers

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly(epsilon-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8%w/v PCL in 7:3 dichloromethane:methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury.     

新型两亲性超枝状大分子聚己内酯／聚缩水甘油醚纳米粒子实验研究

目的选择合适实验条件将两亲性超枝状大分子聚己内酯／聚缩水甘油醚制备成纳米粒子,以进一步应用于靶向药物传递载体。方法称取聚己内酯／聚缩水甘油醚（通过H^1NMR和GPC定性）30mg,溶于3mL丙酮和2mL乙醇混合溶剂中,在搅拌速度为250r／min的条件下。将上述溶液按250uL·min^-1速度注入30mL的蒸馏水中,搅拌过夜,减压挥发除掉溶剂,冷冻干燥得到纳米粒子。结果通过透射电镜观察,所得纳米粒子为球形,形状均匀,粒径约在50—100nm之间。结论本研究通过实验,首先合成分子量2×10^4U左右的聚己内酯／聚缩水甘油醚聚合物,通过×选择合适的溶剂。得到聚己内酯／聚缩水甘油醚纳米粒子。该方法操作简易,环境友好,易于后处理,所得纳米粒子大小均匀,通过后修饰,这种纳米粒子将有望被应用于体内靶向传递。