Tissue engineered heart valves: autologous cell seeding on biodegradable polymer scaffold

We previously reported on the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts then subsequently coated with endothelial cells. Using these methods, autologous cell/polymer constructs were implanted in 6 animals. In 2 additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, using cardiopulmonary bypass, the right-posterior leaflet of the pulmonary valve was resected completely and replaced with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. After 6 h and 1, 6, 7, 9, and 11 weeks, the animals were sacrificed and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. Animals receiving leaflets made from polymer without cell seeding were sacrificed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were degraded completely leaving no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group; 4-hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets. In the tissue-engineered heart valve leaflet, transplanted autologous cells generated proper matrix on the polymer scaffold in a physiologic environment at a period of 8 weeks after implantation.     

Experimental in vitro endothelialization of cardiac valve leaflets.

This study reports our results with vitro endothelialization of fresh nonpreserved homograft valve leaflets compared with mild alternatively preserved valves and valves treated by preservation procedures commonly used for commercially available tissue valves. In vitro lining of biological heart valves with cultured autologous endothelial cells might help prevent the detrimental effects of degeneration on valve durability. To investigate the growth characteristics of endothelial cells on valve bioprostheses, three different methods of storage and preservation were compared. After precoating with fibronectin and seeding of 4.4 x 10(4) endothelial cells/cm2 onto the different leaflet surfaces, primary adherence, growth kinetics, morphology, and maintenance of monolayer integrity were studied over a period of 10 days. On valve leaflet surfaces of group 1 (fresh nonpreserved homograft valve leaflets) and group 2 (mild alternatively preserved valves), endothelial cells grew to persistent monolayers between days 6 and 10. In contrast, endothelial cell proliferation with monolayer growth could not be achieved on the group 3 leaflets (preserved like commercially available biological valve prostheses). In that group, no viable endothelial cells could be found on the valve surfaces 2 days after seeding. These results demonstrate the theoretical feasibility of endothelializing biological heart valve leaflets in vitro if they are not preserved and stored according to commonly used procedures. Provided such an endothelium can withstand the mechanical forces after implantation in vivo, in vitro endothelialization might contribute either to the development of new biological heart valves for modern cardiac surgery or to the improvement of clinical results with homograft valve transplants.     

Further insights into normal aortic valve function: role of a compliant aortic root on leaflet opening and valve orifice area

BACKGROUND: This study aims to find the fundamental differences in the mechanism of opening and closing of a normal aortic valve and a valve with a stiff root, using a dynamic finite element model. METHODS: A dynamic, finite element model with time varying pressure was used in this study. Shell elements with linear elastic properties for the leaflet and root were used. Two different cases were analyzed: (1) normal leaflets inside a compliant root, and (2) normal leaflets inside a stiff root. RESULTS: A compliant aortic root contributes substantially to the smooth and symmetrical leaflet opening with minimal gradients. In contrast, the leaflet opening inside a stiff root is delayed, asymmetric, and wrinkled. However, this wrinkling is not associated with increased leaflet stresses. In compliant roots, the effective valve orifice area can substantially increase because of increased root pressure and transvalvular gradients. In stiff roots this effect is strikingly absent. CONCLUSIONS: A compliant aortic root contributes substantially to smooth and symmetrical leaflet opening with minimal gradients. The compliance also contributes much to the ability of the normal aortic valve to increase its effective valve orifice in response to physiologic demands of exercise. This effect is strikingly absent in stiff roots.     

脱细胞牛心包种植人血管混合细胞构建组织工程心脏瓣膜

目的 探讨在脱细胞牛心包上种植人血管混合细胞构建组织工程心脏瓣膜的方法与效果.方法 将体外培养的人血管混合细胞种植在脱细胞牛心包材料上,观察种子细胞形态学和免疫组织化学变化和牛心包超微结构变化,并测定内皮细胞分泌t-PA和PAI-1的活性.结果 种子细胞全层覆盖脱细胞牛心包表面,并浸润至组织内部生长;牛心包表面为扁平、片状、分层的细胞覆盖,细胞表面有少量绒毛和纤细的纤维结构.免疫组化显示内皮细胞Ⅷ因子、平滑肌细胞α-肌动蛋白、成纤细胞Fibronectin相关抗原呈阳性表达;种子细胞能分泌纤溶活性物质t-PA和PAI-1.结论 人血管混合细胞种植后附着满意,与脱细胞牛心包生物相容性好,生长状况好,并具有内皮功能.     

去细胞猪主动脉瓣移植于犬腹主动脉内构建组织工程瓣

目的 探讨在犬腹主动脉内构建组织工程心脏瓣膜的实验方法。方法 将预种犬血管间质细胞和内皮细胞的去细胞猪主动脉瓣叶(猪瓣),移植于6条犬的腹主动脉内,于术后4,6,8和10周对移植瓣叶进行形态、组织结构及免疫组化染色观察。结果 (1)移植术后4周时瓣叶周边有多层细胞长入,新细胞外基质形成,原支架组织部分吸收。(2)10周末猪瓣组织完全吸收,为宿主细胞及新合成的细胞外基质取代。基质中间质细胞主要为成纤维细胞和肌纤维母细胞。细胞外基质成分主要为Ⅰ、Ⅲ型胶原和少量弹力纤维,并含有中性和酸性黏多糖。(3)内皮细胞覆盖于瓣叶表面。结论 (1)移植于犬腹主动脉内的去细胞猪瓣于移植术后10周末基本构成组织工程瓣叶;(2)腹主动脉内异位移植是一种可供选择的实验研究方法。     

Mechanical stress as cause of aortic valve disease. Presentation of a new aortic root prosthesis

We present an overview of studies on the aortic valve and propose that mechanical stress is a main causative factor in the degenerative valvular disease. In the normal aortic valve, the leaflets have a smooth surface, free of wrinkles and creases, throughout the opening process. This smooth leaflet surface during motion is achieved by the "pull and release" movement of the commissures, which occurs because of the compliance of the aortic root. When the aortic root is stiffened, either by artificial means or by the loss of elasticity due to aging, the leaflet dynamics change significantly. The leaflets develop a significant number of creases and wrinkles during the opening process. In the bileaflet valve, the leaflets develop similar creasing and wrinkling during the opening process. This happens mainly due to the less-than-ideal design of the bileaflet valve and in spite of the compliant aortic root. When the aortic valve is spared using a noncompliant tube graft, a similar phenomenon of leaflet creasing occurs. Because the creasing produces high stresses from bending and buckling, it is damaging to the leaflet tissue and can lead to degenerative and calcific valvular disease. Based on these observations a new aortic root prosthesis with compliant sinuses has been designed for the valve sparing operation.     

组织工程心脏瓣叶的体外培养

目的 探讨瓣叶支架种植细胞后,不同体外培养时间瓣叶的生成情况. 方法杂种猪9只,取主动脉,分离内皮细胞、成纤维细胞和平滑肌细胞,培养扩增.将成纤维细胞、平滑肌细胞和内皮细胞按顺序种植在预湿处理的支架材料上,以首次种植成纤维细胞、平滑肌细胞为起点,取7天、14天和28天样本使用戊二醛固定,用扫描电子显微镜(SEM)检测,对培养28天的样本进行组织学检查. 结果 SEM检测显示:随着培养时间的延长,细胞数量增加,细胞间连接出现,并有基质生成;至28天,瓣膜支架上可见大量细胞黏附,细胞融合成片,表面可见基质形成.支架的细胞覆盖率也随时间的延长明显增加.组织学检测见大量细胞黏附材料,细胞已长入材料中部. 结论体外培养的瓣叶存在生长活性,使用组织工程方法有可能生成组织工程心脏瓣膜.     

Decellularization of rat aortic valve allografts reduces leaflet destruction and extracellular matrix remodeling

OBJECTIVES: Decellularization of aortic valve allografts in advance of transplantation is a promising approach to overcome immune-induced early graft failure. In this study the effects of in vitro cell extraction on extracellular matrix molecules and in vivo remodeling of decellularized aortic valves were investigated in a heterotopic aortic valve rat implantation model. METHODS: Rat aortic valve conduits were decellularized by a 2-step detergent-enzymatic extraction method involving sodium dodecyl sulfate in combination with RNase and DNase. Cellular and acellular allogeneic (2x, n = 4) and syngeneic valve grafts (2x, n = 3) were grafted infrarenally into the descending aorta for 21 days. Immunohistochemical techniques were used to study extracellular matrix constitution (elastin, collagen, fibronectin, and chondroitin sulfate) and cellular infiltration. RESULTS: The decellularization procedure resulted in a complete loss of all cellular structures from the entire valve conduit with minimal damage to the extracellular matrix. All transplanted cellular allografts became deformed, swollen, and acellular with major changes in extracellular matrix structure. The transplanted decellularized allografts, however, retained normal preserved valve leaflets comparable to transplanted cellular and acellular syngeneic grafts. With the exception of cellular syngeneic grafts, all other grafts showed retrovalvular thrombi. CONCLUSIONS: Damage to the valves caused by decellularization technique is much less than the damage caused by the recipient's immune response. In vitro removal of viable cells in (cryopreserved) homografts may decrease graft failure. Seeding with autologous or major histocompatibility complex-matched donor endothelial cells will be necessary to diminish damage induced by an absent blood-tissue barrier.     

Reconstruction of mitral valve chordae and leaflets with one piece of autologous pericardium in extensively destructed mitral valve due to active infective endocarditis

A 20-year-old female patient underwent urgent surgery for extensive mitral valve endocarditis. All marginal chordae and rough zone of A3 leaflet, posterior commissure leaflet, and P3 leaflet down to the annulus became defective after complete debridement of infected tissues. After annular plication, defective leaflets and chordae were reconstructed with a piece of triangular shaped autologous pericardium. Top of the pericardium was directly attached to the posterior papillary muscle, side edges to remnant leaflets, and the base to the annulus, thus substituting for chordae and leaflets at once. No mitral regurgitation was observed during 3 years of follow-up after the operation.     

Preseeding with autologous fibroblasts improves endothelialization of glutaraldehyde-fixed porcine aortic valves

OBJECTIVE: This study represents the development of a treatment and seeding procedure to improve endothelial cellular adhesion on glutaraldehyde-fixed valves. METHODS: Porcine aortic valves were fixed with 0.2% glutaraldehyde. Wall pieces of these valves had either no additional treatment (n = 4), incubation in M199 Earle (1x), with sodium carbonate at 2.2 g/L without l-glutamine for 24 hours (n = 4), or additional pretreatment with 5%, 10%, or 15% citric acid (three groups, n = 4 each). Thereafter the pieces were washed and buffered to a physiologic pH. This was followed by seeding of human endothelial cells (5 x 10(6) cells). On the basis of the results of these pilot tests, complete glutaraldehyde-fixed aortic roots treated with 10% citric acid were subjected to cell seeding. The valves were seeded with endothelial cells (4.3 x 10(6) cells) either alone (n = 4) or in combination with preseeding of autologous fibroblasts (2.4 x 10(7) cells, n = 4). After each seeding procedure specimens of the free wall of the grafts were taken. In addition, one leaflet was taken for histologic examination after endothelial cell seeding, after 7 days, and after 21 days. Finally, two commercially available stentless aortic valve prostheses (Freestyle; Medtronic, Inc, Minneapolis, Minn) were treated with 10% citric acid and seeded with human fibroblasts and endothelial cells. Specimen were taken according to the glutaraldehyde-fixed aortic roots. Specimen of all experiments were examined with scanning electron microscopy. Frozen sections were stained immunohistochemically for collagen IV, factor VIII, and CD31. RESULTS: On untreated glutaraldehyde-fixed aortic wall pieces, only poor adhesion (24%) was seen. No viable cells were found after 1 week. Cellular adhesion was best on aortic wall pieces pretreated with 10% citric acid. After 7 days, the cells formed a confluent layer. Endothelial cell seeding on citric acid-treated complete aortic valves showed 45% adhesion, but no confluent layer was found after 1 week. Preseeding of these valves with autologous fibroblasts resulted in an endothelial cellular adhesion of 76% and a confluent endothelial cell layer after 7 days. The layer remained stable for at least 21 days. Results of staining for collagen IV, factor VIII, and CD31 were positive on the luminal side of these valves, indicating the synthesis of matrix proteins and viability of the cells. Pretreatment of commercially available porcine valves with 10% citric acid and preseeding with autologous fibroblasts followed by endothelial cell seeding resulted in an adhesion of 78%. The cells formed a confluent cell layer after 7 days. CONCLUSIONS: Pretreatment of glutaraldehyde-fixed porcine aortic valves with citric acid established a surface more suitable for cellular attachment. Preseeding these valves with autologous fibroblasts resulted in a confluent endothelial cell layer on the luminal surface. Flow tests and animal experiments are necessary for further assessment of durability and shear stress resistance.     

Cell composition of the human pulmonary valve: a comparative study with the aortic valve--the VESALIO Project. Vitalitate Exornatum Succedaneum Aorticum labore Ingegnoso Obtinebitur

BACKGROUND: Cell populations present in human semilunar valves have not been investigated thoroughly. The aim of this study was to characterize the cell phenotypes in pulmonary valve leaflets (PVL) in comparison with aortic (AVL) valve leaflets. METHODS: AVL and PVL were dissected from hearts (n = 4) harvested from transplanted patients. Leaflets were processed for immunocytochemistry analysis and Western blotting procedures using a panel of monoclonal antibodies specific for cytoskeletal/contractile antigens. RESULTS: The fibrosa and the ventricularis layers of AVL had a higher cellularity than PVL. In PVL and AVL most cells were reactive for vimentin and nonmuscle (NM) myosin, though vimentin-positive cells were more abundant in AVL than in PVL. Sparse cells positive to anti-smooth muscle (SM) alpha-actin, calponin, and anti-SM myosin antibodies were found only at the outer edge of fibrosa. In Western blotting, AVL and PVL extracts were shown to be equally reactive for vimentin, SM alpha-actin, and NM myosin, whereas both valves were negative for SM myosin and SM22. CONCLUSIONS: Three distinct cell phenotypes have been identified in both valves: fibroblasts, myofibroblasts, and fetal-type SM cells whose distribution is specifically related to the valve layers. Although PVL and AVL cell populations differ quantitatively, some minor qualitative differences exist for vimentin and NM myosin distribution. These data are essential for studies aimed at repopulating valve scaffolds by using tissue engineering technology.     

Preservation of aortic leaflets during rapid deployment valve implantation in patients with aortic insufficiency

Development of a new surgical technique for aortic valve replacement with the use of rapid deployment/sutureless valve: a leaflet preservation technique applying imbrication methods to pliable aortic leaflets. We aim to decrease the incidence of paravalvular leak by preserving aortic leaflets in patients with aortic insufficiency and large aortic annulus.     

Kangaroo versus porcine aortic valve tissue--valve geometry morphology, tensile strength and calcification potential

OBJECTIVE: Valve related factors and patient related factors are responsible for calcification of valvular bioprostheses. Recent studies showed different donor and recipient species have different influences on the total calcification rate of bioprostheses. This study was performed to evaluate and compare Kangaroo aortic valve leaflets with porcine aortic valve leaflets. Experimental design. Prospective study. Setting. Cardio-thoracic experimental research of a university department. MATERIALS AND METHODS: Glutaraldehyde-fixed Kangaroo and porcine valve leaflets were evaluated in vitro according to valve geometry (internal diameter and leaflet thickness), morphology (light and electron microscopy) and tensile strength. In vivo evaluation consisted of implantation in a rat model for 8 weeks, Von Kossa stain for calcium and atomic absorption spectrophotometry for total extractable calcium content. RESULTS: Kangaroo valves indicated a smaller internal valve diameter as well as a thinner valve leaflet (p<0.01, ANOVA) at corresponding body weight, less proteoglycan spicules in the fibrosa, increased elasticity (p<0.05) and low calcification potential (p<0.01, confidence interval 95%). CONCLUSIONS: Kangaroo aortic valve leaflets have different valvular qualities compared to porcine valve tissue. Kangaroo valve leaflets are significantly superior to porcine valve leaflets as far as calcification is concerned. These results are encouraging and suggest further in vivo evaluation in a larger animal model before clinical application can be considered.     

Obstruction of St Jude Medical valves in the aortic position: histology and immunohistochemistry of pannus

OBJECTIVE: This study aims to reveal the morphological, histological, and immunohistochemical mechanism of pannus formation using resected pannus tissue from patients with prosthetic valve dysfunction. METHOD: Eleven patients with prosthetic valve (St Jude Medical valve) dysfunction in the aortic position who underwent reoperation were studied. We used specimens of resected pannus for histological staining (hematoxylin and eosin, Grocott's, azan, elastica van Gieson) and immunohistochemical staining (transforming growth factor-beta, transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, epithelial membrane antigen, CD34, factor VIII, CD68KP1, matrix metalloproteinase-1, matrix metalloproteinase-3, and matrix metalloproteinase-9). RESULTS: Pannus without thrombus was observed at the periannulus of the left ventricular septal side; it extended into the pivot guard, interfering with the movement of the straight edge of the leaflet. The histological staining demonstrated that the specimens were mainly constituted with collagen and elastic fibrous tissue accompanied by endothelial cells, chronic inflammatory cells infiltration, and myofibroblasts. The immunohistochemical findings showed significant expression of transforming growth factor-beta, transforming growth factor-beta receptor 1, CD34, and factor VIII in the endothelial cells of the lumen layer; strong transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, and epithelial membrane antigen in the myofibroblasts of the media layer; and transforming growth factor-beta, transforming growth factor-beta receptor 1, and CD68KP1 in macrophages of the stump lesion. CONCLUSIONS: Pannus appeared to originate in the neointima in the periannulus of the left ventricular septum. The structure of the pannus consisted of myofibroblasts and an extracellular matrix such as collagen fiber. The pannus formation after prosthetic valve replacement may be associated with a process of periannular tissue healing via the expression of transforming growth factor-beta.     

Aortic valve reconstruction using self-developed aortic valve plasty system in aortic valve disease

Aortic valve disease is usually treated by prosthetic valve replacement. We have performed aortic valve plasty (AVP) using glutaraldehyde-treated autologous pericardium. AVP was performed for 88 patients from April 2007 through August 2009. Sixty-five patients had aortic stenosis, and 23 patients had aortic regurgitation (AR). Twenty-one patients showed bicuspid aortic valves, and one patient showed quadricuspid valve. There were 43 males and 45 females. Their mean age was 70.6±10.5 years old. First, diseased leaflets excised. Then, the distance between each commissure was measured. The new leaflet were trimmed with an original template from a glutaraldehyde-treated autologous pericardium sample. Finally, the annular margin of the pericardial leaflet was running sutured to each annulus. There was no operative mortality or embolic event. Postoperative echocardiography revealed a mean peak pressure gradient (PG) of 19.0±9.1 mmHg one week after surgery. Thirty-two patients had echocardiography one year after surgery. The peak PG became 12.9±5.8 mmHg. Ten patients showed no AR, 20 patients showed trivial AR, and two patients showed mild AR. Freedom from reoperation is 100% at three years follow-up.     

Leaflet extension technique in a congenital aortic valve regurgitation

A 17-year-old boy who was diagnosed as congenital aortic valve regurgitation underwent the leaflet extension technique in 3 aortic leaflets using glutaraldehyde-preserved autologous pericardium. Intraoperative transesophageal echocardiography and postoperative cardiac catheter revealed grade I regurgitation and the patient has been doing well for more than 4 months after the surgery. The leaflet extension technique is considered to be an acceptable surgical treatment for aortic valve disease especially in young patients to whom valve replacement or Ross procedure might not be suitable. A careful long-term follow-up will be necessary to evaluate the durability of the leaflet extension technique with autologous pericardium.     

Opening and closing characteristics of the aortic valve after valve-sparing procedures using a new aortic root conduit

BACKGROUND: The durability of aortic valve-sparing procedures is negatively affected by increased leaflet stress in the absence of normally shaped sinuses of Valsalva. We compared valve motion after remodeling procedures using a standard conduit and a specifically designed aortic root conduit. METHODS: Echocardiographic studies of the aortic valve dynamics were performed in 14 patients after remodeling of the aortic root (7 standard conduits, group A; 7 new conduits, group B) and in 7 controls (group C). Opening and closing leaflet velocities and percent of slow closing leaflet displacement were measured. Root distensibility and the pressure strain of the elastic modulus were measured at all root levels. RESULTS: Root distensibility and the pressure strain of the elastic modulus were different in group A and B only at the sinuses (p < 0.001). Opening and closing leaflet velocities were not different among groups. Slow closing leaflet displacement was markedly more evident in group B patients (24.2%+/-1.9% versus 2.5%+/-1.9% in group A, p < 0.001) and similar to controls (22.1%+/-7.9%). CONCLUSIONS: The new conduit guarantees dynamic features of the aortic valve leaflets superior to those obtained with standard conduits and more similar to normal subjects.     

Fabrication of a Novel Hybrid Heart Valve Leaflet for Tissue Engineering: An In Vitro Study

The objective of this study was to fabricate biomatrix/polymer hybrid heart valve leaflet scaffolds using an electrospinning technique and seeded by mesenchymal stem cells. Mesenchymal stem cells were obtained from rats. Porcine aortic heart valve leaflets were decellularized, coated with basic fibroblast growth factor/chitosan/poly-4-hydroxybutyrate using an electrospinning technique, reseeded, and cultured over a time period of 14 days. Controls were reseeded and cultured over an equivalent time period. Specimens were examined biochemically, histologically, and mechanically. Recellularization of the hybrid heart valve leaflet scaffolds was significantly improved compared to controls. Biochemical and mechanical analysis revealed a significant increase of cell mass, 4-hydroxyproline, collagen, and strength in the hybrid heart valve leaflets compared to controls. This is the first attempt in tissue-engineered heart valves to fabricate hybrid heart valve leaflets using mesenchymal stem cells combined with a slow release technique and an electrospinning technique.     

Early aortic bioprosthetic valve deterioration in an octogenarian

We experienced extremely early aortic bioprosthetic valve deterioration with leaflet calcification and stiffening 2 1/2 years after aortic valve replacement in a female octogenarian. We could not identify the possible reason for this devastating complication; however, daily calcium supplement consumption may play a role of acceleration of calcium deposition in the leaflets of implanted bioprosthetic heart valves.     

Hammock mitral valve in an adult patient

We describe a case of hammock mitral valve in an adult. A 38-year-old woman presented with recently acquired dyspnea. Echocardiography showed severe mitral stenosis related to a hammock mitral valve characterized by the presence of an overdeveloped papillary muscle that gives short chordae to both mitral leaflets. No other valve insufficiency was noted. At operation, we observed direct continuity accompanying several fenestrations between both leaflets and the huge abnormal muscular band just underneath the mural leaflet. Resection of the anterior leaflet and was successfully replaced with a bioprosthetic valve. Her postoperative course was unremarkable.