Application of Decellularized Scaffold Combined with Loaded Nanoparticles for Heart Valve Tissue Engineering in vitro

The purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-β1（TGF-β1）,by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro.Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method,and their morphology was observed under a scanning electron microscope.Decelluarized valve scaffolds,prepared by using trypsinase and TritonX-100,were modified with nanoparticles by carbodiimide,and then TGF-β1 was loaded into them by adsorption.The TGF-β1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay.Whether unseeded or reseeded with myofibroblast from rats,the morphologic,biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions.The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles.The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds.Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment,which is beneficial for an application in heart valve tissue engineering.

Development of Biofunctionalized Cellulose Acetate Nanoscaffolds for Heart Valve Tissue Engineering

Currently-used mechanical and biological heart valve prostheses have a satisfactory short-term performance, but may exhibit several major drawbacks on the long-term. Mechanical prostheses, based on carbon, metallic and polymeric components, require permanent anticoagulation treatment, and their usage often leads to adverse reactions, e.g. thromboembolic complications and endocarditis. In recent years, there is a need for a heart valve prosthesis that can grow, repair and remodel. The concept of tissue engineering offers good prospects into the development of such a device. An ideal scaffold should mimic the structural and purposeful profile of materials found in the natural extracellular matrix (ECM) architecture. The goal of this study was to develop cellulose acetate scaffolds (CA) for valve tissue regeneration. After their thorough physicochemical and biological characterization, a biofunctionalization process was made to increase the cell proliferation. Especially, the surface of scaffolds was amplified with functional molecules, such as RGD peptides (Arg-Gly-Asp) and YIGSRG laminins (Tyrosine-Isoleucine-Glycine-Serine-Arginine-Glycine) which immobilized through biotin-streptavidin bond, the strongest non-covalent bond in nature. Last step was to successfully coat an aortic metallic valve with CA biofunctionallized nanoscaffolds and cultivate cells in order to create an anatomical structure comparable to the native valve. Promising results have been obtained with CA-based nanoscaffolds. We found that cells grown successfully on the biofunctionalized valve surface thereby scaffolds that resemble the native tissues, elaborated with bioactive factors such as RGD peptides and laminins not only make the valve’s surface biocompatible but also they could promote endothyliazation of cardiac valves causing an anti-coagulant effect

Fabrication of a Novel Hybrid Scaffold for Tissue Engineered Heart Valve

The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells （MSCs） onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly（3-hydroxybutyrate-co-4-hydroxybutyrate） using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically （hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy）, biochemically （DNA and 4-hydroxyproline） and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline （P〉0.05）. However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls （P〈0.05）. This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.

Expression of matrix metalloproteinase-9 was effected by epoxy chloropropan on creating tissue engineered heart valves

Objectives To investigate the effects of epoxy chloropropan on the expression of matrix metalloproteinases-9 （MMP-9）in creating tissue engineered heart valves（TEHV）,on the tissue structures of TEHV,and to study the effects of epoxy chloropropan on the calcification of TEHV.Methods The porcine aortic valve leaflets were digested and decellularized by using detergent and trypsin.Those treated with 0.3% glutaraldehyde for 48 hours were the control group;those treated with 3% epoxy choloropropan for 24 hours were the experimental group.The cultured human bone marrow mesenchymal stem cells（hBMSCs）were seeded onto the decellularized scaffolds of TEHV.The histological studies were done with pathological sections and scanning electron microscopy and reverse transcriptase-polymerase chain reaction（RT-PCR）were used to detect the expression of MMP-9.Results In the experimental group.the histology showed that the BMSCs grew well into the pores and formed a confluent layer in decellularized scaffolds;RT-PCR indicated significantly attenuated expressions of MMP-9,compared with the control（P〈0.05）.Conclusion The decellularized porcine aortic valves treated with 3% epoxy chloropropan may inhibit the expression of MMP-9;therefore epoxy chloropropan may prevent the calcification of tissue engineered heart valves.

Immobilization of Decellularized Valve Scaffolds with Arg-Gly-Asp-containing Peptide to Promote Myofibroblast Adhesion

The cell adhesive properties of decellularized valve scaffolds were promoted by immobilization of valve scaffold with arginine-glycine-aspartic acid （RGD）-containing peptides. Porcine aortic valves were decellularized with trypsin/EDTA, and detergent Triton X-100. With the help of a coupling reagent Sulfo-LC-SPDP, the valve scaffolds were immobilized with glycine-arginine-glycine-aspartic acid-serine-proline-cysteine （GRGDSPC） peptide. X-ray photoelectron spectroscopy （XPS） was used for surface structure analysis. Myofibroblasts harvested from rats were seeded onto the valve scaffolds. Cell count by using microscopy and modified MTT assay were performed to assess cell adhesion. Based on the spectra of XPS, the conjugation of GRGDSPC peptide with decellularized valve scaffolds was confirmed. Both cell count and MTT assay showed that myofibroblasts were much easier to adhere to the modified valve scaffolds, which was also confirmed histologically. Our findings suggest that it is feasible to immobilize RGD-containing peptides onto decellularized valve scaffolds. And the technique can effectively promote cell adhesion, which is beneficial for in vitro tissue engineering of heart valves.

Immobilization of RGD Peptidcs onto Decellularized Valve Scaffolds to Promote Cell Adhesion

Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD （arginine-glycine-aspartic acid） containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

Structural and biomechanical characterizations of acellular porcine mitral valve scaffolds:anterior leaflets,posterior leaflets,and chordae tendineae

Mitral valve(MV)tissue engineering is still in its early stage,and one major challenge in MV tissue engineering is to identify appropriate scaffold materials.With the potential of acellular MV scaffolds being demonstrated recently,it is important to have a full understanding of the biomechanics of the native MV components and their acellular scaffolds.In this study,we have successfully characterized the structural and mechanical properties of porcine MV components,including anterior leaflet(AL),posterior leaflet(PL),strut chordae,and basal chordae,before and after decellularization.Quantitative DNA assay showed more than 90%reduction in DNA content,and Griffonia simplicifolia(GS)lectin immunohistochemistry confirmed the complete lack of porcine𝛼-Gal antigen in the acellular MV components.In the acellular AL and PL,the atrialis,spongiosa,and fibrosa trilayered structure,along with its ECM constitutes,i.e.,collagen fibers,elastin fibers,and portion of GAGs,were preserved.Never-theless,the ECM of both AL and PL experienced a certain degree of disruption,exhibiting a less dense,porous ECM morphology.The overall anatomical morphology of the strut and basal chordae were also maintained af-ter decellularization,with longitudinal morphology experiencing minimum disruption,but the cross-sectional morphology exhibiting evenly-distributed porous structure.In the acellular AL and PL,the nonlinear anisotropic biaxial mechanical behavior was overall preserved;however,uniaxial tensile tests showed that the removal of cellular content and the disruption of structural ECM did result in small decreases in maximum tensile modulus,tissue extensibility,failure stress,and failure strain for both MV leaflets and chordae.