Objective: In tissue engineering the material properties of synthetic compounds are chosen to enable delivery of dissociated cells onto a scaffold in a manner that will result in in vitro formation of a new functional tissue. The seeding of human fibroblasts followed by human endothelial cells on resorbable mesh is a precondition of a successful creation of human tissues such as vessels or cardiac valves. Methods: Polymeric scaffolds (n=18) composed of polyglycolic acid (PGA) with a fiber diameter of 12-15 μm and a polymer density of 70 mg/ml were used as square sheets of 1×1×0.3 cm. Fibroblasts (passage 7) harvested from human foreskin were seeded (3.4×106) and cultured over a 3 week period on a PGA-mesh, followed by seeding of endothelial cells (passage 5, 2.8×106) harvested from human ascending aorta. Thereafter the new tissue was stained for HE, van Gieson, Trichrom-Masson, Factor VIII and CD 34 and proved by scanning electron microscopy. Results: Microscopic examination of the seeded mesh demonstrated that the human fibroblasts were attached to the polymeric fibers and had begun to spread out and divide. The scanning electron microscopic examination demonstrated a homogeneous scaffold resembling a solid sheet of tissue. The seeded endothelial cells formed a monolayer on the fibroblasts and no endothelial cell invasion or new formation of capillaris could be detected. Conclusions: These results are a first step to demonstrate that seeding of human fibroblasts and endothelial cells on PGA-mesh might be a feasible model to construct human tissues such as vessels or cardiac valve

Hoerstrup, Simon P.

Lachat, Mario

Schoeberlein, Andreina

Turina, Marko

Uhlschmid, Georg

Vogt, Paul R.

Zünd, Gregor

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European Journal of Cardio-thoracic Surgery 13 (1998) 160–164Tissue engineering: A new approach in cardiovascular surgery; Seedingof human fibroblasts followed by human endothelial cells onresorbable mesh1Gregor Zu¨nd *, Simon P. Hoerstrup, Andreina Schoeberlein, Mario Lachat, Georg Uhlschmid,Paul R. Vogt, Marko TurinaClinic for Cardio6ascular Surgery, Uni6ersity Hospital, Ra¨mistrasse 100, CH-8091, Zurich, SwitzerlandReceived 30 September 1997; received in revised form 10 November 1997; accepted 19 November 1997AbstractObjective: In tissue engineering the material properties of synthetic compounds are chosen to enable delivery of dissociated cellsonto a scaffold in a manner that will result in in vitro formation of a new functional tissue. The seeding of human fibroblastsfollowed by human endothelial cells on resorbable mesh is a precondition of a successful creation of human tissues such as vesselsor cardiac valves. Methods: Polymeric scaffolds (n18) composed of polyglycolic acid (PGA) with a fiber diameter of 12–15 mmand a polymer density of 70 mg:ml were used as square sheets of 110.3 cm. Fibroblasts (passage 7) harvested from humanforeskin were seeded (3.4106) and cultured over a 3 week period on a PGA-mesh, followed by seeding of endothelial cells(passage 5, 2.8106) harvested from human ascending aorta. Thereafter the new tissue was stained for HE, van Gieson,Trichrom–Masson, Factor VIII and CD 34 and proved by scanning electron microscopy. Results: Microscopic examination of theseeded mesh demonstrated that the human fibroblasts were attached to the polymeric fibers and had begun to spread out anddivide. The scanning electron microscopic examination demonstrated a homogeneous scaffold resembling a solid sheet of tissue.The seeded endothelial cells formed a monolayer on the fibroblasts and no endothelial cell invasion or new formation of capillariscould be detected. Conclusions: These results are a first step to demonstrate that seeding of human fibroblasts and endothelial cellson PGA-mesh might be a feasible model to construct human tissues such as vessels or cardiac valves. © 1998 Elsevier Science B.V.All rights reserved.Keywords: Tissue engineering; Synthetic biodegradable matrix; Endothelial cells1. IntroductionTransplantation of endothelial cells onto the bloodflow surface of a small-diameter vascular conduit, ho-mografts, or valve prosthesis to partially reproduce theantithrombogenic lining of a naturally occuring intimais a logical concept. In our experiences, endothelial celltransplantation has failed to make a significant transi-tion from the animal laboratory to the clinical arena[1]. We believe nowadays that human endothelial cellsneed a living compound for normal growth and sur-vival in the human blood stream.Tissue engineering is a multidisciplinary science thatutilizes basic principles from engineering and life sci-ences to construct tissues from their cellular compo-nents [2]. For the creation of new functional tissue threegeneral strategies have been adopted [3]. First, isolatedcells or cell substitutes can be used; this techniqueallows the replacement of only those cells that supplythe needed function. Second, tissue-inducing substancessuch as growth factors can be used to stimulate the* Corresponding author. Tel.: 41 1 2551111; fax: 41 12554369; e-mail zund@chi.usz.ch1 Presented at 11th Annual Meeting of EACTS, Copenhagen,September 28–October 1, 1997.1010-7940:98:$19.00 © 1998 Elsevier Science B.V. All rights reserved.PII S 1 010 -7940 (97 )00309 -6G. Zu¨nd et al. : European Journal of Cardio-thoracic Surgery 13 (1998) 160–164 161growth of the desired cell population. Third, cellscan be cultured on or within specific matrices.These days, our laboratory has focused on the cre-ation of new functional tissue substitutes utilizing hu-man cells attached to synthetic, biodegradablepolymer scaffolds. This approach involves isolatingcells from a donor and seeding these cells onto apolymeric scaffold, where they attach and grow andfinally form a tissue-like structure. The cell-polymerconstruct is configured to serve as a template toguide this development. Preliminary animal studieshave demonstrated the feasibility of this strategywith the successful formation of a tissue engineeredvalve leaflet and a successful implantation of thisleaflet with good functional results [4–6]. The abilityto isolate cells types is an important component toany tissue engineering project. Furthermore, the seed-ing of human fibroblast followed by human endothe-lial cells on resorbable mesh is a precondition of thecreation of any tissue in the cardio-vascular field.2. Material and methods2.1. ScaffoldPolymeric non-woven scaffold, composed of polyg-lycolic acid (PGA) with a thickness of 3 mm, a fiberdiameter of 12–15 mm and a polymer density of 70mg:ml (Albany International Research, Mansfield,MA, USA; a kind gift from Dr. J. Vacanti, Chil-dren’s Hospital, Harvard Medical School, Boston,MA, USA) were used as square sheets of 0.311cm. The PGA-polymer by hydrolysis has an in vitroresorption rate of 4–6 weeks.2.2. Human fibroblastsHuman foreskins were obtained from phimosisop-erations performed at Children’s Hospital, UniversityZurich. The harvested foreskins were serially washedwith phosphate buffered saline (Gibco BRL-LifeTechnologie, Grand Island, NY) supplemented with1% antibiotic:antimycotic solution saline (GibcoBRL-Life Technologie, Grand Island, NY). Under alaminar flood hood (Forma Scientific, Marietta, OH),the tissue was minced into 1–2 mm pieces andevenly distributed in 1560 mm tissue culture dishes(Becton Dickinson, Lincoln Park, NJ). Medium wasgently added to the tissue culture dishes. Mediumconsisted of Dulbecco’s modified Eagle’s medium(Gibco BRL-Life Technologies) supplemented with10% fetal bovine serum (Gibco BRL-Life Technolo-gies) and 1% antibiotic:antimycoic solution (GibcoBRL-Life Technologies). The explanted tissue wasplaced in a humidified incubator (Forma scientific) at37°C with 5% CO2 for 2–3 weeks. During this pe-riod, the cells were migrated off the explants. There-after cells were serially passaged to obtain sufficientnumbers of cells for cell seeding.2.3. Human aortic endothelial cellsHuman aortic endothelial cells were obtained fromthe ascending aorta of the explanted sick hearts ofheart transplant patients. The harvested piece of tis-sue of ascending aorta was serially washed withphosphate buffered saline (Gibco BRL–Life Tech-nologies, Grand Island, NY) supplemented with 1%antibiotic:antimycotic solution (Gibco BRL-LifeTechnologies). Under a laminar flood hood (FormaScientific, Marietta, OH), the intima surface wascovered with Collagenase Typ I (75 U:ml in PBSpH 7.4, Worthington) and incubated for 30 min atroom temperature. Cells were detached using a rub-ber cell scraper and cultured in 75 cm2 culturedishes (Becton Dickinson Lincoln Park, NJ).Medium 199 with EBS (Amimed, Allschwil, Switzer-land) supplemented with 10% fetal bovine serum, 16U:ml Heparin, 20 mg:ml Endothelial cell growthsupplement (Becton, Dickinson) and 1% antibiotic.The explanted cells were placed in a humidified in-cubator at 37°C with 5% CO2 for 2–3 weeks. Cellswere serially passaged to obtain sufficient numbersof cells for cell seeding. The cells were then labeledwith an acetylated low density lipoprotein (Ac-Dil-LDL) probe (Biomedical Technologies, Stoughton,MA) which selectively fluorescently tags endothelialcells through their LDL receptor. These cells popu-lations were then checked for their purity by fluores-cent microscopy.2.4. SeedingThe PGA polymers (n18, 110.3 cm) wereseeded with human fibroblasts (passage 7). Therewere 8–10 seeding procedures every 90 min. Eachseeding procedure contained 3.4106 human fibrob-lasts. The seeded fibroblasts were cultured over a 21days period. Media consisted of Dulbecco’s modifiedEagle’s medium (Gibco BRL-Life Technologies) sup-plemented with 10% fetal bovine serum (Sigma, St.Louis, MO) and 1% antibiotic solution (Irvine Scien-tific, Santa Clara, CA). The medium was changedevery 4 days. Thereafter this tissue-like structure wasseeded with the pure human aortic endothelial cellculture (passage 5, 2.8106). The constructs werethen fixed with 0.4% paraformaldehyde and stainedby conventional histological methods (HemalaunEosin, Van Gieson, Trichrom-Masson) and examinedby electron microscopy.G. Zu¨nd et al. : European Journal of Cardio-thoracic Surgery 13 (1998) 160–164162Fig. 1. Formation of fibroblast-polymer structure. Scanning electronmicroscopy demonstrates the formation of a continuous fibroblasttissue.Fig. 3. Formation of endothelial monolayer. CD 34 stain demon-strates a monolayer of endothelial cells (A) on the surface of fibro-blasts core (B). After 4 weeks hydrolysis of polymer fibers (C) is inprocess.3. Results3.1. Con6entional microscopyConventional microscopy examination demonstratedthat the seeded human fibroblasts were attached to thepolymeric fibers and had begun to spread out anddevide (Figs. 1 and 2). Furthermore the seeded cellswere forming cell bridges and a homogeneous tissue.The production of collagen matrices was demonstratedby staining the tissue-like structure with Van Gieson.There was evidence of beginning hydrolytic degradationof the polymeric fibers. The seeded endothelial cellsformed a monolayer on the human fibroblasts asdemonstrated by factor VIII and CD 34 (Fig. 3). Inaddition, no invasive growth of endothelial cells or newformation of capillaris could be detected.3.2. Electron microscopyThe scanning electron microscopic examination 7days before seeding with human aortic endothelial cellsdemonstrated attachment of fibroblasts to the poly-meric fibers and the formation of cell bridges betweenpolymeric fibers. In addition a homogeneous distribu-tion of the seeded fibroblasts could be detected. Therewas the same densitiy of fibroblasts in the upper andlower part of the PGA mesh and no gravitiy gradientcould be detected. Some of the polymeric fibers havestarted hydrolytic degradation.4. DiscussionIn tissue engineering, the material properties of syn-thetic compounds are chosen to enable delivery ofFig. 2. Continous formation of fibroblasts. Scanning electron mi-croscopy shows a continuous distribution of fibroblasts throughoutthe ‘polymeric architecture’ and attachment of the fibroblasts (A) tothe polymer fibers (B).G. Zu¨nd et al. : European Journal of Cardio-thoracic Surgery 13 (1998) 160–164 163dissociated cells into a host with successive formationof new functional tissue. The advantage of such tissueengineered products is the ability to grow, repair andremodel, and no requirement for anticoagulation. Fur-thermore, if autologous cells are used for the construc-tion of such products, an increasing durability will beachieved with lack of risk of immunogenicity and rejec-tion. The new field of tissue engineering is emergingexperimentally and tissue substitutes for liver, cartilage,bone, trachea, intestine and urologic tissue are created[7–10]. The first tissue engineered products in clinicaluse are skin replacements. In the field of cardiacsurgery tissue engineering is not unknown. Marelli et al.demonstrated that skeletal muscle satellite cells can beharvested, grown in vitro, and autotransplanted intodamaged heart muscle [11–13]. Preliminary resultsshowed that muscle formation occured at 8 weeks, butnot at 14 weeks after transplantation [12]. Apart fromthat, the group of Vacanti and Mayer at Children’sHospital in Boston has become a leading group incardiovascular tissue engineering [4–6]. The goal oftheir research is to create an autologous, tissue engi-neered aortic valve with the ability to grow, repair andremodel. They created an animal model in which apulmonary valve leaflet was successfully replaced by anautologous tissue engineered valve leaflet. Furthermoreat our institution autologous capillary endothelial cellshave been harvested, grown in vitro and seeded ontoartificial vascular grafts in order to promote hemocom-patibility [14–17]. In our experiences, endothelial celltransplantation has failed to make a significant transi-tion from the animal laboratory to the clinical area. Webelieve nowadays that human endothelial cells need aliving compound for normal growth and survival in thehuman blood stream. Since the seeding of humanfibroblasts on biodegradable mesh is a precondition ofany tissue engineered product in cardiovascular surgerywe first focused upon the development and evaluationof an adequate method. For this reason we started withcreating a living compound of fibroblasts followed byseeding of endothelial cells. Only endothelial cells of thehuman ascending aorta were used suggesting that hu-man capillary endothelial cells thus be angiogenetic andmight form new capillaris. For the development of aliving compound we choose preputial fibroblasts fromnewborns. Since in contrast to adults this cells have ahigher potential for growth and higher cell generationscan be presumed [18]. The most important steps in thisstudy are the attachment of human fibroblasts to thefibers and the formation of cell bridges and in additionthe seeding of endothelial cells forming a monolayerwith no evidence of invasion or formation of capillarystructures into the core. The polymer templates that weutilize are both biocompatible and biodegradable; arapid degradation is desirable in order to minimize anychronic foreign-body inflammatory response [19]. Thenext step in our study will involve the sorting out of amixed cell population obtained from human ascendingaorta to pure cell populations followed by serial passag-ing to obtain sufficient numbers of cells for cell seeding.Cardiovascular tissue engineering has the potential toimprove our ability to treat cardiovascular disease, oneof the most important single cause of mortality in theindustrial world. The preliminary results of this studyrepresent a basic step on the way to construct humancardiovascular tissue and demonstrate that the appliedtechnique of tissue engineering might be a feasibleapproach.References[1] Jarrwll BE, Williams SK. Microvessel derived endothelial cellisolation, adherence, and monolayer formation for vasculargrafts. J Vasc Surg 1991;13:733–4.[2] Vacanti CA, Vacanti JP. Bone and cartilage reconstruction withtissue engineering approaches. Otolaryngol Clin North Am1994;27(1):263–76.[3] Langer R, Vacanti JP. Tissue Eng Sci 1993;260:920–6.[4] Zu¨nd G, Breuer CK, Shinoka T, Ma PX, Langer R, Mayer JE,Vacanti JP. The in vitro construction of a tissue engineeredbioprosthetic heart valve. Eur J Cardio-thoracic Surg1997;11:493–7.[5] Shinoka T, Breuer CK, Tanel RE, Zu¨nd G, Miura T, Ma PX,Langer R, Vacanti JP, Mayer JE. Tissue engineering heartvalves. Ann Thorac Surg 1995;60(6):513–6.[6] Shinoka T, Ma X, Breuer CK, Cusick RA, Shum-Tim D, Zu¨ndG, Langer R, Vacanti JP, Mayer JE. Tissue engineering heartvalves, autologous valve leaflet replacement study in a lambmodel. Circulation 1996;94(suppl II):164–8.[7] Cao Y, Vacanti JP, Ma X, Paige KT, Upton J, Chowanski Z,Schloo B, Langer R, Vacanti CA. Generation of neo-tendonusing synthetic polymers seeded with tendocytes. TransplantProc 1994;26(6):3390–1.[8] Sakata J, Vacanti CA, Schloo B, Healy GB, Langer R, VacantiJP. Tracheal composites tissue engineered from chondrocytes,trachel epithelial cells and synthetic degradable scaffolding.Transplant Proc 1994;26(6):3309–12.[9] Vacanti JP, Morse MA, Saltzman WM, Domb AJ, Perez-Atayade A, Langer R. Selective cell transplantation using bioab-sorbable artificial polymers as matrices. J Pediatr Surg1988;23(1):3–9.[10] Vacanti JP. Beyond transplantation: Third annual Samuael Ja-son Mixter lecture. Arch Surg 1988;123:545–9.[11] Greentreee D, Marelli D, Ma F, Chiu RC-J. Satellite cell trans-plantation for myocardial repair: labeling techniques. TransplantProc 1994;26(6):3357.[12] Marelli D, Desrosiers C, el-Alfy M, Kao RL, Chiu RC. Celltransplantation for myocardial repair: an experimental ap-proach. Cell Transplant 1992;1(6):383–90.[13] Zibaitis A, Greentree D, Ma F, Marelli D, Duong M, ChiuRC-J. Myocardial regeneration with satellite cell implantation.Transplant Proc 1994;26(6):3294.[14] Muller-Glauser W, Lehmann KH, Bittmann PO, et al. A compli-ant small-diameter vascular prosthesis lined with functionalvenous endothelial cells. ASAIO Trans 1988;34(3):528–31.[15] Pasic M, Muller-Glauser W, von Segesser L, Odermatt B,Lachat M, Turina M. Endothelial cell seeding improves patencyof synthetic vascular grafts: manual versus automatized method.Eur J Cardiothorac Surg 1996;10(5):372–9.G. Zu¨nd et al. : European Journal of Cardio-thoracic Surgery 13 (1998) 160–164164[16] Pasic M, Muller-Glauser W, Odermatt B, Lachat M, Seifert B,Turina M. Seeding with omental cells prevents late neointimalhyperplasia in small-diameter Dacron grafts. Circulation1995;92(9):2605–16.[17] Pasic M, Muller-Glauser W, von Segesser L, Lachat M, Mi-haljevic T, Turina M. Superior late patency of small-diameterDacron grafts seeded with omental microvascular cells: An ex-perimental study. Ann Thorac Surg 1994;58:677.[18] Matsouko H, Furusawa M, Kono A. Growth potential ofhuman fibroblasts on contact sensitive confluent monolayer fixedwith 3% glutaraldehyde. Gan To Kag R 1994;21:563–4.[19] Mooney DJ, Organ G, Vacanti JP, Langer R. Design andfabrication of biodegradable polymer devices to engineer tubulartissues. Cell Transplant 1994;3(2):203–10.Appendix A. Conference discussionDr T. Ferguson (St. Louis, USA): You caught me a little bysurprise because the abstract does not refer specifically to using thisapproach on valves, which to me is the most difficult experimentalenvironment. I guess my question would be, is this technique applica-ble to vascular grafts and other biologic situations?Dr Zund: Yes. I do agree with you. In one of our animal studies wewere just replacing a pulmonary leaflet.Dr Ferguson: Well, if it works in a valve, it will work anywhere inthe body, of that you can be sure.Dr C. Muneretto (Padua, Italy): We have already developed aresearch program on tissue engineering. And in our experience we usehuman vessel treated to suppress host-immune response, and we usethis vessel as a geometrical and mechanical support for the seedingand growth of fibroblasts and endothelial cells. We found two mainproblems. The first one, the endothelial growth; and the second one,membrana basalis formation.I have two questions for you. Did you use any method to increasespeed in endothelial cell growth? And second question, did you findany formation of membrana basalis in you conduit?Dr Zund: First of all, we don’t use any additional factors forendothelial growth. We just are culturing them, and then, after somepassages, we are seeding them on the fibroblast core. Second, we werenot yet looking for the basilar membrane.Dr Ferguson: Were you able to show by low microscopic scans, thattotal coverage of the surface is achieved?Dr Zund: Yes, sir. The fibroblasts were totally confluent andformed a new tissue..

Tissue engineering: A new approach in cardiovascular surgery; Seeding of human fibroblasts followed by human endothelial cells on resorbable mesh

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