Tissue engineered heart valves based on human cells
Minireview S W I S S M E D W K LY 2 0 0 5 ; 1 3 5 : 6 1 8 – 6 2 3 · w w w . s m w . c h 618Peer reviewed article Tissue engineered heart valves based on human cells Dörthe Schmidt, Simon P. Hoerstrup Division of Regenerative Medicine (Tissue Engineering and Cell Transplantation), Department of Surgical Research and Clinic for Cardiovascular Surgery, University Hospital, Zurich, Switzerland Summary Valvular heart disease is still a significant cause by biomimetic in vitro conditioning enabling the of morbidity and mortality worldwide. Clinically development of the neo-heart valve tissue. Here, used valve replacements including mechanical we review different human cell sources such as ves- valves as well as fixed biological xeno- or homo- sels, bone marrow, umbilical cord tissue and blood, grafts are associated with several major disadvan- and chorionic villi with particular regard to cell tages. Alternatively, tissue engineering aims at the phenotypes and their suitability for extracellular fabrication of autologous living cardiovascular matrix production for tissue engineering purposes. replacements with the potential to grow and to repair, particularly for paediatric applications. Key words: tissue engineering; heart valves; bone Therefore, autologous cells are harvested and marrow cells; umbilical cord cells; chorionic villi cells seeded onto three-dimensional matrices followed Introduction Progress in medical treatment of cardiovascu- the repair of congenital defects. Regarding this lar diseases and defects has been significant; par- major limitation, there is a special need for living, ticularly tissue substitution has shown that func- growing cardiovascular replacements for paedi- tional replacements of tissue and organs could be atric applications. Paediatric treatment of cardiac lifesaving. In the United States, for example, defects commonly requires non-autologous valves 60,000 heart valve replacements are performed an- or conduits [4, 5] with many disadvantages includ- nually . Nevertheless, heart valve disease is still ing obstructive tissue ingrowths and calcification a significant cause of morbidity and mortality of the replacements . This typically causes re- worldwide and leads in approximately 20,000 cases operations over the lifetime of paediatric patients per year to death . Furthermore, 60% of substi- with cardiovascular defects, associated with in- tute valve recipients develop serious prosthesis- creasing morbility and mortality. Living tissue related complications within 10 years postopera- replacements with the capacity of growth and re- tively . Thromboembolisms as well as increased generation would have fundamental advantages rates of infections and immunological reactions over the currently available cardiovascular replace- against the foreign material are the major prob- ments. lems with currently available heart valve replace- The above mentioned limitations have moti- ments, which are either artificial valves or chemi- vated the exploration of novel approaches towards cally treated biological xeno- or homografts. Often valve replacement. A series of studies have been lifelong anti-coagulation therapy is necessary, bur- undertaken to determine if tissue engineering dened with a substantial risk of spontaneous bleed- principles could be used to develop viable, valve ing and embolism, particularly in patients over substitutes with a thromboresistant surface and a 70 years . viable interstitium with repair, remodelling and Remaining inherently different from the tis- growth capabilities. Several groups demonstrated sue it replaces, the currently available prostheses the feasibility of creating living cardiovascular This work was supported by do not actively adapt to the physiological environ- structures by cell seeding on synthetic polymers, the NFP46 grant of ment such as to pressure changes and mechanical collagen, or xenogeneic scaffolds [7–10]. As to the Swiss National Science Founda- demands as they represent non-living materials. heart valve tissue engineering the first milestone tion (NFP 46 Tis- Furthermore, when implanted into the immature was the successful replacement of a single pul- sue Engineering heart of a child, these materials do not grow with monary valve leaflet by a tissue engineered autol- 4046-101116). the paediatric patient, which is a disadvantage for ogous leaflet .
S W I S S M E D W K LY 2 0 0 6 ; 1 3 6 : 6 1 8 – 6 2 3 · w w w . s m w . c h 619 Strategies in heart valve tissue engineering Two strategies have been used to generate liv- larised tissues derived from pericardium or valves, ing autologous heart valve replacements. One re- cell free porcine small intestine submucosa  or quires an in vitro phase generating the replacement synthetic biodegradable polymeric scaffold such as ex vivo . The other bypasses the in vitro tissue collagen or fibrin gel. However, decellularised culture phase by direct implantation of natural tis- scaffolds implanted in humans demonstrated in- sue-derived heart valve matrices for potential cell growth of host cells, no calcification but a strong ingrowth and remodelling in vivo . Matrices inflammatory response . The structural failure used for the latter approach included decellu- of these materials inhibited further use. Concept of in vitro heart valve tissue engineering Following the approach of in vitro tissue engi- After expansion in vitro, cells are seeded onto a neering, the successful fabrication of autologous biodegradable heart valve scaffold, which should living cardiovascular replacements similar to their ideally be at least 90% porous . Scaffolds could native counterparts is supported by three main el- be fabricated from polymers and the use of these ements: (1) autologous cells that resemble their na- different synthetic materials has already been tive counterparts in phenotype and functionality, broadly demonstrated for cardiovascular tissue en- (2) a temporary supporter matrix (biodegradable gineering. An overview about the most common scaffold) which promotes tissue strength until the scaffold types applied in cardiovascular tissue en- extracellular matrix produced by the autologous gineering is given in table 1. After seeding, the cells guarantees functionality on its own, and (3) constructs are cultured in nutrient media supple- culture conditions enabling tissue formation and mented with ascorbic acid and 10% foetal calf maturation by in vitro conditions similar to a phys- serum in a pulse duplicator in vitro system (bio- iological environment. reactor) mimicking the in vivo environment. In Figure 1 summarises the concept of in vitro order to improve cell migration, proliferation and tissue engineering. Concretely, in a first step cells extracellular matrix production mechanical load are harvested from an autologous donor structure. by means of shear stress have been applied to the seeded valves in pulsatile flow bioreactors [16–18]. There, the tissue formation takes placeFigure 1 and after several days the constructs are ready forConcept of in vitroheart valve tissue implantation.engineering. Auto- This concept for in vitro heart valve tissue en-logous cells areharvested from the gineering was earlier applied in an animal modelpatient and expanded using completely autologous tissue engineeredin vitro (1). When heart valves based on polyglycolic acid coated withsufficient numbersare reached, cells are poly-4-hydroxybutyrate (PGA/P4HB) (figure 2)seeded onto a starter matrices . In this “proof of principle”biodegradable heartvalve scaffold (2). study trileaflet heart valve scaffolds were fabricatedConstructs are posi- from PGA/P4HB bioabsorbable polymers andtioned in a bioreactor(3) and conditioned. sequentially seeded with autologous ovine myo-When tissue forma- fibroblasts and endothelial cells. The constructstion is sufficient, were grown for 14 days in a pulse duplicator in vitrotissue engineeredheart valves are system under gradually increasing flow andready for implanta- pressure conditions and implanted into a growingtion (4). sheep model (n = 6 lambs; mean weight at cellTable 1 Scaffold Source Examples ReferenceExamples of synthetic biocompatible polyglycolic acid (PGA) Shinoka T, et al., Circulation 1996different scaffolds and biodegradable polylactic acid (PLA) Shinoka T, et al., JTCS 1998for heart valve polymers polyhydroxyalkanoates (P3HB) Sodian R, et al., Circulation 2000tissue engineering. PGA and PLA (PGLA) Zund G, et al., EJCTS 1997 PGA and P4HB Hoerstrup SP, et al., Circulation 2000 biological xenogenic or decellularized porcine pulmonary heart valves Schenke-Layland K, et al. allogenic pulmonary heart valves on allogenic acellular Cardiovasc Research 2003 matrix conduits Steinhoff G, et al., Circulation 2000 gels fibrin heart valves based on fibrin-myofibroblast cell suspension Jockenhövel S, et al., EJCTS 2001 hybrid decellularised heart porcine aortic heart valves dip coated Stamm C, et al., Ann Thorac Surg 2004 valves coated with with biodegradable poly(hydroxybutyrate) synthetic polymer
Tissue engineered heart valves based on human cells 620 harvest 9 ± 2.8 kg). Echocardiography demon- in vivo and resembled normal heart valves as to strated mobile, functioning leaflets without steno- microstructure, mechanical properties and extra- sis, thrombus or aneurysm. These autologous cellular matrix formation. tissue engineered valves functioned up to 5 monthsFigure 2Example ofa biodegradablescaffold (PGA/P4HB)before seeding. Evaluation of human cell sources for tissue engineered heart valves Following extensive studies using ovine vascu- seeding on biodegradable scaffolds as aortic cells lar derived cells [9, 19, 20] and regarding future  and mammary artery cells (unpublished data). human application the suitability of human cells Saphenous vein cells can be obtained by a minor derived from various human cell sources have been surgical intervention in local anaesthesia and investigated. Among the most promising are vas- therefore represent an attractive cell source for cular-derived cells, bone marrow-derived cells, cardiovascular tissue engineering. In recent exper- blood-derived cells, umbilical cord-derived cells iments we studied the influence of the cell donor and chorionic villi-derived cells, particularly for age on the suitability of human myofibroblasts paediatric application (table 2). derived from saphenous vein for tissue engineer- ing purposes. Neither the growth as monolayer Human vascular-derived cells cell culture nor the three-dimensional growth as Experiments with human aortic myofibro- tissue engineered constructs was influenced by the blasts and endothelial cells demonstrated easy iso- age of the cell donor (unpublished data). Extracel- lation and in vitro culture [9, 21]. Sequentially lular matrix protein production and mechanical seeded on biodegradable scaffolds, the human aor- properties of the tissue engineered constructs tic cells showed layered tissue formation . Cells were comparable among the different age groups from saphenous veins showed comparable excel- (unpublished data). Based on these results we lent growth properties and tissue formation after conclude that the cardiovascular tissue engineer-Table 2 Human cell source Cardiovascular construct ReferenceExamples of different dermal fibroblast (covered with bovine endothelium) valve leaflets Sinoka T, et al. Ann Thorac Surg 1995;cell sources for Circulation 1996heart valve tissueengineering. foreskin fibroblast (covered with human endothelial) Patch Zund G, et al. EJCTS 1998 marrow stromal cells trileaflet heart valve Hoerstrup SP, et al. Circulation 2002 aortic myofibroblasts patch/trileaflet heart valve Ye Q, et al, EJCTS 2000 Jockenhoevel S, et al. EJCTS 2001 aortic myofibroblasts and venous cells Patch Hoerstrup SP, et al. ASAIO 2002 Schnell A, et al. Thorac Cardiovasc Surg 2001 venous myofibroblast Leaflets Mol A, et al. Thorac Cardiovasc Surg 2003 umbilical cord myofibroblast from vein pulmonary artery conduits Hoerstrup SP, et al. Ann Thorac Surg 2002 umbilical cord myofibroblast Patch Schmidt D, et al. EJCTS 2005 (covered with human endothelial cells derived from umbilical cord blood endothelial progenitor cell) chorionic villi-derived cells Leaflets Schmidt D, et al. Circulation 2006 (covered with human endothelial cells derived from umbilical cord blood endothelial progenitor cell)
S W I S S M E D W K LY 2 0 0 6 ; 1 3 6 : 6 1 8 – 6 2 3 · w w w . s m w . c h 621 ing concept may be independent of cell donor age valve tissue such as collagen I and III, and gly- and therefore be suitable also for elder patient cosaminoglycans. However, the typical three- populations. layered structural composition of native valve leaflets comprising a ventricularis, spongiosa and Human marrow stromal cells fibrosa layer was not achieved. The ultra-struc- With regard to future routine clinical realisa- tural analysis of the tissue engineered heart tion of the tissue engineering concept, human valves supported this observation demonstrating marrow stromal cells are a promising cell source. cell elements typical of viable, secretionally active In contrast to vascular cells, these cells can be ob- myofibroblasts such as actin/myosin filaments as tained without surgical interventions representing well as collagen fibrils and elastin. The quantita- an easy-to-access cell source in a possible routine tive extracellular matrix protein analysis revealed clinical scenario. The usage of marrow stromal values significantly lower compared to human cells may offer several advantages in i) easy collec- native valve tissue. tion by a simple bone marrow puncture avoiding the sacrifice of intact vascular structures, ii) show- Human umbilical cord-derived cells ing the potential to differentiate into multiple In order to provide tissue engineered con- cell lineages, and iii) demonstrating unique im- structs for congenital heart defects, alternative cell munological characteristics allowing persistence sources have been investigated, with particular at- in allogenic settings. Recently, human marrow tention to preserving the intact donor structure of stromal cells have been used for the fabrication of the newborn patients. Human umbilical cords are trileaflet heart valves (figure 3) . Histology readily available, easy to obtain and by means of of the tissue engineered valve leaflets revealed modern cell and tissue banking technologies they viable tissue organised in a layered fashion with ex- might be used as an individual cell pool for the pa- tracellular matrix proteins characteristic of heart tient’s lifetime. Furthermore, the presence of mes- enchymal progenitor cells in the Wharton’s jelly ofFigure 3 human umbilical cords with multilineage potential [25, 26] and the possibility to obtain these cells pre-Tissue engineeredtrileaflet heart valve natally using ultrasound guided sampling technol-based on human ogy make this cell source even more attractive. Instromal cells.Reprinted with culture, human umbilical cord cells demonstratedpermission from: excellent growth properties. Recently, umbilicalHoerstrup SP, et al.Circulation. cord-derived cells have been successfully utilised2002;106:I-143–50. to generate paediatric tissues in vitro demonstrat- ing excellent production of extracellular matrix [27, 28]. Blood derived endothelial progenitor cells It has been shown that the presence of en- dothelium on heart valves reduces the risk for both coagulation and inflammatory complications. Therefore, to improve the functional capacities, the tissue engineered heart valve constructs are covered with a layer of autologous human en- dothelial cells. Endothelial cells from different vascular sources have been investigated demon-Figure 4 strating promising results [21, 22]. When typicalUmbilical cord blood- endothelial antigen expression of endothelial cellsderived endothelialprogenitor cells. from different sources including arteria radialis,21 days after isola- arteria mammaria, vena saphena, umbilical cordtion, endothelial pro-genitor cells demon- was compared by flowcytometry, no significantstrated cobble-stone cell-source dependent differences were detectedmorphology (A) and (unpublished data). Also functional assays such asup-take for acetylatedhuman Low Density adhesion assays using peripheral blood-derivedLipoprotein (ac-LDL; mononuclear cells have shown comparable func-B). Furthermore, cellsshowed endothelial tionality (unpublished data). However, the harvestphenotype express- of endothelial cells from vessels requires an inva-ing cluster of differ- sive procedure. Furthermore, for paediatric appli-entiation 31 (CD31;C) and von Wille- cation prenatal endothelial cell harvest from ves-brand factor (vWF; sels would not be possible without substantial risksD). Reprinted withpermission from: for the unborn child. Blood-derived endothelialSchmidt D, et al. progenitor cells are an attractive alternative cellAnn Thorac Surg.2002;78:2094–8. source as they can be isolated from peripheral blood as well as from umbilical cord blood. The
Tissue engineered heart valves based on human cells 622 latter one can already be obtained during preg- its chorionic villi provide extra-embryonically sit- nancy using the well-established method of ultra- uated foetal mesenchymal cells including pro- sound guided percutaneous blood sampling. Thus, genitor cells. The obtained tissue samples could harvesting endothelial progenitor cells prior birth also serve as a cell source for tissue engineering. is possible without substantial risks. The feasibil- Theoretically, one specimen could then be used ity of using human umbilical cord blood-derived for both diagnostics and the tissue engineering endothelial progenitor cells for tissue engineering application. Recently, the successful use of chori- of cardiovascular replacements for paediatric ap- onic villi-derived myofibroblast-fibroblast like plication has been demonstrated (figure 4) [27, 28]. cells obtained from routine prenatal tissue sam- When differentiated endothelial progenitor cells pling combined with umbilical cord blood- were co-cultured with non-endothelial cells as well derived endothelial progenitor cells for heart valve as when exposed to mechanical stimuli they tissue engineering has been demonstrated . showed stabile phenotypes . The extracellular The generated heart valve tissues showed cell matrix production of undifferentiated endothelial phenotypes similar to their native counterparts progenitor cells was demonstrated to be insuffi- and production of glycoaminoglycans and collagen cient whereas the differentiation into endothelial as major extracellular matrix elements of native cells on biodegradable scaffolds was observed . heart valves. In the overall tissue engineering concept, endo- thelial progenitor cells represent a promising cell Limitations source for the endothelialisation of heart valves. Besides a few occasional pilot studies based Since endothelial progenitor cells are easily acces- on decellularised heart valves [14, 32] no systemic sible current research aims at their transdifferen- evidence that the heart valve tissue engineering tation into myofibroblast cells in order to enable concept can be applied in the clinical routine has blood as a sole cell source for paediatric applica- been reported so far. However, as shown by Simon tions. et al.  some of the decellularised scaffolds im- planted in humans exhibited a strong inflamma- Chorionic villi-derived cells tory response resulting in dramatic failure. Of particular importance is cell-harvesting at Regarding the use of biodegradable scaffolds, early perinatal stage that allows having the tissue- local inflammation and systemic toxicity due to engineered replacement ready for implantation at degradation products is a possible problem. Fur- the birth of the patient in order to prevent second- thermore, there is a potential risk that ex vivo treat- ary damage to the immature heart. As prenatal tis- ment of cells or the use of immature cells such as sue harvest from human placenta by chorionic villi progenitors might lead to tumour development sampling is already a routine procedure for prena- by uncontrolled cell growth or differentiation via tal genetic diagnostics the human placenta repre- genetic alterations. Future in vivo studies will focus sents a promising prenatal cell source. Particularly, on these important aspects. Conclusion In summary, various cells seem to be suitable However, heart valve tissue engineering is a for tissue engineering purposes. Among the promising approach for living, functional autolo- most promising are progenitor cells either ob- gous replacements. Particularly paediatric patients tained from bone marrow, umbilical cord or will benefit from growing replacement materials chorionic villi, particularly for paediatric appli- for the repair of congenital heart defects. Never- cations. This review was undertaken to provide theless, before clinical application of the tissue more detailed understanding of cell phenotype and engineering heart valve concept will be routine extracellular matrix development during the tissue several issues will have to be addressed. engineering process, in order to define quality criteria for future clinical use. Although having first indications as to the influence of age, cell Correspondence: sources and in vitro conditions, knowledge on bio- Simon Philipp Hoerstrup, MD., PhD chemical and immunological characteristics of Clinic for Cardiovascular Surgery the cells / tissues undergoing in vitro growth is still University Hospital of Zurich very limited. In addition, little is known about Raemistrasse 100 the influence of endothelial cells on extracellular CH-8091 Zurich matrix formation and on the quality of in vitro Switzerland engineered tissue. E-Mail: email@example.com
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