It has been hypothesized that leaflet substrates with a trilayer structure and anisotropicmechanical properties could be useful for the production of functional and long-lasting tissue-engineered leaflets.To investiga...It has been hypothesized that leaflet substrates with a trilayer structure and anisotropicmechanical properties could be useful for the production of functional and long-lasting tissue-engineered leaflets.To investigate the influence of the anisotropic structural and mechanical characteristics of a substrate on cells,in this study,we electrospun trilayer anisotropic fibrous substrates and randomly oriented isotropic fibrous substrates(used as controls)from polycaprolactone polymers.Consequently,the random substrates had higher radial and lower circumferential tensile properties than the trilayer substrates;however,they had similar flexural properties.Porcine valvular interstitial cells cultured on both substrates produced random and trilayer cell-cultured constructs,respectively.The trilayer cell-cultured constructs had more anisotropic mechanical properties,17%higher cellular proliferation,14%more extracellular matrix(i.e.,collagen and glycosaminoglycan)production,and superior gene and protein expression,suggesting that more cells were in a growth state in the trilayer constructs than in the random constructs.Furthermore,the random and radial layers of the trilayer constructs had more vimentin,collagen,transforming growth factor-beta 1(TGF-ß1),transforming growth factor-beta 3(TGF-ß3)gene expression than in the circumferential layer of the constructs.This study verifies that the differences in structural,tensile,and anisotropic properties of the trilayer and random substrates influence the characteristics of the cells and ECM in the constructs.展开更多
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 ...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.展开更多
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 ...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 chloropropan 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.展开更多
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 matri...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.展开更多
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 an...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展开更多
Existing therapies for the treatment of chronic heart failure still have some limitations and there is a pressing need for the development of new therapeutic modalities. The amniotic membrane has been used for the tre...Existing therapies for the treatment of chronic heart failure still have some limitations and there is a pressing need for the development of new therapeutic modalities. The amniotic membrane has been used for the treatment of various diseases, such as conjunctive defects;however, the mechanisms behind its repair functions are still unclear. Regenerative medicine is seeking newer alternatives and among them, biomaterials have emerged in recent years for developing and manipulating molecules, cells, tissues or organs grown in laboratories in order to replace human body parts. Many such materials have been used for this purpose, either synthetically or biologically, in order to provide new medical devices. This review provides a wider view of the regeneration potential of the use of amniotic membrane as a potential biomaterial to facilitate the implementation of new research in surgical procedures. Amniotic membrane appears to be an alternative source of stem cells as well as an excellent biomaterial for cell-based therapeutic applications in engineering heart tissue.展开更多
Background Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilica...Background Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds. Methods Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 × 10^6 cells/cm^2 and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).Results EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells. Conclusions EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.展开更多
Interstitial Cajal-like cells are a distinct type of interstitial cell with a wide distribution in mammalian organs and tissues,and have been given the name"telocytes".Recent studies have demonstrated the po...Interstitial Cajal-like cells are a distinct type of interstitial cell with a wide distribution in mammalian organs and tissues,and have been given the name"telocytes".Recent studies have demonstrated the potential roles of telocytes in heart development,renewal,and repair.However,further research on the functions of telocytes is limited by the complicated in vivo environment.This study was designed to construct engineered heart tissue(EHT)as a three-dimensional model in vitro to better understand the role of telocytes in the architectural organization of the myocardium.EHTs were constructed by seeding neonatal cardiomyocytes in collagen/Matrigel scaffolds followed by culture under persistent static stretch.Telocytes in EHTs were identified by histology,toluidine blue staining,immunofluorescence,and transmission electron microscopy.The results from histology and toluidine blue staining demonstrated widespread putative telocytes with compact toluidine blue-stained nuclei,which were located around cardiomyocytes.Prolongations from the cell bodies showed a characteristic dichotomous branching pattern and formed networks in EHTs.Immunofluorescence revealed positive staining of telocytes for CD34 and vimentin with typical moniliform prolongations.A series of electron microscopy images further showed that typical telocytes embraced the cardiomyocytes with their long prolongations and exhibited a marked appearance of nursing cardiomyocytes during the construction of EHTs.This finding highlights the great importance of telocytes in the architectural organization of EHTs.It also suggests that EHT is an appropriate physical and pathological model system in vitro to study the roles of telocytes during heart development and regeneration.展开更多
Background Currently used heart valve prostheses are associated with anticoagulation complications or limited durability. The advancement of stem cell study and tissue-engineered heart valve research may offer a relat...Background Currently used heart valve prostheses are associated with anticoagulation complications or limited durability. The advancement of stem cell study and tissue-engineered heart valve research may offer a relatively ideal solution to these problems. Methods Bone marrow was aspirated from sternum of lamb goats to isolate BMCs. Cells were identified by flow cytometry and its capacity of differentiation. Cellular viability was assessed with Rhdomine 123 staining. 1 × 10^7cells were seeded on a patch of PGA sheet. After two-day in vitro culture, autologous cell/ scaffold sheets were used to replace the right posterior pulmonary valve leaflets under cardiopulmonary bypass. The leaflets were explanted at 2 days, 2, 6, 8 and 10 weeks after implantation. The samples were examined macroscopically, histologically, immunohistochemically, and by Scanning Electron Microscope (SEM). Two goats were implanted with acellular sheets and established as a control group. Results BMCs exhibited fibroblastoid morphology with good viability. Flow cytometry showed negative CD14 and CD45 expression. In vitro cultured BMCs demonstrated the potential to differentiate into adipocytes. The explanted leaflets resembled the characteristics of native extracellular matrix was leaflets macroscopicaIly in the cellular group. Histology showed synthesized and cells were distributed in the single-layered leaflets.Immunohistochemistry revealed positive staining for yon Willebrand factor, α-SMA, vimentin. A confluent cell surface was formed on the explanted TEHLs. No calcium deposited on the leaflets. In control group, the acellular scaffolds were completely degraded, without leaflet remained at 8 weeks. Conclusions It is possible to create tissue-engineered heart valves in vivo using autologous bone marrow-derived cells.展开更多
Tissue engineering heart valves(TEHV)may be the most promising valve substitute,but the study has been relatively stagnant in the recent five years due to the special position,function and mechanical property of heart...Tissue engineering heart valves(TEHV)may be the most promising valve substitute,but the study has been relatively stagnant in the recent five years due to the special position,function and mechanical property of heart valves.It is one of the key factors to select an ideal scaffold material in the construction of TEHV.And this article will briefly review the current research and progress on the scaffolds of TEHV,especially based on Chinese works.展开更多
The lifespan of biological heart valve prostheses available in the market is limited due to structural alterations caused by calcium phosphate deposits formed from blood plasma in contact with the tissues.The objectiv...The lifespan of biological heart valve prostheses available in the market is limited due to structural alterations caused by calcium phosphate deposits formed from blood plasma in contact with the tissues.The objective of this work is to present a comparative methodology for the investigation of the formation of calcium phosphate deposits on bioprosthetic and tissue-engineered scaffolds in vitro and the influence of mechanical forces on tissue mineralization.Based on earlier investigations on biological mineralization at constant supersaturation,a circulatory loop simulating dynamic blood flow and physiological pressure conditions was developed.The system was appropriately adapted to evaluate the calcification potential of decellularized(DCV)and glutaraldehyde-fixed(GAV)porcine aortic valves.Results indicated that DCV calcified at higher,statistically nonsignificant,rates in comparison with GAV.This difference was attributed to the tissue surface modifications and cell debris leftovers from the decellularization process.Morphological analysis of the solids deposited after 20 h by scanning electron microscopy in combination with chemical microanalysis electron-dispersive spectroscopy identified the solid formed as octacalcium phosphate(Ca8(PO4)6H2·5H2O,OCP).OCP crystallites were preferentially deposited in high mechanical stress areas of the test tissues.Moreover,GAV tissues developed a significant transvalvular pressure gradient increase past 36 h with a calcium deposition distribution similar to the one found in explanted prostheses.In conclusion,the presented in vitro circulatory model serves as a valuable prescreening methodology for the investigation of the calcification process of bioprosthetic and tissue-engineered valves under physiological mechanical load.展开更多
Ischemic heart disease and dilated cardiomyopathy followed by heart failure are a worldwide problem,which seriously challenge clinical outcomes and quality of life of patients. Heart failure is one of the major causes...Ischemic heart disease and dilated cardiomyopathy followed by heart failure are a worldwide problem,which seriously challenge clinical outcomes and quality of life of patients. Heart failure is one of the major causes of morbility and mortality. The human heart cannot regenerate significantly because adult cardiomyocytes are terminally differentiated and cannot replicate after injury. The loss of cardiomyocytes accounts for a decrease in myocardial function, which leads to heart failure. Conservative treatment for cardiac conditions has focused on the reduction of workload, and protection from risk factors and has little therapeutic effect on patients in end-stage heart failure. Heart transplantation represents a life-saving and life-extending treatment modality for end-stage heart failure. In spite of advances in surgical techniques, the shortage of availability of donor organs has limited this treatment modality and has prompted researchers to develop alternative approaches. Cardiomyocyte regeneration is a prospective treatment modality, of which, in vitro engineering of myocardial tissue has had promising outcomes that should help cope with failing hearts in the future. Over the past years, much progress has been made to replace infarcted, non-functioning myocardium with newly formed tissue by means of cell-grafting techniques. Our country has made substantial progress in this field and promises a bright future for clinical management of heart failure.展开更多
基金supported by the National Institute of Health(No.NIH R00HL134823).
文摘It has been hypothesized that leaflet substrates with a trilayer structure and anisotropicmechanical properties could be useful for the production of functional and long-lasting tissue-engineered leaflets.To investigate the influence of the anisotropic structural and mechanical characteristics of a substrate on cells,in this study,we electrospun trilayer anisotropic fibrous substrates and randomly oriented isotropic fibrous substrates(used as controls)from polycaprolactone polymers.Consequently,the random substrates had higher radial and lower circumferential tensile properties than the trilayer substrates;however,they had similar flexural properties.Porcine valvular interstitial cells cultured on both substrates produced random and trilayer cell-cultured constructs,respectively.The trilayer cell-cultured constructs had more anisotropic mechanical properties,17%higher cellular proliferation,14%more extracellular matrix(i.e.,collagen and glycosaminoglycan)production,and superior gene and protein expression,suggesting that more cells were in a growth state in the trilayer constructs than in the random constructs.Furthermore,the random and radial layers of the trilayer constructs had more vimentin,collagen,transforming growth factor-beta 1(TGF-ß1),transforming growth factor-beta 3(TGF-ß3)gene expression than in the circumferential layer of the constructs.This study verifies that the differences in structural,tensile,and anisotropic properties of the trilayer and random substrates influence the characteristics of the cells and ECM in the constructs.
基金supported by grants from National Natural Sciences Foundation of China (No.30571839,30600608 and 30872540)National High Technology Research and Development Program ("863" Program) of China (No.2009AA-03Z420)
文摘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.
文摘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 chloropropan 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.
基金supported by grants from the National Natural Sciences Foundation of China (No. 30571839, No. 30600608,No. 30872540)the National High Technology Research and Development Program of China (863 Program) (No. 2009AA03Z420)
文摘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.
文摘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
文摘Existing therapies for the treatment of chronic heart failure still have some limitations and there is a pressing need for the development of new therapeutic modalities. The amniotic membrane has been used for the treatment of various diseases, such as conjunctive defects;however, the mechanisms behind its repair functions are still unclear. Regenerative medicine is seeking newer alternatives and among them, biomaterials have emerged in recent years for developing and manipulating molecules, cells, tissues or organs grown in laboratories in order to replace human body parts. Many such materials have been used for this purpose, either synthetically or biologically, in order to provide new medical devices. This review provides a wider view of the regeneration potential of the use of amniotic membrane as a potential biomaterial to facilitate the implementation of new research in surgical procedures. Amniotic membrane appears to be an alternative source of stem cells as well as an excellent biomaterial for cell-based therapeutic applications in engineering heart tissue.
基金the grants from Shanghai Science Committee Fund for Key Research Project(No.04JC14012)Fudan University Med-X Fund Abstract
文摘Background Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds. Methods Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 × 10^6 cells/cm^2 and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).Results EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells. Conclusions EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.
基金supported by the National High Technology Research and Development Program of China(2012AA020506)Key Program of National Natural Science Foundation of China(31030032)+1 种基金National Natural Science Funds for Distinguished Young Scholar(31025013)the National Natural Science Foundation of China(31100697)
文摘Interstitial Cajal-like cells are a distinct type of interstitial cell with a wide distribution in mammalian organs and tissues,and have been given the name"telocytes".Recent studies have demonstrated the potential roles of telocytes in heart development,renewal,and repair.However,further research on the functions of telocytes is limited by the complicated in vivo environment.This study was designed to construct engineered heart tissue(EHT)as a three-dimensional model in vitro to better understand the role of telocytes in the architectural organization of the myocardium.EHTs were constructed by seeding neonatal cardiomyocytes in collagen/Matrigel scaffolds followed by culture under persistent static stretch.Telocytes in EHTs were identified by histology,toluidine blue staining,immunofluorescence,and transmission electron microscopy.The results from histology and toluidine blue staining demonstrated widespread putative telocytes with compact toluidine blue-stained nuclei,which were located around cardiomyocytes.Prolongations from the cell bodies showed a characteristic dichotomous branching pattern and formed networks in EHTs.Immunofluorescence revealed positive staining of telocytes for CD34 and vimentin with typical moniliform prolongations.A series of electron microscopy images further showed that typical telocytes embraced the cardiomyocytes with their long prolongations and exhibited a marked appearance of nursing cardiomyocytes during the construction of EHTs.This finding highlights the great importance of telocytes in the architectural organization of EHTs.It also suggests that EHT is an appropriate physical and pathological model system in vitro to study the roles of telocytes during heart development and regeneration.
基金supported by the grant from Guangdong Nature Science Foundation(7001117)
文摘Background Currently used heart valve prostheses are associated with anticoagulation complications or limited durability. The advancement of stem cell study and tissue-engineered heart valve research may offer a relatively ideal solution to these problems. Methods Bone marrow was aspirated from sternum of lamb goats to isolate BMCs. Cells were identified by flow cytometry and its capacity of differentiation. Cellular viability was assessed with Rhdomine 123 staining. 1 × 10^7cells were seeded on a patch of PGA sheet. After two-day in vitro culture, autologous cell/ scaffold sheets were used to replace the right posterior pulmonary valve leaflets under cardiopulmonary bypass. The leaflets were explanted at 2 days, 2, 6, 8 and 10 weeks after implantation. The samples were examined macroscopically, histologically, immunohistochemically, and by Scanning Electron Microscope (SEM). Two goats were implanted with acellular sheets and established as a control group. Results BMCs exhibited fibroblastoid morphology with good viability. Flow cytometry showed negative CD14 and CD45 expression. In vitro cultured BMCs demonstrated the potential to differentiate into adipocytes. The explanted leaflets resembled the characteristics of native extracellular matrix was leaflets macroscopicaIly in the cellular group. Histology showed synthesized and cells were distributed in the single-layered leaflets.Immunohistochemistry revealed positive staining for yon Willebrand factor, α-SMA, vimentin. A confluent cell surface was formed on the explanted TEHLs. No calcium deposited on the leaflets. In control group, the acellular scaffolds were completely degraded, without leaflet remained at 8 weeks. Conclusions It is possible to create tissue-engineered heart valves in vivo using autologous bone marrow-derived cells.
基金supported by the National Natural Science Foundation of China(Grant Nos.30371414,30571839,30600608).
文摘Tissue engineering heart valves(TEHV)may be the most promising valve substitute,but the study has been relatively stagnant in the recent five years due to the special position,function and mechanical property of heart valves.It is one of the key factors to select an ideal scaffold material in the construction of TEHV.And this article will briefly review the current research and progress on the scaffolds of TEHV,especially based on Chinese works.
基金This research was funded by the People Program(Marie Curie Actions)of the European Union’s Seventh Framework FP7/2007–2013/under REA grant agreement n°317512.
文摘The lifespan of biological heart valve prostheses available in the market is limited due to structural alterations caused by calcium phosphate deposits formed from blood plasma in contact with the tissues.The objective of this work is to present a comparative methodology for the investigation of the formation of calcium phosphate deposits on bioprosthetic and tissue-engineered scaffolds in vitro and the influence of mechanical forces on tissue mineralization.Based on earlier investigations on biological mineralization at constant supersaturation,a circulatory loop simulating dynamic blood flow and physiological pressure conditions was developed.The system was appropriately adapted to evaluate the calcification potential of decellularized(DCV)and glutaraldehyde-fixed(GAV)porcine aortic valves.Results indicated that DCV calcified at higher,statistically nonsignificant,rates in comparison with GAV.This difference was attributed to the tissue surface modifications and cell debris leftovers from the decellularization process.Morphological analysis of the solids deposited after 20 h by scanning electron microscopy in combination with chemical microanalysis electron-dispersive spectroscopy identified the solid formed as octacalcium phosphate(Ca8(PO4)6H2·5H2O,OCP).OCP crystallites were preferentially deposited in high mechanical stress areas of the test tissues.Moreover,GAV tissues developed a significant transvalvular pressure gradient increase past 36 h with a calcium deposition distribution similar to the one found in explanted prostheses.In conclusion,the presented in vitro circulatory model serves as a valuable prescreening methodology for the investigation of the calcification process of bioprosthetic and tissue-engineered valves under physiological mechanical load.
文摘Ischemic heart disease and dilated cardiomyopathy followed by heart failure are a worldwide problem,which seriously challenge clinical outcomes and quality of life of patients. Heart failure is one of the major causes of morbility and mortality. The human heart cannot regenerate significantly because adult cardiomyocytes are terminally differentiated and cannot replicate after injury. The loss of cardiomyocytes accounts for a decrease in myocardial function, which leads to heart failure. Conservative treatment for cardiac conditions has focused on the reduction of workload, and protection from risk factors and has little therapeutic effect on patients in end-stage heart failure. Heart transplantation represents a life-saving and life-extending treatment modality for end-stage heart failure. In spite of advances in surgical techniques, the shortage of availability of donor organs has limited this treatment modality and has prompted researchers to develop alternative approaches. Cardiomyocyte regeneration is a prospective treatment modality, of which, in vitro engineering of myocardial tissue has had promising outcomes that should help cope with failing hearts in the future. Over the past years, much progress has been made to replace infarcted, non-functioning myocardium with newly formed tissue by means of cell-grafting techniques. Our country has made substantial progress in this field and promises a bright future for clinical management of heart failure.