Steady and pulsatile flow studies on a trileaflet heart valve prosthesis

The need for better and longer lasting trileaflet valves has led to the design and development of the ABIOMED polymeric trileaflet valve prosthesis. In vitro fluid dynamic studies in the aortic position indicate that overall it has improved leaflet motion characteristics and pressure drop characteristics compared to the Carpentier-Edwards porcine and Ionescu-Shiley pericardial tissue valves in current clinical use. The ABIOMED valve is, however, more stenotic compared to the St. Jude and Medtronic-Hall low profile mechanical valves, at normal cardiac outputs. Steady and pulsatile flow velocity measurements with a laser-Doppler anemometer system indicate that the flow field downstream of the ABIOMED valve is jet-like and leads to elevated shear stresses. These shear stresses are, however, lower than those observed with the Ionescu-Shiley and Carpentier-Edwards tissue valves. The ABIOMED valves tested had been originally configured for use in valved conduits, and it is therefore our opinion that further improvements can be made to the valve and stent design which would enhance its fluid dynamic performance.     

小口径组织工程血管体外构建的研究

目的 探讨间充质干细胞体外构建组织工程血管的可行性.方法 将体外培养扩增的犬骨髓间充质干细胞(MSCs)定向分化为平滑肌样细胞和内皮样细胞,接种于ε-己内酯/L-丙交酯(PCLA)支架上,将其置于生物反应器内,在搏动性力学(100±20/55±20)mm Hg(1 mm Hg=0.133 kPa)刺激条件下培养.3 d后行血管组织学检测.结果 血管支架拉伸强度6.1 MPa;骨髓间充质干细胞成功定向分化为平滑肌样细胞和内皮样细胞;血管腔内表面完全为细胞覆盖,表面的细胞沿液体流动的方向分布;种植的部分细胞已经渗透入血管壁内.结论 骨髓间充质干细胞可作为种子细胞,与PCLA支架在生物反应器内构建组织工程血管.     

Thoracic Surgery Directors Association Award. Bone marrow as a cell source for tissue engineering heart valves

BACKGROUND: This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach. METHODS: Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing. RESULTS: Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including alpha-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets. CONCLUSIONS: Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form "tissue" that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.     

种植人体活性细胞的生物心脏瓣膜

目前,种植人体活性细胞的生物心脏瓣膜主要有组织工程心脏瓣膜和种植人体活性细胞的猪主动脉瓣两种。组织工程心脏瓣膜是在人体可吸收的聚二醇酸纤维支架上种植人体同种活性细胞,先种植成纤维细胞,再种植单层内皮细胞包裹瓣叶。种植人体活性细胞的猪主动脉瓣是在清除原有细胞的组织内重建人体同种活性细胞。清除新鲜猪主动脉瓣呐原有细胞的方法是将瓣膜先经高、低渗溶液处理,然后用酶溶液处理。细胞经培养分离后,将成纤维细胞植入经处理的瓣膜组织,再植入内皮细胞。种植人体活性细胞的生物心脏瓣膜不会促使受者产生有害的免疫反应,并具有再生能力。     

In Vitro Comparison of Velocity Profiles and Turbulent Shear Distal to Polyurethane Trileaflet and Pericardial Prosthetic Valves

A comparative study of flow dynamics past biomer trileaflet valves and a pericardial bioprosthetic valve under steady and physiological pulsatile flow conditions in vitro is reported in this paper. The velocity profiles and the turbulent shear stresses distal to the valves were measured using laser Doppler anemometry. The authors' results showed that the velocity profiles distal to the trileaflet valves were similar to that measured distal to the pericardial valve. Higher magnitudes of absolute turbulent shear stresses were measured distal to the synthetic valves in comparison to the pericardial valves. However, when the stresses were nondimensionalized with respect to the orifice diameter at the inlet aspect, the stresses were comparable for all of the three valves. With design modifications to increase the orifice diameter at the inlet aspect of the polyurethane valves, the turbulent stresses distal to the valves can be minimized. Such in vitro studies on the flow dynamics past the polyurethane valves can provide information towards design changes to improve the performance characteristics of these valves. Polyurethane valves with flow characteristics comparable to the pericardial valves can be manufactured relatively inexpensively compared to mechanical or tissue valve prosthesis. Hence, the synthetic valves may be a viable alternative for short-term use in total artificial heart devices as a bridge to transplant.     

In vitro endothelialization of bioprosthetic heart valves provides a cell monolayer with proliferative capacities and resistance to pulsatile flow

OBJECTIVES: Degeneration of bioprosthetic heart valves has been suggested to be at least partly an immunogenic reaction toward the xenogeneic tissue. An autologous endothelial lining has been proposed to overcome this problem. We examined in vitro endothelialization of such tissue and retention of endothelial cells after exposure to flow resembling the in vivo situation. METHODS: Cultured human saphenous vein endothelial cells were used to in vitro endothelialize photo-oxidized bioprosthetic heart valves. The endothelialized valves were mounted in a specially designed flow device, creating a pulsatile flow through the valve. Maintenance of a confluent cell layer and deposition of basement membrane markers were determined with immunohistochemical labeling. RESULTS: Labeling of the main components of the basement membrane, laminin and collagen type IV, was verified within 6 hours after in vitro endothelialization. Under static conditions, 4-mm wide denudations were completely re-endothelialized in 4 days, which was similar to the growth rate on gelatin-coated cell culture plastic, which served as a control material. After exposure of endothelialized valves to pulsatile flows for 24 hours (80 beats/min, 3.4 L/min), there were minimal cell losses from the bioprostheses. The cell layer adapted to the pulsatile flow, as verified by rearrangement of morphology and intracellular stress fibers. CONCLUSIONS: This study shows the feasibility of in vitro endothelialization of photo-oxidized bioprosthetic heart valves. The cells are able to withstand a pulsatile flow in vitro, to develop basement membrane-like structures, and to re-endothelialize denuded areas. This technology may be used to enhance the performance of bioprosthetic heart valve prostheses.     

Design of a new pulsatile bioreactor for tissue engineered aortic heart valve formation

Evidence has been gathered that biomechanical factors have a significant impact on cell differentiation and behavior in in vitro cell cultures. The aim of this bioreactor is to create a physiological environment in which tissue engineered (TE) aortic valves seeded with human cells can be cultivated during a period of several days. The bioreactor consists of 2 major parts: the left ventricle (LV) and the afterload consisting of a compliance, representing the elastic function of the large arteries, and in series a resistance, mimicking the arterioles and capillaries. The TE aortic valve is placed between the LV and the compliance. With controllable resistance, compliance, stroke volume and frequency, and hydrodynamic conditions can be changed over a wide physiological range. This study resulted in a prototype of a compact pulsatile flow system for the creation of TE aortic valves. In addition a biocompatibility study of the used materials is performed.     

Bone marrow as a cell source for tissue engineering heart valves

Background

This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach.

Methods

Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing.

Results

Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including α-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets.

Conclusions

Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form “tissue” that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.     

体外组织工程血管支架内皮化的实验研究

目的：研究兔血管内皮细胞种植于组织工程血管支架内腔面的生长状况。方法：（1）将聚羟基乙酸（Plyglycolic acid,PGA)纤维无纺网和胶原纤维相混合，设计构建组织工程血管支架材料。（2）采用酶消化法从兔主动脉中分离培养兔血管内皮细胞并传代，纯化，接种于组织工程血管支架的内腔面，体外培养，并行电镜等观察。结果：该支架具有一定弹性和韧性，内皮细胞在其内表面形成较完整内皮细胞层，生长状况良好。结论：胶原包埋处理的PGA支架可以作为组织工程人工血管研究的较理想支架材料。为组织工程方法构筑具有分层结构的组织工程血管打下实验基础。     

Mesenchymal stem cell-based tissue engineering of small-diameter blood vessels

The aim of the study was to construct small-diameter vascular grafts using canine mesenchymal stem cells (cMSCs) and a pulsatile flow bioreactor. cMSCs were isolated from canine bone marrow and expanded ex vivo. cMSCs were then seeded onto the luminal surface of decellularized arterial matrices, which were further cultured in a pulsatile flow bioreactor for four days. Immunohistochemical staining and scanning electron microscopy was performed to characterize the tissue-engineered blood vessels. cMSCs were successfully seeded onto the luminal surface of porcine decellularized matrices. After four-day culture in the pulsatile flow bioreactor, the cells were highly elongated and oriented to the flow direction. Immunohistochemistry demonstrated that the cells cultured under pulsatile flow expressed Von Willebrand factor, an endothelial cell marker. In conclusion, cMSCs seeded onto decellularized arterial matrices could differentiate into endothelial lineage after culturing in a pulsatile flow bioreactor, which provides a novel approach for tissue engineering of small-diameter blood vessels.     

In Vitro Hydrodynamic Evaluation of a Scaffold for Heart Valve Tissue Engineering

Although prosthetic heart valves have saved many lives, the search for a living substitute continues with the aid of tissue engineering. Much progress has been made so far, but the translation of this technology to clinical reality remains a challenge, especially due to the structural complexity of heart valves and the harsh environment they are in. In a joint effort, researchers from Federal University of ABC and Institute Dante Pazzanese of Cardiology have conceived a new bioresorbable scaffold for heart valve tissue engineering (HVTE), whose hydrodynamic performance was first assessed and described in this work. The scaffold was studied at the mitral position of a left heart simulator from Escola Politécnica of the University of São Paulo, under 60 bpm and with no cell seeding. In this condition, two‐dimensional particle image velocimetry was performed to investigate the flow during diastolic and systolic phases. The results indicate that the scaffold can withstand the required intraventricular pressures for a simulated normal physiologic condition in a bioreactor. Furthermore, the averaged (N = 150) velocity vector maps showed a smooth and well‐distributed flow during diastole and qualitatively demonstrated no‐significant regurgitation at systole.     

去细胞猪主动脉瓣叶的获取和内皮细胞的种植

目的　探讨猪主动脉瓣叶去细胞后作为组织工程心脏瓣膜支架的可行性。　方法　经胰酶 - EDTA、表面活性剂和核酸酶处理 ,去除猪主动脉瓣叶的细胞成分 ,测定瓣叶去细胞前、后的生物力学特性 ,并在其表面种植新生牛主动脉内皮细胞 (BAECs) ;分别行大鼠皮下包埋实验。　结果　猪主动脉瓣叶中的细胞成分能完全去除 ,获得完整无细胞的纤维网状支架 ,断裂强度和断裂伸长率无明显变化 ;种植的 BAECs在去细胞瓣叶表面可形成一层连续的细胞层 ,其分泌前列环素 (PGI2 )的能力同直接种植在 2 4孔板中的比较 ,差异无统计学意义 (P>0 .0 5 )。　结论　猪主动脉瓣去细胞后获得的纤维支架可以用来构建组织工程瓣膜 ,适宜于血管内皮细胞的生长。     

Heart valve tissue engineering

Valvular heart disease is a significant cause of morbidity and mortality world-wide. Classical replacement surgery involves the implantation of mechanical valves or biological valves (xeno- or homografts). Tissue engineering of heart valves represents a new experimental concept to improve current modes of therapy in valvular heart surgery. Various approaches have been developed differing either in the choice of scaffold (synthetic biodegradable polymers, decellularised xeno- or homografts) or cell source for the production of living tissue (vascular derived cells, bone marrow cells or progenitor cells from the peripheral blood). The use of autologous bone marrow cells in combination with synthetic biodegradable scaffolds bears advantages over other tissue engineering approaches: it is safe, it leads to complete autologous prostheses and the cells are more easily obtained in the clinical routine. Even though we demonstrated the feasibility to construct living functional tissue engineered heart valves from human bone marrow cells, so far their general potential to differentiate into non-hematopoietic cell lineages is not fully exploited for tissue engineering applications.     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的:研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架.方法:应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液,接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物.应用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性,并用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态.结果:网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性.尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞;长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架紧密贴附,适度伸展并有基质分泌.结论:网状尿道支架适合尿道移行上皮细胞黏附生长,可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道.     

Tissue engineering with chondrocytes

Tissue engineering of cartilage, using chondrocytes based on the use of synthetic biodegradable polymer cell delivery vehicles (scaffolds), is an alternate treatment modality for replacing missing cartilage. Cartilage tissue engineering has an important role to play in the generation of graft material for head and neck reconstruction. It is an approach to fabricate cartilage constructs in vitro, which could be used in reconstructive surgery. Methods involve (1) harvesting septal cartilage during septoplasty, (2) isolating chondrocytes through enzymatic digestion of the septal cartilage, (3) expanding the cell number in a two-dimensional monolayer culture, using serum-free media, (4) seeding the cells onto a biodegradable polymer scaffold, and (5) cultivating the seeded scaffolds in a rotating bioreactor. In this article we briefly outline the methodology and clinical applications of cartilage grown ex vivo.     

Tissue-Engineered Heart Valve: Future of Cardiac Surgery

Background

Heart valve disease is currently a growing problem, and demand for heart valve replacement is predicted to increase significantly in the future. Existing “gold standard” mechanical and biological prosthesis offers survival at a cost of significantly increased risks of complications. Mechanical valves may cause hemorrhage and thromboembolism, whereas biologic valves are prone to fibrosis, calcification, degeneration, and immunogenic complications.

Methods

A literature search was performed to identify all relevant studies relating to tissue-engineered heart valve in life sciences using the PubMed and ISI Web of Knowledge databases.

Discussion

Tissue engineering is a new, emerging alternative, which is reviewed in this paper. To produce a fully functional heart valve using tissue engineering, an appropriate scaffold needs to be seeded using carefully selected cells and proliferated under conditions that resemble the environment of a natural human heart valve. Bioscaffold, synthetic materials, and preseeded composites are three common approaches of scaffold formation. All available evidence suggests that synthetic scaffolds are the most suitable material for valve scaffold formation. Different cell sources of stem cells were used with variable results. Mesenchymal stem cells, fibroblasts, myofibroblasts, and umbilical blood stem cells are used in vitro tissue engineering of heart valve. Alternatively scaffold may be implanted and then autoseeded in vivo by circulating endothelial progenitor cells or primitive circulating cells from patient’s blood. For that purpose, synthetic heart valves were developed.

Conclusions

Tissue engineering is currently the only technology in the field with the potential for the creation of tissues analogous to a native human heart valve, with longer sustainability, and fever side effects. Although there is still a long way to go, tissue-engineered heart valves have the capability to revolutionize cardiac surgery of the future.     

In vitro analysis of performance of porcine xenografts with inward bending of stent posts: real-time measurement of valve orifice area using an area meter

The influence of inward bending of the stent posts on bioprosthetic valve function was assessed in a hydromechanical simulation of the left heart. A Carpentier-Edwards mitral xenograft (31 mm) and an aortic xenograft (27 mm) were used. Valve function was evaluated before and after the stent posts were bent inward 15 degrees by suture constriction of the tops of the three posts. To evaluate the effects of the stent-post deformity on valve performance, the mean transvalvular pressure drop during steady flow, the bioprosthetic valve orifice area, and the maximum valve opening and closing speeds during pulsatile flow were measured using an area meter. Steady-flow data showed identical transvalvular pressure drops, and no significant difference in valve performance was detected in the pulsatile-flow study under the two experimental conditions (i.e., normal valve and deformed valve). We conclude that a 15-degree inward bending of the stent posts does not appreciably affect valve function in vitro.     

Tissue engineering of cardiac valves on the basis of PGA/PLA Co-polymers

The limitations of currently used heart valve devices are well known. For prosthetic valves they include infection risk and thrombembolic complications; biologic devices have limited durability. Particularly for pediatric cardiac patients the problem of a lack of growth potential remains a serious issue. The multidisciplinary field of tissue engineering potentially offers an attractive pathway to overcome these disadvantages. The basic concept of tissue engineering is to build a new "tissue" from individual cellular components in vitro using a scaffold to provide an architecture upon which the cells can organize and develop into the desired "tissue" prior to implantation. The scaffold provides the biomechanical profile for the replacement tissue until the cells produce their own extracellular matrix. This newly generated matrix would then ultimately provide the structural integrity and biomechanical profile for the newly developed tissue structure. This work focuses on the concept of using a synthetically produced co-polymer (polyglycolic acid/polylactid acid) as the scaffold for the development of a new generation of heart valves.     

The Dura Mater Valve: In Vitro Characteristics and Pathological Changes After Implantation in Calves

Human dura mater valves of various sizes with rigid and flexible stents were tested in an in vitro pulsatile mock circulatory system. A 22-mm flexible stent valve incorporating a new fabrication technique showed almost the same pressure gradient as a 28-mm rigid stent valve. The backflow/stroke volume ratio was about 4% at a net flow of 10 L/min. One hundred and five rigid stent-mounted dura mater valves were used in 51 pump implantations for up to 316 days. Collagen fiber degeneration began three months after implantation. Microscopic and macroscopic calcification of the valve tissue was seen in eight out of 105 valves, giving an overall incidence of 7.6%. The calcified degeneration was dystrophic in nature, not accompanied by cellular reactions, and was seen in the areas of the valve under stress. The degenerative changes were more severe in the left side than in the right side of the total artificial heart. These findings suggest that mechanical damage to the tissue plays an important role in the pathogenesis of calcification.     

Effects of Pulsatile Bioreactor Culture on Vascular Smooth Muscle Cells Seeded on Electrospun Poly (lactide‐co‐ε‐caprolactone) Scaffold

Electrospun nanofibrous scaffolds have several advantages, such as an extremely high surface‐to‐volume ratio, tunable porosity, and malleability to conform over a wide variety of sizes and shapes. However, there are limitations to culturing the cells on the scaffold, including the inability of the cells to infiltrate because of the scaffold's nano‐sized pores. To overcome the limitations, we developed a controlled pulsatile bioreactor that produces static and dynamic flow, which improves transfer of such nutrients and oxygen, and a tubular‐shaped vascular graft using cell matrix engineering. Electrospun scaffolds were seeded with smooth muscle cells (SMCs), cultured under dynamic or static conditions for 14 days, and analyzed. Mechanical examination revealed higher burst strength in the vascular grafts cultured under dynamic conditions than under static conditions. Also, immunohistology stain for alpa smooth muscle actin showed the difference of SMC distribution and existence on the scaffold between the static and dynamic culture conditions. The higher proliferation rate of SMCs in dynamic culture rather than static culture could be explained by the design of the bioreactor which mimics the physical environment such as media flow and pressure through the lumen of the construct. This supports regulation of collagen and leads to a significant increase in tensile strength of the engineered tissues. These results showed that the SMCs/electrospinning poly (lactide‐co‐ε‐caprolactone) scaffold constructs formed tubular‐shaped vascular grafts and could be useful in vascular tissue engineering.