Currently available tissue valve prostheses based on aldehyde-fixed xenogenic tissue are inevitably subject to calcium phosphate deposition and degeneration. The tanning process effectively devitalizes the native cell population, denaturizes antigenic protein domains, and changes the scaffold protein architecture rendering in vivo repopulation with recipient cells impossible. Furthermore, none of the currently available heart valve prostheses has the potential for growth, limiting their use in infants and children. Goal of the current heart valve tissue engineering efforts is therefore the development of a valve prosthesis that combines unlimited durability with physiologic blood flow pattern and biologically inert surface properties. Decellularized heart valve matrix was suggested as a scaffold for tissue engineering, providing the natural valve architecture and ideal conditions for repopulation with recipient cells.1 However, there is a major problem: the mechanical tissue properties deteriorate when cells are removed and the tertiary structure of fibrous valve tissue constituents is altered during the decellularization process. To address this issue, matrix/polymer hybrid scaffold based on decellularized heart valve leaflet scaffold that are coated with biodegradable polymers using an electrospinning technique were developed.
The biodegradable polymer used in this study was poly-4-hydroxybutyrate (P4HB), which is biocompatible, resorbable, processable, strong and ductile. The degradation product 4HB is a natural human metabolite present in the brain and several other organs including the heart.2 P4HB has a much higher modulus (stiffer) and much lower strain to failure compared with either poly(glycerol-sebacate) or vulcanized rubber.3 The mechanical properties of P4HB, such as pliability and elasticity, make it very suitable for application in soft tissue engineering.4
Electrospinning is a useful technique for producing nonwoven, porous, three-dimensional scaffolds containing fibers, ranging in diameter from tens of microns to tens of nanometers.5–7 This technology offers the potential for controlling the composition, structure, and mechanical properties of biomaterials. The matrix content and distribution of the electrospun scaffolds can be controlled to achieve various matrix compositions depending on the need. The ability to reproducibly fabricate a biological scaffold with characteristics similar to a native tissue has many advantages over existing scaffolds, because biomechanical properties and composition of a native tissue vary between the type and site, as well as between individuals of different age. In addition, favorable interactions between cells and the scaffold were demonstrated.5 This scaffold may promote tissue biosynthesis to guide cell growth and accelerate subsequent tissue regeneration faster than any other known form of gel or sponge.8–10
Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their unique biological properties of self-renewal and plasticity. The usage of MSCs may offer several advantages by: 1) showing characteristics of multipotent progenitor cells which are able to differentiate into a variety of mesenchymal cell types, 2) by avoiding the sacrificing of intact cardiovascular structures due to easy collection and isolation methods, and 3) demonstrating immunological unique characteristics allowing persistence in an allogenic setting.11–13
In this study, hybrid scaffold was fabricated from decellularized porcine aortic valve leaflet scaffold and coated with P4HB using an electrospinning technique. Then MSCs were seeded onto the scaffold and cultured in vitro for 14 days. The hypothesis that this hybrid scaffold exhibits improved biomechanical properties and its biological characteristics are comparable with decellularized heart valve leaflet scaffold in vitro was tested.
Materials and Methods
All animal experiments and surgical procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the local animal care committees.
Cell Isolation and Cultivation
Whole bone marrow was aspirated from the tibia and femur of adult S-D rats (body weight, 150–200 g). MSCs were isolated from rat bone marrow by washing in phosphate buffered saline (PBS, Gibco) for 10 min at 1,500 rpm. The cells were recovered after centrifugation and resuspended in PBS. A low-density cell fraction of bone marrow was obtained by centrifugation of the cell suspension over a Ficoll step gradient (density 1.077 g/ml) (Ficoll-Histopaque 1077, Sigma, St. Louis, MO) at 1,500 rpm for 10 min. The nucleated cells were collected from the interface, diluted with two volumes of PBS and centrifuged at 1,500 rpm for 10 min. After, the cells were resuspended, counted and plated at 2 × 105 cells/cm2.
The isolated cell fraction was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, HyClone), penicillin (Gibco), and streptomycin (Gibco) in tissue flasks and left to adhere at 37°C for 4–5 hours. The nonadherent cells floated off, while mesenchymal cells adhered, spread, and grew. Medium was replaced at 24 and 72 hours and every 3 days after. Daily growth progress was monitored by phase- contrast microscopy. The cells were serially passaged and expanded in a humidified incubator (37°C, 5% CO2). Sufficient cell numbers for cell seeding on scaffolds were obtained after 21–28 days. Cells were characterized before valve leaflet implantation.
Preparation of Decellularized Scaffolds
Decellularization was performed as described previously.1 However, in this study, several modifications were applied. Briefly, the aortic valve leaflets were placed in a solution of 1% tert-octylphenylpolyoxyethylen (Triton ×−100, Sigma) with 0.02% ethylenediaminetetraacetic acid (Sigma) in PBS without Ca2+ and Mg2+ for 24 hours, together with RNase A (0.02 mg/ml) (Sigma) and DNase (0.2 mg/ml) (Sigma). All steps were conducted in a 5%CO2/95% air atmosphere at 37°C under continuous shaking. The decellularized valve leaflets were washed with PBS supplemented with penicillin and streptomycin served for removal of cell debris, and were then stored in PBS at 4°C before further processing and seeding.
Preparation of Hybrid Scaffolds
Hybrid scaffolds were fabricated from decellularized scaffolds and coated with biodegradable polymer using an electrospinning technique. Solution was prepared by dissolving the purified P4HB (MW: 1 × 106, PHA 4400, TEPHA Inc., Cambridge, MA) (0.2 g) in dichloromethane (10 ml) at room temperature with stirring for 3 hours. Electrospinning was carried out with a high voltage power supply set (DWP503-1AC, Dong-Wen High Voltage power supply Factory, Tianjin, China) at 20 kV with a collection distance between 15 and 20 cm. A 5 ml glass syringe with a tip-blunt needle (0.9 mm in diameter) was used to store the polymer solution for electrospinning, and the solution was delivered at a flow rate of 3.0 ml/h.5,14 Polymeric fibers were collected on the outflow side (arterialis) of the decellularized scaffolds until the required thickness of membrane was achieved. Uniform thickness of membrane were created using 2.4 ml of solution. In the end, the decellularized scaffolds were coated with P4HB fibers and hybrid scaffolds were created.
Cell Seeding and in vitro Culture
MSCs were seeded onto the outflow side (arterialis) of each scaffold with an approximate cell density between 4.5 and 5.5 × 106 cells/cm2. Then the reseeded hybrid scaffolds and the reseeded decellularized scaffolds were cultured in DMEM supplemented with 10% FBS. All reseeded scaffolds were cultured in vitro for 14 days in the humidified incubator.
A single cell suspension of MSCs was prepared for flow cytometry. 1 × 106 cells in 100 ml PBS plus bovine serum albumin (BSA, Sigma) were incubated with saturating concentrations of monoclonal antibodies CD29-FITC (Caltag, San Francisco, CA), CD 44-FITC, CD 14-FITC and CD 45-FITC (Beckon Dickinson, San Jose, CA). Samples were analyzed with the flow cytometer FACS-Calibur (Becton Dickinson Immunocytometry Systems, San Jose, CA). Data analysis was performed with the CELL QUEST software program (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Immunocytochemistry was conducted as previously described.15 MSCs were characterized by incubation with monoclonal mouse antibodies for a-smooth muscle actin (a-SMA, clone 1A4, 1:400; Sigma) and vimentin (clone V9, 1:50; Dako). For the primary antibodies a FITC-conjugated rabbit antimouse antibody (1:20) (Dako) served as secondary antibodies. Negative controls with the absence of the primary antibodies were demonstrated. Cells were examined by fluorescence microscopy (IX71-A12FL/PH, Olympus, Japan).
Histology and Immunohistochemistry
Specimens of each scaffold construct were embedded in paraffin. For general morphology serial sections were stained with hematoxylin–eosin (HE) stain. Immunohistostaining was carried out using immunofluorescence techniques. Monoclonal primary antibodies were a-SMA (1:250). An FITC-conjugated rabbit antimouse antibody served as secondary antibodies (1:20). Negative controls with the absence of the primary antibodies were demonstrated. Sections were analyzed and documented by fluorescence microscopy.
Scanning Electron Microscopy
Specimens were examined by scanning electron microscopy (SEM) (Hitachis-520 ESEM, Hitachi, Japan) for microstructure and surface morphology.
Biochemical assays were performed for analysis of cellular and extracellular components of a representative number of specimens. Tissue sample extraction was prepared as previously described.16 All samples were normalized according to equivalent dry weight. Total DNA was isolated and purified by sequential organic extractions with phenol (Carl Roth, Karlsruhe, Germany) and phenol/chloroform/isoamyl alcohol (Roth) and quantified by spectrophotometry (Helios h, Thermo Spectronic, Rochester, NY). Total 4-hydroxyproline (4-OHP) determination was performed as described before.16 Total collagen were quantified using SIRCOL assays (Biocolor, Belfast, Northern Ireland).
To assess the biomechanical behavior of each scaffold, specimens were subjected to tensile testing. Specimens of 1.5 mm wide were cut out from each scaffold. The tensile tests were performed on a Dynamic Mechanical Analyzer DMA 2980 (TA-Instruments, New Castle, DE) at ambient temperature. The tests were carried out with a force ramping of 1 N/min until specimen fracture. The elastic modulus, tensile strength, and elongation to break were assessed.
All values are shown as mean ± standard deviation. The significance of differences between specimens was estimated by analysis of variance (Independent-Samples t test) using the commercially available software package SPSS for windows, version 10.0. p values <0.05 were considered significant.
Characterization of the MSCs
Flow cytometry characterization of rat MSCs demonstrated that these cells expressed CD29 (94.82%) and CD44 (93.59%), and did not express the leukocyte common antigen CD14 (0.49%) or CD45 (2.00%) (Figure 1).
MSCs at low density displayed the spindle-shaped morphology characteristic of fibroblast-myofibroblast cell lineage. Immunocytochemistry of isolated and fixed MSCs revealed a strong expression of a-SMA and vimentin (Figure 2).
Histological analysis of the HE stained decellularized valve leaflets sections showed that the decellularization process converted native porcine aortic valve leaflets into an almost cell free scaffold (Figure 3A, B). After decellularization process, the surface structure appears eroded and the fiber architecture becomes very prominent (Figure 4A). The ultrathin fibers produced from P4HB have magnitude similarity to the fibrils in extracellular matrix of tissue (Figure 4B). A complete and comparable cell lining of the entire surface of the decellularized scaffolds and hybrid scaffolds was demonstrated by HE and immunostaining (Figure 3C–F). Cells from all specimens showed a strong expression of a-SMA throughout the entire surface of the scaffolds (Figure 3E, F). A confluent MSCs cluster of the entire surface of the decellularized scaffolds and hybrid scaffolds could be further demonstrated by SEM (Figure 4C, D). MSCs were seen well attached to the electrospun membrane, and the electrospun membrane of P4HB firmly combined with the surface of the decellularized scaffold and showed uniform structure (Figure 3D, F).
Quantitative tissue analysis data are summarized in Table 1. DNA content was comparable with those of decellularized scaffolds and hybrid scaffolds. In addition, decellularized scaffolds and hybrid scaffolds revealed almost comparable amounts of 4-OHP and collagen.
In comparison with decellularized scaffolds, hybrid scaffolds revealed a significant increase of tensile strength and elastic modulus. Elongation to break was comparable with those of decellularized scaffolds and hybrid scaffolds. Biomechanical testing of hybrid scaffolds demonstrated a significant increase in stability, compared with decellularized scaffolds. The mechanical properties of each scaffold are shown in Table 2.
The approach described herein to improve decellularized heart valve leaflet scaffold design consisted of the following steps: 1) extraction of a porcine aortic valve leaflet and removal of all xenogenic cells by decellularization process without altering the biological properties of the leaflet components (no tanning process), 2) coating of the decellularized valve leaflet scaffold with biodegradable polymer to enhance the mechanical characteristics of the porous leaflet scaffold. It was shown that coating with P4HB does indeed improve mechanical properties in vitro, and the biological properties of such hybrid scaffold were comparable with decellularized valve leaflet scaffold.
The electrospinning technology represents an attractive approach to scaffold fabrication for tissue engineering, because the ultrathin fibers produced have magnitude similarity to the fibrils in extracellular matrix of tissue.17 The fibers produced from P4HB randomly deposited on the surface of decellularized scaffolds and formed membrane structure with uniform thickness. The membranes showed acceptably well-blended structures, because there was no visual phase separation appearing on the surfaces or inside of the membranes. Our results showed that the electrospun membrane firmly combined with the surface of the decellularized scaffold and showed uniform fibers and microstructure.
The mechanical tests showed that P4HB coating has a reinforcing effect on the decellularized scaffold properties. This is expressed in higher values observed for the tensile strength and elastic modulus. For the decellularized scaffold, however, the tensile tests showed weakened mechanical properties. We assume that the behavior of these specimens is dominated by the adverse effects of the decellularization process. Decellularization of the specimens involved macroscopic shrinking processes presumably causing an impairment of the three-dimensional fiber architecture and thereby altering the structural properties of the leaflets. Together with the assessed leaflet thickness reduction after decellularization, our observations support the theory of how decellularization changes the biomechanical behavior of soft tissue by altering the structural organization of the matrix fibers. Similar effects can be observed comparing the behavior of fresh and dehydrated vegetables18 or native and enzymatically digested tendon tissue.19 The observed increase in tensile strength and elastic modulus in the hybrid scaffold can be attributed largely to the following reasons: 1) After the coating with P4HB, the thickness of the decellularized scaffold was increased, and the increase in tensile strength was partly due to increase in thickness. 2) The P4HB based tissues showed supraphysiologic tensile strength and elastic modulus.4 3) The anisotropy of the P4HB fibers that carried part of the stress and led to an increase of tensile strength under load, and P4HB would provide the decellularized scaffold with some comprehensive improvements related to stiffness (elastic modulus) when these two components were well blended together.
The use of uncoated decellularized matrix with or without in vitro autologous preseeding for heart valve replacement remains controversial. We believe that reseeding of decellularized or acellular matrices before implantation is crucial. However, the ideal cell source for tissue engineering of heart valves is still not well defined. Accordingly, a broad variety of cells were used for the reseeding in prior investigations.20–22 The decision to use MSCs was based on the knowledge that the phenotypical characteristics of differentiated MSCs seeded onto PGA/P4HB composite scaffold and cultured in vitro were similar to those of interstitial cells of heart valve leaflets.23 Our study showed the similar results. Flow cytometric analysis and immunocytochemistry demonstrated that MSCs isolated from rat bone marrow could be used to provide cells of fibroblast-myofibroblast lineage for the interstitial cell fraction of a substitute heart valve leaflet.
Cells on the surface of each scaffold stained positively for a-SMA, confirming the presence of MSCs. And a comparable layer of MSCs that expressed a-SMA in the decellularized scaffold and hybrid scaffold was demonstrated. A confluent cell layer of the surface of the decellularized scaffold and hybrid scaffold was further proved by HE staining and SEM. Overall, morphology and ultra-structure analysis showed cell anchor to both scaffolds equally. These results demonstrated that hybrid scaffolds were supportive for cell growth, and coating with P4HB did not weaken the excellent cellular adhesive property of decellularized scaffold. The complete repopulation was further proved by DNA analysis. The decellularized scaffold and hybrid scaffold revealed comparable amounts of DNA content. In addition, the decellularized scaffold and hybrid scaffold showed almost comparable amounts of 4-OHP and collagen. Our results showed that, compared with decellularized scaffolds, the hybrid scaffolds revealed the similar effects on the cell proliferation and the formation of extracellular matrix.
In conclusion, hybrid scaffold was fabricated from decellularized porcine aortic valve leaflet and coated with P4HB using an electrospinning technique. Then MSCs were seeded onto the hybrid scaffold. We demonstrated that hybrid scaffold has improved biomechanical characteristics, and its biological characteristics are comparable with decellularized scaffold. This is the first attempt in tissue engineered heart valves to fabricate hybrid scaffold using an electrospinning technique combined with MSCs as seeded cells. We believe that the problem of impaired biomechanics in the decellularized heart valve matrix can be overcome by coated with P4HB. Altogether this study demonstrates the feasibility and improved biomechanical characteristics of a novel hybrid heart valve leaflet scaffold for an application in tissue engineering.
Supported by grants from the National Natural Science Foundation of China (Nos. 30371414, 30571839, and 30600608).
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