PLCL Scaffolds Research Articles

The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells

Articular cartilage is subjected to complex loading, which plays a major role in its growth, development and maintenance. Previously, we found that mechanical stimuli enhanced the development and function of engineered cartilage tissues in elastic mechano-active poly(lactide-co-caprolactone) (PLCL) scaffolds. In addition, it is well known that the three-dimensional spatial organization of cells and extracellular matrices in hydrogels is crucial to chondrogenesis. This study was conducted to enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs) in the hybrid scaffolds of fibrin gels and PLCL scaffolds in dynamic environments by compression. A highly elastic scaffold was fabricated from very elastic PLCL with 85% porosity and a 300–500 µm pore size using a gel-pressing method. A mixture of rabbit BMSCs and fibrin gels was then seeded onto the PLCL scaffolds and subjected to continuous compressive deformation of 5% strain at 0.1 Hz for 10 days in a chondrogenic medium containing 10 ng ml−1 TGF-β1. The BMSCs-seeded scaffold constructs were then implanted subcutaneously into nude mice. As a control, the cell–PLCL scaffold constructs were cultured under dynamic conditions or the cell–PLCL/fibrin hybrid scaffold constructs and the cell–PLCL scaffold constructs were cultured under static conditions for 10 days in vitro. The results revealed that cells adhered onto the hybrid scaffolds of fibrin gels and PLCL scaffolds cultured under dynamic conditions. In addition, the accumulation of the extracellular matrix of cell–scaffold constructs, which was increased through mechanical stimulation, showed that chondrogenic differentiation was sustained and enhanced significantly in the stimulated hybrid scaffold constructs. Overall, the results of this study indicate that the proper periodic application of dynamic compression and the three-dimensional environments of the hybrid scaffolds composed of fibrin gels and elastic PLCL can encourage BMSCs to differentiate into chondrocytes, maintain their phenotypes and enhance GAGs production, thereby improving the quality of cartilaginous tissue formed in vitro and in vivo.

In situ chondrogenic differentiation of human adipose tissue-derived stem cells in a TGF-β1 loaded fibrin–poly(lactide-caprolactone) nanoparticulate complex

When conducting cartilage tissue engineering with stem cells, it is well known that chemical and physical stimulations are very important for the induction and maintenance of chondrogenesis. In this study, we induced chondrogenic differentiation of human adipose tissue-derived stem cells (hASCs) in situ by effective stimulation via the continuous controlled release of TGF-β1 from a heparin-functionalized nanoparticle–fibrin–poly(lactide-co-caprolactone) (PLCL) complex. PLCL scaffolds were fabricated with 85% porosity and 300–500μm pore size by a gel-pressing method. Heparin-functionalized nanoparticles were prepared by a solvent-diffusion method, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then TGF-β1 was loaded to the nanoparticles. A mixture of hASCs, fibrin gels and TGF-β1 loaded nanoparticles was then seeded onto PLCL scaffolds and cultured in vitro, after which they were subcutaneously implanted into nude mice for up to five weeks. The results of in vitro and in vivo studies revealed that chondrogenic differentiation of the hASCs on the complex was induced and sustained by continuous stimulation by TGF-β1 from the heparin-functionalized nanoparticles. In addition, there was no significant difference between the predifferentiation condition prior to incubation in chondrogenic medium and the proliferation condition, which suggests that in situ chondrogenic differentiation of hASCs was induced by the TGF-β1 loaded nanoparticles. Consequently, the hybridization of fibrin and PLCL scaffolds for three-dimensional spatial organization of cells and the effective delivery of TGF-β1 using heparin-functionalized nanoparticles can induce hASCs to differentiate to a chondrogenic lineage and maintain their phenotypes.

Transplantation of mesenchymal stem cells within a poly(lactide‐co‐ɛ‐caprolactone) scaffold improves cardiac function in a rat myocardial infarction model

Cardiac tissue engineering has been proposed as an appropriate method to repair myocardial infarction (MI). Evidence suggests that a cell with scaffold combination was more effective than a cell-only implant. Nevertheless, to date, there has been no research into elastic biodegradable poly(lactide-co-epsilon-caprolactone) (PLCL) scaffolds. The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) with elastic biodegradable PLCL scaffold transplants in a rat MI model. Ten days after inducing MI through the cryoinjury method, a saline control, MSC, PLCL scaffold, or MSC-seeded PLCL scaffold was transplanted onto the hearts. Four weeks after transplantation, cardiac function and histology were evaluated. Transplanted MSCs survived and differentiated into cardiomyocytes in the injured region. Left ventricular ejection fraction in the MSC+PLCL group increased by 23% compared with that in the saline group; it was also higher in the MSC group. The infarct area in the MSC+PLCL group was decreased by 29% compared with that in the saline group; it was also reduced in the MSC group. Mesenchymal stem cells plus PLCL should be an excellent combination for cardiac tissue engineering.

A Collagen/Smooth Muscle Cell-Incorporated Elastic Scaffold for Tissue-Engineered Vascular Grafts

Biodegradable tubular scaffolds have been developed for vascular graft application. This study was focused to improve the adhesion and proliferation of vascular smooth muscle cells (SMCs) in a tubular scaffold. Tubular scaffolds (ID 4 mm, OD 6 mm) were fabricated from a biodegradable elastic polymer, poly(L-lactide-co-ε-caprolactone) (PLCL) (50:50, M n 1.58 × 105), by an extrusion/particulate leaching method. SMCs suspended in a collagen solution were infiltrated in tubular PLCL scaffolds under vacuum and incubated for 1 h at 37°C to form a collagenous gel. Results from SEM image analysis showed that collagen was infiltrated into the inside of the scaffolds. Cell adhesion and proliferation rate increased in collagen/SMC-incorporated tubular PLCL scaffolds as compared with the scaffolds in which only SMCs were seeded. From SEM image and histological analysis, we further found that SMCs grew on the inside as well as on the surface of collagen/SMCs-incorporated scaffolds and the cells continued to grow as a monolayer on collagen fibers. In particular, cell proliferation and elastin contents were the highest in a PLCL scaffold with 50–100 μm pore size than any other scaffolds used in this experiment. A collagen/SMC-incorporated PLCL scaffold may support SMC growth and functions and can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.

Cartilage regeneration with highly-elastic three-dimensional scaffolds prepared from biodegradable poly( l-lactide- co-ɛ-caprolactone)

Compressive mechanical stimuli are crucial in regenerating cartilage with tissue engineering, which creates a need for scaffolds that can maintain their mechanical integrity while delivering mechanical signals to adherent cells during strain applications. With these goals in mind, the aim of this study was to develop a mechano-active scaffold that facilitated effective cartilaginous tissue formation under dynamic physiological environments. Using a gel-pressing method, we fabricated a biodegradable and highly-elastic scaffold from poly( l-lactide- co-ɛ-caprolactone) (PLCL; 5:5), with 85% porosity and a 300–500-μm pore size, and we compared it to control scaffolds made of rigid polylactide (PLA) or poly(lactide- co-glycolide) (PLGA). After tensile mechanical tests and recovery tests confirmed the elasticity of the PLCL scaffolds, we seeded them with rabbit chondrocytes, cultured them in vitro, and subcutaneously implanted them into nude mice for up to eight weeks. The PLCL scaffolds possessed a completely rubber-like elasticity, were easily twisted and bent, and exhibited an almost complete (over 97%) recovery from applied strain (up to 500%); the control PLA scaffolds showed little recovery. In vitro and in vivo accumulations of extracellular matrix on the cell–PLCL constructs demonstrated that they could not only sustain but also significantly enhance chondrogenic differentiation. Moreover, the mechanical stimulation of the dynamic in vivo environment promoted deposition of the chondral extracellular matrix onto the PLCL. In contrast, on the PLA scaffolds, most of the chondrocytes had de-differentiated and formed fibrous tissues. In a rabbit defect model, the groups treated with PLCL scaffolds exhibited significantly enhanced cartilage regeneration compared to groups harboring an empty control or PLGA scaffolds. These results indicated that the mechano-active PLCL scaffolds effectively delivered mechanical signals associated with biological environments to adherent chondrocytes, suggesting that these elastic PLCL scaffolds could successfully be used for cartilage regeneration.

Mechanical and Biological characterization of A Porous Poly‐L‐Lactic Acid‐Co‐ϵ‐Caprolactone scaffold for Tissue Engineering

This article presents a method for making highly porous biodegradable scaffold that may ultimately be used for tissue engineering. Poly(L‐lactic‐co‐ϵ‐caprolactone) acid (70∶30) (PLCL) scaffold was produced using the solvent casting/leaching out method, which entails dissolving the polymer and adding a porogen that is then leached out by immersing the scaffold in distillated water. Tensile tests were performed for three types of scaffolds, namely pre‐wetted, dried, and UV‐irradiated scaffolds and their mechanical properties were measured. The pre‐wetted PLCL scaffold possessed a modulus of elasticity 0.92±0.09 MPa, a tensile strength of 0.12±0.03 MPa and an ultimate strain of 23±5.3%. No significant differences in the modulus elasticity, tensile strength, nor ultimate strain were found between the pre‐wetted, dried, and UV irradiated scaffolds. The PLCL scaffold was seeded by human fibroblasts in order to evaluate its biocompatibility by Alamar blue® assays. After 10 days of culture, the scaffolds showed good biocompatibility and allowed cell proliferation. However, the fibroblasts stayed essentially at the surface. This study shows the possibility to use the PLCL scaffold in dynamic mechanical conditions for tissue engineering

Application of an elastic biodegradable poly(L-lactide-co-ε-caprolactone) scaffold for cartilage tissue regeneration

In cartilage tissue regeneration, it is important that an implant inserted into a defect site can maintain its mechanical integrity and endure stress loads from the body, in addition to being biocompatible and able to induce tissue growth. These factors are crucial in the design of scaffolds for cartilage tissue engineering. We developed an elastic biodegradable scaffold from poly(L-lactideco-ε-caprolactone) (PLCL) for application in cartilage treatment. Biodegradable PLCL co-polymer was synthesized from L-lactide and ε-caprolactone in the presence of stannous octoate as a catalyst. A highly elastic PLCL scaffold was fabricated by a gel-pressing method with 80% porosity and 300–500 μm pore size. The tensile mechanical and recovery tests were performed in order to examine mechanical and elastic properties of the PLCL scaffold. They could be easily twisted and bent and exhibited almost complete (over 94%) recoverable extension up to breaking point. For examining cartilaginous tissue formation, rabbit chondrocytes were seeded on scaffolds. They were then cultured in vitro for 5 weeks or implanted in nude mice subcutaneously. From in vitro and in vivo tests, the accumulation of extracellular matrix on the constructs showed that chondrogenic differentiation was sustained onto PLCL scaffolds. Histological analysis showed that cells onto PLCL scaffolds formed mature and well-developed cartilaginous tissue, as evidenced by chondrocytes within lacunae. From these results, we are confident that elastic PLCL scaffolds exhibit biocompatibility and as such would provide an environment where cartilage tissue growth is enhanced and facilitated.

The effect of cyclic strain on embryonic stem cell-derived cardiomyocytes

Cardiomyocytes in the body are subjected to cyclic mechanical strain induced by the rhythmic heart beating. In this study, we tested the hypothesis that cyclic strain promotes cardiomyogenesis of embryonic stem cell-derived cardiomyocytes (ESCs). ESCs cultured on elastic polymer [poly(lactide- co-caprolactone), PLCL] scaffolds subjected to cyclic strain in vitro displayed elevated cardiac gene expression compared to unstrained controls. Six weeks after implantation into infarcted rat myocardium, the elastic cardiac patches (ESC-seeded PLCL scaffolds) showed reduced fibrotic tissue formation, likely due to a combination of lower apoptotic activity, higher vascular endothelial growth factor (VEGF) expression, and more extensive angiogenesis in the strained versus unstrained control [ESC-seeded, non-elastic poly(lactide- co-glycolide) scaffolds] patches. Importantly, cardiac gene expression was upregulated in the elastic patches compared to control, with evidence for cardiomyocyte-specific microstructures including myofibrillar bundles and Z-lines. This study shows that the use of an elastic polymer scaffold designed to permit mechanical strain transduction as a cell transplantation vehicle significantly increases cardiomyogenesis of the implanted ESCs.

Cartilaginous Tissue Formation with an Elastic PLCL Scaffold and Human Adipose Tissue-Derived Stromal Cells

In cartilage tissue engineering, as a cell source, adult stem cells are very attractive for clinical applications. Recent studies suggest that human adipose tissue-derived stromal cells (ASCs) have multilineage potential similar to bone marrow-derived stromal cells (BMSCs). ASCs are obtained from adipose tissue easily isolated by suction-assisted lipectomy in various body parts. Also, as one of major factors of cartilage tissue engineering, scaffolds have an important role in cartilage formation. Poly(L-lactide-co-ε-carprolactone) scaffolds have physiological activity, biodegradability, high cell affinity, and mechano-activity. The object of this study is cartilaginous tissue formation using highly elastic PLCL scaffolds and ASCs in vitro and in vivo. Poly(L-lactide-co-ε-carprolactone) copolymers were synthesized from lactide and ε-carprolactone in the presence of stannous octoate as catalyst. The scaffolds with 85% porosity and 300-500μm pore size were fabricated by gel-pressing method. ASCs were seeded on scaffolds and cultured for 21days in vitro. Cell/polymer constructs were characterized by reverse transcriptase-polymerase chain reaction for confirming differentiation to chondrocytes onto PLCL scaffolds. Also, for examining cartilaginous tissue formation in vivo, ASCs seeded scaffolds which were induced chondrogenesis for 2 weeks were implanted in nude mice subcutaneously for up to 8weeks. Histological studies showed that implants partially developed cartilaginous tissue within lacunae. And there was an accumulation of sulfated glycoaminoglycans. Immunohistochemical analysis revealed that implants were positively stained for specific extracellular matrix. These results indicate that ASCs and PLCL scaffols could be used to cartilage tissue engineering.

Characterization of the Gel-Spun Tubular Scaffold for Cardiovascular Tissue Engineering

A tubular and fibrous scaffold was fabricated from an elastic polymer, poly (L-lactideco- ε-caprolactone) (PLCL; Mn 193,813, Mw 538,623) 50:50 by using a novel gel spinning apparatus. To characterize the gel-spun scaffold, we investigated morphology, tensile property, tissue in-growth rate and degradation rate. From SEM images, fibrous structure in the scaffold wasn’t fabricated well in the condition of 4% gel concentration. In general, the thickness level of microfibers increased as the gel concentration increased. In addition, the gel-spun scaffolds showed stronger tensile properties in the circumferential direction than the longitudinal direction. 5%, 7.5%, 10% and 12.5% scaffolds were analyzed in both directions: circumferential direction and longitudinal direction. On the other hand, the gel-spun scaffolds have been implanted in mouse to examine the degradation rate in vivo and tissue in-growth aspects, compared to extruded scaffolds. Both shows very similar degradation rates, but the aspect in tissue in-growth was different. In conclusion, gel-spun PLCL scaffolds have good characteristics as a plausible scaffold for cardiovascular tissue engineering.

Cartilage Tissue Engineering using a Elastic Poly (L-Lactide-co-ε-Caprolactone) Scaffold

Mechano-active scaffolds were fabricated from very elastic poly(L-lactide-co-ε-carprolactone). The scaffolds with 80 % porosity and 300~500 μm pore size were prepared by a gel-pressing method. As a control group for elastic properties of polymer scaffolds, rigid poly L-lactide scaffolds were fabricated. The scaffolds were seeded with chondrocytes and cultured to evaluate the effect of elastic properties of polymer scaffolds for the differentiation and the ECM secretion of chondrocytes. Also, the chondrocytes-seeded constructs were implanted in nude mice subcutaneously to investigate their biocompatibility and cartilage formation. From the biochemical analyses, chondrogenic differentiation was sustained and enhanced significantly and chondral extracellular matrix was increased through mechanical stimulation of dynamic environment in the dynamic body systems. Histological analysis showed that implants of PLCL constructs formed mature and well-developed cartilaginous tissue, as evidenced by chondrocytes within lacunae. Consequently, the elastic PLCL scaffolds could be used to engineer cartilage in mechanically dynamic environments

Optimization of Scaffold for a Successful Hydrogel-Seeding Method for Vascular Tissue Engineering

Porosity and pore size are needed for successful cell seeding and proliferation into porous scaffolds. This study was focused on a hydrogel-seeding method to improve cell adhesion and proliferation in tubular porous scaffolds for vascular grafts application. Tubular scaffolds were fabricated from a biodegradable elastic polymer, poly(L-lactide-co-ε-caprolactone) (PLCL) (50:50, Mn 1.58×105), by an extrusion-particulate leaching method. Vascular smooth muscle cells (VSMCs) were dispersed in collagen hydrogel and then seeded into the tubular PLCL scaffolds having various pore sizes, 50-100 μm, 100-200 μm, and 300-500 μm, respectively. As a result, the efficiency of cell adhesion and proliferation was dependent on the pore size of the scaffolds. Especially, the cell proliferation efficiency was improved by using the hydrogel-seeding method as compared with by using a previously established method. In summary, this study demonstrates that the efficiency of cell adhesion and proliferation was dependent on the pore size of the scaffolds in the hydrogel-seeding method.

Preliminary experience with tissue engineering of a venous vascular patch by using bone marrow–derived cells and a hybrid biodegradable polymer scaffold

Currently available synthetic polymer vascular patches used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. These problems may be avoided by vascular patches tissue-engineered with autologous stem cells and biodegradable polymeric materials. The objective of this study was to develop a tissue-engineered vascular patch by using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. Tissue-engineered vascular patches (15 mm wide x 30 mm long) were fabricated by seeding vascular cells onto PGA/PLCL scaffolds and implanted into the inferior vena cava of bone marrow donor dogs. Compared with PLCL scaffolds, PGA/PLCL scaffolds exhibited tensile mechanical properties more similar to those of dog inferior vena cava. Eight weeks after implantation of vascular patches tissue-engineered with BMCs and PGA/PLCL scaffolds, the vascular patches remained patent with no sign of thrombosis, stenosis, or dilatation. Histological, immunohistochemical, and scanning electron microscopic analyses of the retrieved vascular patches revealed regeneration of endothelium and smooth muscle, as well as the presence of collagen. Calcium deposition on tissue-engineered vascular patches was not significantly different from that on native blood vessels. Immunofluorescent double staining confirmed that implanted BMCs survived after implantation and contributed to regeneration of endothelium and vascular smooth muscle in the implanted vascular patches. This study demonstrates that vascular patches can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.

Fabrication of a new tubular fibrous PLCL scaffold for vascular tissue engineering

Biodegradable macroporous scaffolds have been developed for tissue-engineering applications. We fabricated and characterized a new tubular, macroporous, fibrous scaffold using a very elastic biodegradable co-polymer, poly(L-lactide-co-caprolactone) (PLCL, 5:5) in a gel-spinning process. A viscous PLCL solution was spun as a gel-phase under swirl-flow conditions and was subsequently fabricated to produce a tubular fibrous scaffold on a rotating cylindrical shaft in a methanol solution. The porosity and median pore size of the fibrous PLCL scaffolds were 55–75% and 120–150 μm, respectively, using a 5–10% PLCL solution. The use of a 7.5% (w/v) solution resulted in scaffolds with tensile strength and elastic modulus of 3.39 MPa and 1.22 MPa, respectively. The scaffolds exhibited 500–600% elongation-at-break. The tensile strength and modulus of fibrous PLCL scaffolds were proven to decrease on lowering the concentration of the PLCL spinning solution; however, the tensile strength and modulus of fibrous PLCL scaffolds, produced from 5% solutions, are approximately 4- and 5-times higher than those of extruded PLCL scaffolds. These properties indicated that the fibrous PLCL scaffolds were very elastic and mechanically strong. The scaffolds appeared to be well interconnected between the pores as determined by SEM imaging analysis. In addition, the cell-seeding efficiency was 2-fold higher using gel-spun scaffolds than using extruded scaffolds. These results suggest that the gel-spun fibrous PLCL scaffold is an excellent matrix for vascular tissue-engineering applications.

Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds

Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow. Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype. In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro. To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL [poly(lactide- co-caprolactone), 50:50] scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks. As control experiments, VSMCs were cultured on PLCL scaffolds statically. The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production. In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned. Importantly, the expression of SM α-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition. This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype.

Morphology of elastic poly(L-lactide-co-epsilon-caprolactone) copolymers and in vitro and in vivo degradation behavior of their scaffolds.

Very elastic PLCL [poly(L-lactide-co-epsilon-caprolactone), 50:50] copolymers were synthesized and extruded into porous tubular scaffolds (pore size 150 +/- 50 microm, porosity 90%) for the application to tissue engineering. The copolymers were basically random and amorphous. However, two T(g)'s (glass transition temperatures) were observed in dynamic mechanical thermal analysis and also in differential scanning calorimetry thermograms. Furthermore, microdomains (about 17 nm in size) were indicated on the small-angle X-ray scattering profile and finally confirmed by transmission electron microscopy. Therefore, the PLCL copolymer was probably composed of a soft matrix of mainly epsilon-caprolactone moieties and hard domains containing more L-lactide units to exhibit a rubberlike elasticity in virtue of the physically cross-linked structure. The smooth muscle cells seeded scaffolds were implanted into nude mice subcutaneously for up to 15 weeks to monitor the in vivo degradation. In addition, they were degraded in vitro in phosphate buffer solution (pH 7.4) for up to 1 year to compare the results each other. All the scaffolds degraded slowly in vivo and in vitro even in the form of a highly porous thin membrane. However, the degradation rate was somewhat faster for in vivo than for in vitro. This should be explained by enzymes that might have played a certain role in the degradation in the body. In addition, the epsilon-caprolactone moieties degraded faster than the L-lactide units did in these PLCL scaffolds, although their hydrophilicities are in the opposite order. This behavior appeared more prominently in the in vivo case. This should result from that the amorphous regions composed of mainly epsilon-caprolactone units might have been first attacked by water because water can penetrate into the amorphous regions easier than the hard domains containing more L-lactides.

In vivo biocompatibilty and degradation behavior of elastic poly( l-lactide- co- ε-caprolactone) scaffolds

Tubular scaffolds were fabricated from very elastic poly( l-lactide- co- ε-caprolactone) (PLCL, 50:50). The scaffolds were seeded with smooth muscle cells (SMCs) and implanted in nude mice to investigate the tissue compatibility and in vivo degradation behavior. Histological examination of all the implants with haematoxylin and eosin staining, masson trichrome staining, SM α-actin antibody, and CM-DiI labeling confirmed that the regular morphology and biofunction of the SMCs seeded and the expression of the vascular smooth muscle matrices in PLCL scaffolds. The implanted PLCL scaffolds displayed a slow degradation on time, where caprolactone units were faster degraded than lactide did. This could be explained by the fact that amorphous regions composed of mainly CL moieties degraded earlier than hard domains where most of the LA units were located. From these results, the scaffolds applied in this study were found to exhibit excellent tissue compatibility to SMCs and might be very useful for vascular tissue engineering.

Manufacture of elastic biodegradable PLCL scaffolds for mechano-active vascular tissue engineering

A soft and very elastic poly(lactide-co-ε-caprolactone) (PLCL)(50:50, M n 185 × 103) was synthesized. Tubular scaffolds were prepared by an extrusion-particulate leaching method for mechano-active vascular tissue engineering. The copolymer was very flexible but completely rubber-like elastic. Even the high porous PLCL scaffolds (90% salt wt) exhibited 200% elongation, but recovery over 85% in a tensile test. Moreover, the PLCL scaffolds maintained their high elasticity also in culture media under cyclic mechanical strain conditions. The highly porous scaffold (90% salt wt) withstood for an initial 1 week without any deformation and sustained for 2 weeks in culture media under cyclic stress of 10% amplitude and at 1 Hz frequency which are similar to the natural vascular conditions. Vascular smooth muscle cells (VSMCs) were seeded on to the PLCL scaffolds. The cell adhesion and proliferation on the scaffolds of various pore-size were increased with increasing pore size. For the pore sizes of 50-100 μm, 100-150 μm, 150-200 μm and 200-250 μm, the ratios of cell numbers were about 1:1.2:1.9:2.2, respectively, at both 12 h and 5 days. Similarly, the higher porous scaffolds exhibited more cell adhesion and proliferation compared to lower porous one, where the effect was more pronounced in the longer proliferation period. SMC-seeded scaffolds were implanted subcutaneously in athymic nude mice to confirm the biocompatibility. Such a high elastic property and proper biocompatibility to SMCs of PLCL scaffolds prepared in this study will be very useful to engineer SM-containing tissues such as blood vessels under mechanically dynamic environments (mechano-active tissue engineering).