A method has been developed to induce and retain a contractile phenotype for vascular smooth muscle cells, as the first step towards the development of a biomimetic blood vessel construct with minimal compliance mismatch. Melt spun PCL fibers were deposited on a mandrel to form aligned fibers of 10
As research in implantable biomaterial advances, the understanding and manipulation of cell-substrate interactions have increased in importance. One approach is to produce a more biomimetic construct that can recruit and control the patterning of functional cells to mimic the native tissue organization. For example, the aligned orientation of cells on extracellular matrix (ECM) plays an important role in several tissues including corneal stroma, tendons, bones, skeletal muscle, and, with significance to the present study, the vasculature [
The development of a small diameter vascular prosthesis (>6 mm diameter) for arterial disease has been hampered by the mechanical compliance mismatch of the prosthesis and the native blood vessel. The mismatch is the key factor for the relatively rapid loss of patency compared to larger-diameter prostheses. In turn the mismatch is due to the fact that artificial prostheses do not mimic the layered structure of the native vessel, in which one of the layers has circumferentially aligned vascular smooth muscle cells (VSMCs) as well as extracellular matrix (ECM) [
The two predominant cell types within blood vessels, fibroblasts and VSMCs, could both functionally benefit cell-seeded prosthesis if recruited in an aligned orientation [
Of particular interest to this study is the alignment of VSMCs. These cells are integral to the vascular functioning through regulation of vessel tone and lumen diameter. Interestingly, these cells exist as two very distinct and changeable phenotypes: the contractile, characterized by a spindle shape and the abundant presence of alpha-smooth muscle actin (
During the culturing of VSMC, freshly seeded VSMCs exist primarily in the contractile state, but over time the population shifts predominantly towards the secretory phenotype. For long-term patency, it is important to preserve the contractile phenotype, as only VSMCs in the contractile state are beneficial in fabricating cellularized tissue engineered blood vessel (TEBV) because this phenotype minimizes the compliance mismatch. Furthermore, the cells must be circumferentially orientated to direct their function [
Previous studies already have demonstrated that the aligned orientation and phenotypic characteristics of cells can be guided by surface cues generated through micropatterning a surface with channels [
Here, we construct a scaffold of parallel melt spun PLC fibers. As the fiber width is 10
Early alignment studies employed nondegradable polymers such as channels on PDMS films to produce aligned cell-orientating scaffolds. More recently, however, biodegradable polymers have been studied, since such scaffolds can be slowly replaced by native tissue and ECM which minimizes the issue of thrombosis. Such polymers have included P[(LLA-CL)] [
In this work, we have successfully produced a highly aligned melt spun PCL fiber scaffold that allows fibroblast and VSMC aligned attachment and also preserves the contractile
Melt spinning of polycaprolactone was performed as described in An et al. [
Melt spinning apparatus depositing fiber on a turning mandrel, demonstrating the slide rail, z-stage, melt holder, and mandrel; for more details see An et al. [
The fibers were adhered into place by dip coating in 5% chitosan (w/v) in acetic acid and then incubated for 5 days at room temperature to allow solvent evaporation. As a control, chitosan films were formed by coating the base of 6-well plates with 500
A PLC vascular graft was formed by dip coating a 5 mm diameter stainless steel mandrel in a 12% (w/v) PLC solution dissolved on chloroform using a Nima DC-Mono 300 dip coating apparatus. The PLC coating was incubated at room temperature for 48 hours to allow solvent evaporation. Subsequently the mandrel was placed into the melt spinning apparatus and a layer of circumferentially aligned PCL fibers was applied. The fibers were adhered using 5% chitosan solution as an adhesive as described above; following solvent evaporation the scaffold was coated in 1% gelatin to enhance cell attachment on the tubular surface.
FTIR analysis was used to qualitatively characterize the functional groups introduced presented on the surface of the construct. FTIR spectra were collected with Frontier FT-IR spectrometer (PerkinElmer) at resolution of 4 cm−1 and signal average of 16 scans in each interferogram over the range of 4000–600 cm−1. Two measurements were retrieved on two random locations per sample for each group. Results were analyzed by plotting % transmission against wavelength (nm).
Intermittent contact mode atomic force micrographs were obtained on JPK Instruments Nanowizard III (Aufgang C, Germany) with a nanoprobe of 100
Smooth muscle cells (SMCs, Lonza) or human fibroblasts were cultured up to the 8th passage in smooth muscle cell basal medium (SmGM-2 Media (Lonza Bioscience)) or FibroGRO Complete Media (Millipore), respectively. PCL fiber films (1 × 1 cm3) were sterilized with 70% ethanol for 1 h and then were washed away with PBS (three times). The cells were seeded on the PCL fiber films and a control surface at a density of 5 × 104 cells/cm2. Cell-seeded fibers were cultured in a flat bottom 24-well plate for 7 days.
A 5 cm section of either uncoated or aligned PCL fiber coated conduits was placed in a 6 cm diameter dish. Subsequently, 3 mLs of a concentrated SMC suspension (5 × 106 cells/mL) was slowly applied to the upper surface; after 10 min incubation at room temperature the tube was turned over and another 3 mLs of the cell suspension was applied. The constructs were incubated over night at 37°C in 5% CO2; then the cells morphology was examined using fluorescence microscopy.
The seeded fibroblasts were recorded after 14 days. The fluorescence was generated by 30-minute incubation with 2
SMCs in cell-seeded fibers at day 3 and day 7 were fixed in 4% paraformaldehyde for 30 min in room temperature. Following fixation, fibers were washed 3x with PBS, permeabilized with 0.1% Triton X-100, and blocked using 2% BSA in PBS for 1 h at 4°C. After washing 3x at room temperature in PBS and immunohistochemistry labeling on fibers and control (chitosan film) samples was performed, applying primary antibody against alpha-smooth muscle actin (monoclonal mouse anti-human), at 1 : 100 dilutions in PBS/BSA/buffer at room temperature for 2 h. After washing 3 times for 10 min in PBS/BSA buffer, secondary antibodies (AF 488 goat anti-mouse, Invitrogen) were applied at 1 : 200 dilutions in PBS/BSA buffer at room temperature for 1 h. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and PCL fibers were then imaged via confocal microscopy (Leica, Wetzlar, Germany).
The melt spin method produced a thin layer of highly aligned PCL fibers of 10
The fiber mat was examined by AFM (Figure
Surface analysis of the aligned chitosan covered fibers: AFM of fibers bonded by chitosan (a). FTIR of the chitosan film and PCL coated chitosan (b).
Fibroblasts cultures on aligned fiber (a) and on tissue culture plastic (b) for 14 days; cells are stained with calcein AM. SMCs orientated on melt spun aligned fiber (c) and cultured on chitosan film (d) for several days. The cells are immunostained for F-actin and DAPI nuclear stain.
Fibroblasts were seeded onto the fiber surface and readily aligned with the parallel fibers and became noticeably elongated compared to the control (Figures
The recently passaged smooth muscle cells (up to day 3) demonstrated positive staining by immunochemistry for the contractile phenotype marker protein
SMC expression of
The melt spun fibers could be readily used to pattern the surface of a prototype PLC vascular conduit (Figure
SMC seeding of a PLC prosthesis. A PLC tube was coated with PCL melt spun fiber (a), SMCs were seeded at high concentration (5 × 106 cells/mL) on fiberless (b) and fiber coated (c) tubes, and images were recorded 5 days after seeding.
PCL based conduits are seen to have potential for the future fabrication of cellularized vascular prosthesis, with some successful clinical implantation [
Fibers removed from the mandrel without the chitosan or gelatin bonding separated very readily. Furthermore polycaprolactone without modification has poor cell adhering properties, whereas chitosan with high deacetylation (85–95% in this case) has very good cell recruiting qualities. The higher the deacetylation, the greater the amount of free cationic amino groups that encourage cell adhesion to the surface [
Previous studies have demonstrated that SMCs are often seeded in a strongly contractile phenotype; then following a prolonged period of culture, the cells gradually become predominantly synthetic [
The contractile phenotype in this study was assessed by the elongated cell morphology and
The intracellular mechanism by which the contractile phenotype can be promoted in SMC elongating with cell aligning channels has been examined. There is a milieu of signal transduction pathways affecting SMC phenotype (as reviewed by [
Fibroblasts were able to align and become confluent on the fibers whereas SMCs differentiating towards contractile phenotype have a greatly reduced rate of proliferation [
Several groups have created aligning scaffolds by electrospinning polymers. For example, Xu et al. [
Unlike most work on cell aligning fiber scaffolds, the current study employed micron-diameter scaffolds. It has been demonstrated that PCL can be melt spun into fibers with range of diameters from 10 to 200
The melt spun fibers can be used to circumferentially align the SMCs on a prototype PLC vascular prosthesis in an elongated contractile-like phenotype thus demonstrating the effectiveness of the technique to orientate the cell both on flat 2D film and also circumferentially on the outer surface of a tube.
This ability of fabricating and controlling alignment patterns on tissue engineering scaffolds will aid the production of a more physiologically relevant representation of natural tissue [
We aim to eventually achieve the coating of fully functional, vasoresponsive SMCs for the structure. However, SMCs can exist at various points of differentiation between the two phenotypes. Through contact guidance features we have successfully promoted a more contractile-like phenotype. To achieve fully functional contractile SMCs, we expect to involve additional steps [
In summary, the combination of aligned melt spun micron-sized fibers with a polymeric “adhesive” (chitosan) is a very promising substrate that enables the retention of the desired contractile phenotype of smooth muscle cells. Such a construct may be advantageously applied onto tubular constructs for forming a biomimetic blood vessel that will potentially have superior compliance matching with the native vessel.
The technology described in this paper is included in a patent application filed by authors Scott A. Irvine, Animesh Agrawal, Jia An, Chee Kai Chua, and Subbu S. Venkatraman.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Animesh Agrawal and Bae Hoon Lee contributed equally to this paper.
The authors would like to thank Professor Mark Featherstone, School of Biological Studies, NTU, for his support. This research is supported by the Singapore National Research Foundation under CREATE programme (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and the