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。     

Viability Properties in Autologous Fascia Lata Heart Valve Grafts: A Transmission Electron Microscopical Study

Four frame-mounted autologous fascia lata heart valve grafts in situ for 18 to 41 months as well as one free-mounted fascia cusp in situ for 10 years were examined by transmission electron microscopy (TEM).In all cusps, areas of well-preserved subcellular structures were found as well as areas with degeneration and fragmentation of both nuclear and collagen fibers. Fibrocytes, fibroblasts, and a special cell form, the myofibroblast, were encountered. The appearance of this last cell form may explain the shrinkage phenomenon encountered in these valves, all retrieved at reoperation because of valve dysfunction. Some of the fascial cells thus stayed alive within the bloodstream for up to 10 years.     

Autologous fascia lata transplantation for heart valve replacement: A two-year experience at the National Heart Hospital, London

Between April 1969 and December 1970, 201 patients underwent 262 valve replacements with frame-mounted autologous fascia lata valves in the aortic, mitral, and/or tricuspid positions at the National Heart Hospital. A single fascia lata valve was inserted in 146 patients and multi-valve replacement was performed in 55 instances. Hospital and late mortality are comparable with that following other procedures of heart valve replacement. One hundred and fifty-four patients with 193 fascia lata valves were available for follow-up. A retrospective analysis of this method of valve replacement has been undertaken to assess the suitability of fascia lata as a heart valve substitute as well as the clinical results achieved by this method of valve replacement.     

Postoperative changes in autologous fascia lata heart valve grafts. A pathological study

In a series of 45 consecutive patients undergoing insertion of frame-mounted autologous fascia lata heart valve grafts, 11 of the grafts were examined microscopically following different durations of implantation, 8 of them at postmortem examination, in order to study the reaction of the implanted fascia tissue. Three of these grafts were also examined by radioautography.Signs of infection were observed in 5 of the 11 grafts studied microscopically. One important reason for infection in these patients must be that the fascia is exposed to a considerable risk of contamination during dissection and preparation. Strict sterility precautions must therefore be observed during tailoring of the grafts. On the other hand, it was found that fascia lata cells were able to remain viable and retain their metabolic activity for at least eighteen months after implantation of the tissue.     

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.     

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.     

The effect of steroids on Dupuytren's disease: role of programmed cell death

This study compared the rates of proliferation and apoptosis of cells within nodules of Dupuytren's disease and nodules from patients that had been injected preoperatively with steroid (Depo-Medrone). It also compared the effects of steroids in apoptosis in cultured Dupuytren's cells and control fibroblasts from palmar fascia and fascia lata. Steroids reduced the rate of fibroblast proliferation and increased the rate of apoptosis of both fibroblasts and inflammatory cells in Dupuytren's tissue. Steroids also produced apoptosis of cultured Dupuytren's cells but not of palmar fascia and fascia lata cells.     

Replacement of heart valves with frame-mounted tissue grafts

Ionescu, M. I., Pakrashi, B. C., Mary, D. A. S., Bartek, I. T., and Wooler, G. H. (1974). Thorax, 29, 56-67. Replacement of heart valves with frame-mounted tissue grafts. Between April 1969 and March 1973, 213 patients had heart valve replacement with frame-mounted autologous or homologous fascia lata or with heterologous pericardial grafts. There were 111 single aortic, 95 single mitral, and seven tricuspid valve replacements.     

Fascia lata valves: a clinicopathological study.

Sixteen frame-mounted fascia lata valves removed from the mitral, aortic or--in one patient--pulmonary position have been detailed histologically. These valves had remained in 15 patients (11 men and four women) for periods varying between 10 and 44 months. The reason for the original transplantation was either chronic rheumatic endocarditis or calcific aortic disease. In the mitral position, the leaflet in position nearest the site of the original anterior mitral valve cusp showed the least changes. The remaining two leaflets of the fascia lata valve in the mitral position, as well as those removed from the aortic or pulmonary position, showed more severe changes; these consisted of degeneration of collagen tissue and often a severe decrease of nuclei belonging to the fibroblastic series. These changes, as well as superimposition of fibrin or fibrous tissue, tended to become more pronounced the longer the valve had remained in the patient. Viability studies in valves removed from two patients have also been undertaken showing very greatly reduced activity. The possible causes for valve dysfunction have been reviewed, and the findings in this study suggest that contraction of fibrous tissue, which sandwiches the fascia lata valve cusps, may contribute to failure of satisfactory valve function. It is concluded that fascia lata forms a poor substitute for replacement of diseased cardiac valves.     

A fascial frustum valve for aortic valve replacement

The promising qualities of autologous fascia lata in heart valve replacement have resulted in a search for improved methods of fascial heart valve manufacture. This paper describes a simply made and inserted and reliably competent unsupported fascial valve for aortic valve replacement.     

Connective tissue structures in clubfoot: a morphologic study.

A morphologic study of connective tissue structures in clubfoot, with special emphasis on the presence of myofibroblasts, was undertaken to investigate the theory of retracting fibrosis as an etiologic factor. Nine idiopathic clubfeet from six patients were studied. Specimens from the medial and lateral capsule, medial and lateral fascia, spring and lacinate ligaments, and flexor digitorum longus, flexor hallucis longus, posterior tibialis, and Achilles tendon sheaths were investigated. Fifty specimens were examined using light microscopy and hematoxylin and eosin staining, and 26 were examined by transmission electron microscopy. Light microscopy failed to reveal any myofibroblast-like cells or any qualitative differences between specimens from capsule, fascia, ligaments, and tendon sheath. Using transmission electron microscopy, the authors identified two cell types: fibroblasts and mast cells. Some fibroblasts contained a network of microfilaments but all lacked microbundles, basal lamina, or plasmalemmal attachment plaques seen in typical myofibroblasts. Mast cells were rarely identified in capsular specimens. The absence of myofibroblast-like cells or typical myofibroblasts in clubfoot connective tissue structures does not support the theory of retracting fibrosis as a likely cause of contracture in idiopathic clubfoot.     

Androgen receptors in Dupuytren's contracture.

Palmar fascia tissue and cultured cells from patients with Dupuytren's contracture and from normal subjects were characterized and analyzed for androgen receptor expression. Androgen receptors have never been studied in Dupuytren's myofibroblasts and may have a role in its high male predominance. Surgical samples were collected from eight patients undergoing surgery for Dupuytren's contracture and from four patients with carpal tunnel syndrome, used as control tissue. Immunohistochemical analysis was performed on tissue samples and on cell cultures with anti-androgen receptor, anti-alpha-smooth muscle actin, anti-fibronectin, and anti-type I and III collagen antibodies using the biotin avidin peroxidase method as revelatory system. Immunostaining for androgen receptors in tissue samples and cultured cells revealed nuclear reaction in many Dupuytren's myofibroblasts, but in few fibroblasts of the normal palmar fascia. In a double-labeling study, androgen receptors were seen to co-localize with alpha-actin in both cell cultures and tissue samples. We present the first evidence that the palmar fascia is a target tissue for androgen action and that the expression of androgen receptors in Dupuytren's contracture is considerably higher than in the normal palmar fascia. Further studies will need to evaluate whether the androgen-responsive state of the tissue is related to the high incidence of Dupuytren's contracture in the male sex.     

Tissue engineering of heart valves: in vitro experiences

BACKGROUND: Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS: It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS: Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.     

An electron microscopic study of the fascia from the medial and lateral sides of clubfoot

Two cell types were observed in fascia from the lateral side of the clubfoot: a cell resembling the fibroblast of hypertrophic scar and a fibroblast-like cell with dilated rough endoplasmic reticulum. Three cell types were observed in fascia from the medial side of the clubfoot: typical fibroblasts, cells resembling myofibroblasts, and mast cells. The contracture of the medial side may be due to the myofibroblast-like cells, and this contracture may be enhanced by histamine released from the mast cells. The stretching of the lateral side is probably due to a compensatory fibrosis that maintains the tissue density.     

Autologous fascia lata for heart valve replacement

To obviate the drawbacks of prosthetic devices and in search of a better substitute for diseased heart valves a technique of using autologous fascia lata grafts attached to a support frame was developed. The technique of preparing mounted fascia lata grafts is briefly outlined and their surgical implantation into the mitral, tricuspid, and aortic areas is described. Since April 1969, 83 fascial grafts have been inserted into 76 patients with very good immediate and short-term results. Ten patients succumbed within the operative period. None of these deaths was due to failure of the graft per se. Thromboembolic manifestations have been completely absent although anticoagulants were not used. All sixty-six survivors are very much improved. Data concerning the structure and function of fascia are presented and the experimental and clinical use of fascia lata in extracardiac and cardiac surgery is reviewed. The use of autologous, living, fascia lata valves is considered to be a better approach for heart valve replacement because autologous fascia maintains its structure unchanged after transplantation and, being under mechanical stimulation, it retains its functional properties.     

222. Gegenwärtiger Stand der autologen Fascia lata-Transplantation beim Herzklappenersatz

Zusammenfassung Die Resultate nach Implantation von 327 külichen Herzklappen aus autologer Fascia lata bei 262 Patienten am National Heart Hospital London (April 1969–Dezember 1970) enthüllen bei vergleichbaren Frühergeb-nissen ein differentes Verhalten gleichartiger Prothesen in unterschiedlichen Klappenpositionen: Früitige Degeneration der Klappensegel macht Fascie für den Klappenersatz im rechten Herzen unbrauchbar. Im linken Herzen sind die Spätergebnisse nach Aortenklappenersatz wesentlich günstiger als nach Mitralklappenersatz. Mitralklappen sind nach Häufigkeit und Schweregrad stärker von Insuffizienzsymptomen betroffen als Aortenprothesen. Die Ursachen für diese unterschiedliche Verhaltensweise könnten mechanischer Natur sein (Klappen modell, unterschiedliche Hämodynamik, Klappenöffnungsdruck) oder in den Gewebeeigenschaften lebender Fascie (Elastizitätsarmut, Orientierung der kollagenen Fasern bei unterschiedlichem Stress) begründet liegen. Diesen Problemen könnte z. T. in der Verwendung freier Fascientransplantate mit natürlichem Klappenbauplan begegnet werden (wie am NHH bei 62 Klappen erfolgt). Gewebsreaktionen sind evtl. durch die Benutzung nichtlebender Fascie vermeidbar.

---

The present state of autologous fascia lata transplants for the replacement of cardiac valves

Summary The results obtained with implantation of 327 artificial cardiac valves fashioned from autologous fascia lata in 262 patients treated at the National Heart Hospital, London, between April 1969 and December 1970 show that although the early results are comparable, similar protheses can behave differently according to the position of the valves. Early degeneration of the cusps of the valves rules out the use of fascia for the substitution of valves in the right heart. In the left heart, later results obtained with aortic valve replacement are substantially better than in the case of mitral valve substitution. Mitral valves, both with regard to incidence and degree of severity, are more likely to develop signs of insufficiency than aortic protheses. The causes for this different behaviour are probably of a mechanical nature (valve-model, variable haemodynamics, valve-opening pressure) or are to be found in the tissue characteristics of living fascia (poor elasticity, orientation of collagen fibres under variable stresses).These problems could probably be overcome to a certain extent by the use of free fascia transplants in the shape of normal valves (this was done at the N.H.H. with 62 valves).Tissue reactions can possibly be avoided by using nonliving fascia.

     

The effect of 5-fluorouracil on Dupuytren fibroblast proliferation and differentiation

INTRODUCTION: Dupuytren's disease is a proliferative disease with contractile properties, prone to recur after surgery. Intra-operatively applied 5-fluorouracil has been used to avoid scar problems in the eye after glaucoma filtration surgery and was therefore investigated as a means to inhibit proliferation and myofibroblast differentiation in Dupuytren fibroblasts in vitro. METHOD: Primary cell lines were obtained by explants from Dupuytren's tissue (n = 6), non-diseased palmar fascia from patients with Dupuytren's disease (n = 3) and carpal ligament from patients undergoing carpal tunnel release (n = 3). The effect of 5-fluorouracil on proliferation was assessed by cell counting. Myofibroblast differentiation, an intergral part of Dupuytren's contracture, was investigated by staining for alpha smooth muscle actin, a marker for contractile cells, using immunohisto-chemical methods. RESULTS: A single exposure to 5-fluorouracil caused a sustained inhibition of proliferation in Dupuytren's and non-diseased fascia cultures, whilst the effect on carpal ligament cultures was transient. Untreated Dupuytren's fibroblasts exhibited the highest myofibroblast differentiation, whilst differentiation in non-diseased fascia cultures was shown to be proportional to cell density and virtually non-existent in carpal ligament cultures. After 5-fluorouracil exposure, the differentiation was significantly reduced in Dupuytren's fibroblasts cultures, reduced at high cell densities in non-diseased fascia and unchanged in carpal ligament cell cultures. DISCUSSION: 5-fluorouracil inhibits both proliferation and myofibroblast differentiation in Dupuytren's cell cultures and may have a potential use as an adjuvant treatment to Dupuytren surgery in order to reduce the rate of recurrence and contracture.     

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。     

In-vivo-Züchtung von Herzklappengewebe

Currently pursued tissue engineering principles of heart valves require tissue or stem cell-derived autologous cells with subsequent in vitro incubation on matrix scaffolds. Limitations of this approach are a long in vitro culture, a constantly accompanied risk of infection, and the requirement of a sophisticated, cost intensive infrastructure. An “off-the-shelf” heart valve with in vivo endothelialization and tissue regeneration potential represents an attractive alternative to overcome these limitations. Particularly for the pediatric patients, the development of heart valves with growth potential would significantly improve current treatment options. This article discusses different starter matrices, homing and immobilization strategies of host cells, and masking approaches of inflammation for in vivo surface and tissue engineering of heart valves. A novel concept based on highly specific DNA aptamers immobilized on the heart valve surface as capture molecules for endothelial progenitor cells (EPCs) circulating in the blood stream is presented.     

组织工程心脏瓣膜构建现状与发展

为了克服目前临床应用的人工心脏瓣膜在抗凝和衰坏方面的缺点，随着组织工程学的发展、构建组织工程心脏瓣膜的研究已了得初步进展。综述近年来组织工程心脏瓣膜在种植材料、种子细胞、动物实验和基础研究方面取得的进展，并探讨组织工程心脏瓣膜在种植材料、种子细胞、动物实验和基础研究方面取得的进展，并探讨组织工程心脏瓣膜的优越性和可行性。