Tissue Engineering

Author(s): Robert Langer and Joseph P. Vacanti

Source: Science, New Series, Vol. 260, No. 5110 (May 14, 1993), pp. 920-926
Published by: American Association for the Advancement of Science
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10

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Engineering

Robert Langer*and Joseph P. Vacanti

The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly
problems in human health care. A new field, tissue engineering, applies the principles of
biology and engineering to the development of functional substitutes for damaged tissue.
This article discusses the foundations and challenges of this interdisciplinaryfield and its
attempts to provide solutions to tissue creation and repair.

Every year, millions of Americanssuffer

tissueloss or end-stageorganfailure(Table
1). The total national health care cost for
these patientsexceeds$400 billion peryear
(1, 2). Approximately8 million surgical
proceduresare performedannuallyin the
United States to treat these disordersand
40 to 90 million hospitaldaysare required
(2). Physicianstreatorganor tissueloss by
transplantingorgans from one individual
into another, performingsurgical reconstruction,or usingmechanicaldevicessuch
as kidney dialyzers (3). Although these
therapieshave saved and improvedcountless lives, they remainimperfectsolutions.
Transplantationis severely limited by a
criticaldonorshortage.Forexample,fewer
than 3,000 donors are availableannually
for the approximately30,000 patientswho
die fromliver failure(4). Donor shortages
worsenevery year and increasingnumbers
of patients die while on waiting lists for
needed organs(5). Surgicalreconstruction
can result in long-termproblems.For example, colon cancers often develop after
surgicaltreatmentof incontinencethat directs urineinto the colon (6). Mechanical
devicescannot performall of the functions
of a single organ and therefore cannot
preventprogressivepatientdeterioration.
Tissue engineering is an interdisciplinary field that applies the principles of
engineeringandthe life sciencestowardthe
developmentof biological substitutesthat
restore,maintain, or improvetissue function (7). Threegeneralstrategieshave been
adoptedfor the creationof new tissue:
1) Isolatedcells or cell substitutes.This
of surgery,
approachavoidsthe complications
allowsreplacementof only those cells that
supplythe neededfunction,andpermitsmanipulationof cellsbeforeinfusion.Its potenR. Langer is in the Department of Chemical Engineering and the Harvard-M.I.T.Division of Health, Sciences and Technology, Massachusetts Institute of Technology, E25-342, Cambridge, MA 02319 and the Department of Surgery, Children's Hospital, Boston, MA
02115. J. P. Vacanti is in the Department of Surgery,
Harvard Medical School and Children's Hospital, Boston, MA 02115.
*To whom correspondence should be addressed at
Massachusetts Institute of Technology, E25-342.

920

tial limitationsincludefailureof the infused

cells to maintaintheirfunctionin the recipient, andimmunological
rejection.
2) Tissue-inducingsubstances.The success of this approachdependson the purification and large-scaleproductionof appropriatesignal molecules, such as growth
factors, and, in many cases, the development of methodsto deliverthese molecules
to their targets.
3) Cells placed on or within matrices.
In closedsystems,the cells areisolatedfrom
the bodyby a membranethat allowspermeation of nutrientsand wastesbut prevents
largeentities such as antibodiesor immune
cells fromdestroyingthe transplant.These
systemscan be implantedor used as extracorporealdevices (Fig. 1). In open systems,
cells attachedto matricesareimplantedand
become incorporatedinto the body (Fig.
2). The matricesarefashionedfromnatural
materialssuch as collagenor fromsynthetic
polymers.Immunologicalrejectionmay be
preventedby immunosuppressive
drugsor
by usingautologouscells.
Investigatorshave attempted to engineer virtuallyevery mammaliantissue. In
the followingsummary,we discussreplacement of ectodermal,endodermal,and mesodermal-derived
tissue.

Ectoderm
Nervoussystem.Braindiseasessuch as Parkinson's disease, where there is a loss of
dopamineproduction,representan important target for tissue engineering. Transplantation of normal fetal dopamine-producingcells by stereotaxically'
guidedinjection into the brainhas producedsignificant
reversalof debilitatingsymptomsin humans
(8). Altemative methodshave been tested
in animalmodels.PC12 cells, an immortalized cell line derivedfromrat pheochromocytoma,have been encapsulatedin polymer
membranesand implantedin the guineapig
striatum(Fig. 3A). Dopaminereleasefrom
the capsuleswasdetectablefor6 months(9).
bovineadrenalchroSimilarly,encapsulated
maffincells have been implantedinto the
subarachnoidspace in rats, where through
SCIENCE * VOL. 260 *

their continuousproductionof enkephalins

and cathecholaminesthey appearedto relieve chronicintractablepain (10).
Nerveregenerationhasalsobeen studied.
Peripheral
nervesarecapableof regeneration
after transectioninjury.Transectednerves
can sometimesbe clinicallyrepairedby endto-end approximationof the stumpswith
fine sutures.When nerve injuryresultsin
gaps that are too wide for healing, autologousnerve graftsare used as a bridge.Synthetic nerveguides(conduits)couldhelp in
these cases by protectingthe regenerating
nerve from infiltratingscar tissue or by directingnew axonstowardtheir target.Severallaboratories
have shownin animalmodels that syntheticguidescomposedof natural
polymers (laminin, collagen, chondroitin
sulfate)or syntheticpolymerscan enhance
nerve regeneration(11). Initial resultssuggestthat this processcan be aidedby placing
Schwanncells derivedfromsciaticnervesin
Matrigel? seeded in polymer membranes
(12). In addition,polymerscan be designed
so that they slowly releasegrowthfactors,
which may allow regrowthof the damaged
nerveover a greaterdistance(13).
Comea. More than 10 million people
worldwidesufferfrombilateralcomealblindness.Not onlyaretransplantdonorslimited,
but there is a riskof infectiousagent transmission. Ideally,an artificialcomea would
consist of materialsthat supportadhesion
and proliferationof comeal epithelialcells
so that an intact continuousepitheliallayer
forms. These materialsshould also have
nutrientand fluidpermeability,
appropriate
light transparency,
and no toxicity.Comeal
epithelialcells havebeen preseededon polyvinyl alcohol hydrogelsand transplanted
into rabbit comeas, where they remained
adherentand proliferatedfor 1 to 2 weeks
(14). Long-termstudiesof suchmaterialsare
warranted;safe and effective methods of
attachingthesematerialsto the comea must
also be developed.
Skin. Approximately150,000 individuals are hospitalizedand 10,000 die each
yearin the United Statesbecauseof burns.
Severalnew types of tissue transplantsare
being studied for the treatmentof burns,
skin ulcers, deep wounds, and other injuries. In some cases, patientsare implanted
with a composite material whose upper
layer consists of silicone (which prevents
fluidloss) and whose lowerlayerconsistsof
chondroitin-sulfate
and collagen(whichinduces new blood vessels and connective
tissue ingrowth). In essence, the patients

14 MAY 1993

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receive a new dermis. After 3 weeks, the

upperlayer is replacedwith an extremely
thin epidermalgraft.Clinical studieshave
showngood graftacceptancewith minimal
scarring(15). In a refinementof the procedure, the second skin graftwas eliminated
by seedingepidermalcells obtainedfroma
smallskingraft(0.003 inch thick) onto the
lower layer priorto placementon the patient (16).

A secondapproachto skingraftsinvolves
the in vitro cultureof epidermalcells (keratinocytes). Small skin biopsies(1 cm2) are
harvestedfrombum patientsand expanded
10,000-fold-a sizecomparableto an adult's
body surfacearea.This expansionhas been
achievedby cultivatingkeratinocyteson a
feeder layer of irradiatedNIH 3T3 fibroblasts, which, in conjunctionwith certain
addedmedia components,stimulatesrapid
cell growth.An advantageof this approach
is the abilityof the graftsto coverextremely
large wounds;a disadvantage
-is the 3- to
Table 1. Incidence of organ and tissue defi4-week periodrequiredfor cell expansion,
ciencies, or the numberof surgicalprocedures
related to these deficiencies, in the United whichmaybe too long for a severelybumed
States. Thisis a partiallistcompiledfromsourcpatient. Cryopreserved
allograftsmay help
es that includethe AmericanDiabetes Associto
circumvent
the
problem
(17).
ation, American Liver Foundation, Muscular
Another
promising
approach
useshuman
DystrophyAssociation, American Red Cross,
grownon degradAmerican Kidney Foundation,The Wilkersonr neonataldermalfibroblasts
able polyglycolicacid mesh (Fig. 3B). BeGroup,Cowen and Co., AmericanAcademy of
OrthopedicSurgery,AmericanHeartAssociacausefibroblasts
areeasyto cryopreserve
and
tion,NationalInstituteof NeurologicalDisorders grow,a uniformstock of cells can be mainand Stroke, Source Book of Health Insurance tained for these grafts.In deep injuriesin(Health Assurance Association of America),
volving all layers of skin, the grafts are
1991, Federal Register, and Departmentof
Healthand HumanServices (Medicare-based placedonto the woundbed and a skin graft
is placedon top. The graftthen vascularizes,
information).
resultingin the formationof organizedtissue
Procedures resembling dermis. Clinical trials have
or patients
Indication
shown good graftacceptancewith no eviper year
dence of immunerejection(18). Fibroblasts
havealsobeenplacedon a hydratedcollagen
Skin
gel. Upon implantation,the cells migrate
Burns*
2,150,000
throughthe gel by enzymaticdigestionof
Pressuresores
1,500,000
Venous stasis ulcers
500,000
collagen,which resultsin reorganization
of
Diabeticulcers
600,000
collagenfibrils(19). This approachhas unNeuromusculardisorders
200,000
dergonelimitedclinicaltesting.
Spinalcord and nerves
40,000
Bone
Jointreplacement
558,200
Bone graft
275,000
Internalfixation
480,000
Facialreconstruction
30,000
Cartilage
Patellaresurfacing
216,000
Chondromalaciapatellae
103,400
Meniscalrepair
250,000
Arthritis(knee)
149,900
Arthritis(hip)
219,300
Fingersand small joints
179,000
Osteochondritisdissecans
14,500
Tendonrepair
33,000
Ligamentrepair
90,000
Bloodvessels
Heart
754,000
Largeand small vessels
606,000
Liver
Metabolicdisorders
5,000
Livercirrhosis
175,000
Livercancer
25,000
Pancreas (diabetes)
728,000
Intestine
100,000
Kidney
600,000
Bladder
57,200
Ureter
30,000
Urethra
51,900
Hernia
290,000
Breast
261,000
Blood transfusions
18,000,000
Dental
10,000,000
*Approximately
150,000of these individuals
arehospitalized
and10,000die annually.

Fig. 1. There are three common

closed-system configurationsfor
cell transplantdevices (69, 70)
[figure adapted with permission
from (70)]. In vascular devices,
the cells are placed in an extracellularcompartmentsurrounding
a tubularmembrane(i.d. -1 mm)
throughwhich blood can flow. In
macrocapsule systems, the cells
are placed in sheaths, rods, or
disks (diameter?0.5 to 1.0 mm).
In microcapsule systems, the
cells are placed in injectable
spherical beads (diameter <0.5

Endoderm
Liver. Most liver supportsystems remove
toxins normallymetabolizedby the liver
through dialysis, charcoal hemoperfusion,
immobilizedenzymes,or exchangetransfusion (20). None of these systems,however,
can offer the full spectrum of functions
performedby a healthy liver. Investigators
are now endeavoringto achieve liver replacementwith isolated hepatocytes.The
hepatocyteshave been placed in suspensions, encapsulatedin microcapsules
or hollow fibers, placed on substratessuch as
microcarriers
coated with extracellularmatrix proteins, or attached to polymernetworks (20, 21). In animal models, the
transplantedhepatocyteshave producedalbumin and other liver function markers,
and have clearedproductsof bilirubinand
ureametabolism.
Hepatocytesystemsarebeingstudiedfor
both extracorporealand implantableapplications. Extracorporealsystems, which
wouldbe usedwhen a patient'sown liver is
recoveringor as a bridgeto transplant,offer
severaladvantages:(i) bettercontrolof the
medium surroundingthe cell system (for
example, the ability to achieve improved
transportof oxygen, nutrients,and wastes);
(ii) better control of the timing and duration of use; and (iii) a decreasedchance of
immune rejection because the patient's
white cells can be separatedfrom hepatocytes by plasmapheresis.
Implantablehepatocyte systems,on the otherhand, offerthe
e

Mt
Blood

flow

t t t

rac
coff_partments

IS

Macrocapsules

esheaths,rods,discs

tttt

mm). Device biocompatibility is

critical because tissue reaction

can blockthe flowof nutrientsand
wastes to and from the capsule.
Microcapsules

are

commonly

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.'

sle
Microcapsules
spherical

made of hydrogels-in particular,

j
'
dispersions
the polysaccharidealginate-be'
Pores
cause of the extremelymild conditions requiredfor gel formation.The alginate can be furthercoated with polyanions,such as
polylysine,and again with alginate if desired. Such coatings can affect the flow of nutrientsand
wastes, mechanicalstrength,and biocompatibility.
Results of in vivo studies with alginate are not
alwaysconsistent,possiblybecause of variationsinalginatepurity(71). Macrocapsulesand vascular
devices often consist of acrylonitrile-vinyl
chloride copolymers (69, 70). Microcapsules have
advantages over macrocapsulesin thatthey impose fewer limitationson diffusionalflowof nutrients
and wastes and they can be administeredby injection.Macrocapsules,on the otherhand,are easier
to retrieveshould complicationsoccur and are physicallymore stable than microcapsules.
SCIENCE * VOL. 260 * 14 MAY 1993

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921

possibilityof permanentliver replacement

if properlyintegratedinto the patient. Furthermore,the vascularaccess requiredfor
extracorporeal
use, which is sometimesassociated with thromboemboliccomplications, may not be necessarywith an implantablesystem(20).
Successful hepatocyte transplantation
dependson a numberof criticalsteps. First,
the hepatocytesmust be culturedin vitro
priorto transplantation.Hepatocytemorphologycan be maintainedby sandwiching
the cells between two hydratedcollagen
layers.Underthese conditions,the hepatocytes secretefunctionalmarkersat physiological levels for at least 6 weeks (22).
Second, the hepatocytesmust be attached
to the polymersubstrataso that they maintain their differentiatedfunction. By controllingthe densityof the extracellularmatrix substrateused to coat microcarriers
or
polymerfilms, both the extent of differentiatedfunctionandcell proliferationcan be
regulated(23). Third, the viabilityof the
transplantedhepatocytes must be maintained. This step can be accomplishedby
vascularizingthe cell transplantregion to
provide oxygen and nutritional factors
(24). Fourth, a sufficientmass of hepatocytesmustbecomeengraftedandfunctional
to achieve metabolicreplacement.Forimplantablesystems, this problemhas been
addressedin animalmodelsin which large
numbers of hepatocytes are placed into
vascularizedareasof the body and supplied
with hepatotrophicfactorsfrom the portal
circulation(25). Finally,hepatocytetransplantationper se does not provideall cell
types nor the delicate and complex structural featuresof the liver. For example,
products normally excreted through bile
may accumulatebecauseof the difficultyin
reconstructingthe biliarytree solely from
hepatocytes. This problem is not lifethreatening,however,and resinsthat bind
suchproductscouldbe incorporatedinto an
artificialliver (20). Hepatocytesplacedon
appropriatepolymerscan formtissuestructuresresemblingthose in the naturalorgan
(Fig. 2) and have shown evidence of bile
ductsand bilirubinremoval(25).
Pancreas.Each year, over 728,000 new
casesof diabetesarediagnosedand 150,000
Americans die from the disease and its
complications;the total yearlycost in the
United States is over $20 billion. Diabetes
is characterized
by pancreaticislet destruction, leading to loss of glucose control.
Tissue-engineering
approachesto treatment
have focusedon transplantinghealthypancreatic islets, usually encapsulatedin a
membrane to avoid immune rejection.
Three general approaches(Fig. 1) have
been testedin animalmodels.In the first,a
tubularmembranewas coiled in a housing
that contained islets. The membranewas
922

connected to a polymergraftthat in turn

connectedthe device to bloodvessels.The
membranehad a 50-kD molecular mass
cutoff, which allowedfree diffusionof glucose and insulin but blocked passage of
antibodiesand lymphocytes.In pancreatectomizeddogs treatedwith this device, normoglycemiawas maintainedfor more than
150days(26). In a secondapproach,hollow
fiberscontainingrat isletswereimmobilized
in the polysaccharidealginate. When the
device was placed intraperitoneally
in diabetic mice, blood glucoselevels were loweredfor more than 60 daysand good tissue
wasobserved(27). Finally,
biocompatibility
islets have been placed in microcapsules
composedof alginate or polyacrylates.In
some cases, rodentstreatedwith these microcapsulesmaintainednormoglycemiafor
over 2 years(28). All of these transplantation strategiesrequirea large,reliablesource
of donor islets. Porcine islets are used in
many studies, although genetically engineeredcellsthatoverproduce
insulinarealso
being examined.
Tubularstructures.The currentpractice
of usingpartsof otherorgansforreconstruction of the ureter, bladder, and urethra
often leadsto urinaryreflux,infection, and

dilation of the upperurinarytract, and, in

some instances, electrolyte disturbance.
Polymeror metal implantshave been used
to replaceuretersbut have generallyfailed
because of poor biocompatibility,lack of
peristalticactivity,andaccumulationof salt
deposits. Because the ureter has a good
regenerative capacity, cell-polymer implants have also been exploredas replacement therapy. In an initial study, cells
derivedfroma bladdercell carcinomawere
culturedon collagenspongesand implanted
in rats and dogs for over 3 months. Althoughthe rat implantsshowedsubstantial
salt depositson the spongesurface,the dog
implants showed extensive urothelial cell
regenerationon the collagen graft inner
surface(29). Morerecently,urothelialcells
were seeded onto degradablepolyglycolic
acid tubes and implantedin rats and rabbits. After 20 days, two to three layersof
urothelialcells lined the polymers(30).
The concept of using tubularstructures
is being studiedfor other tissuessuch as the
trachea,esophagus,intestine, and kidney.
A diseasedesophagus,for example, can be
treatedclinically with autograftsfrom the
colon, stomach, skin, or jejunalsegments.
However, such proceduresmay result in

Fig. 2. In one approach to opensystem implants, three-dimensional highly porous scaffolds

composed of synthetic polymers
serve as cell transplantdevices.
These devices may facilitateformationof structuraland functional
tissue units by the transplanted
cells. This approach is based on
the followingobservations:(i) Ev-

Biodegradable
polymer
scaffold

Invtrotissueculture

(ii) Isolated cells tend to reform Oslasts

the appropriate tissue structure

s
Chondrocytes195e

under appropriate experimental

conditions. For example, when
capillary endothelial cells are

Hepatocytes
Enterocytes
nrotes

placed on the proper substrate in

Urothelial
cells

Invivo implantation

vitro,they formtubularstructures.
(iii)Althoughisolated cells have
New
j
the capacity to formthe appropriate tissue structure,they do so
Bone
only to a limited degree when
Cartilage
placed as a suspension into tisiver
sue. Such cells begin withoutany
Iver
intrinsicorganizationand have no
template to guide restructuring.
Ureter
(iv)Tissue cannot be implantedin
large volumes-cells will not survive if they are located morethan
a few hundredmicrometersfrom
the nearest capillary. Thus, the
open-system implants are designed so that the polymerscaffold guides cell organizationand growthand allows diffusionof nutrientsto the transplantedcells
(32). Ideally,the cell-polymermatrixis prevascularizedor would become vascularizedas the cell
mass expands afterimplantation.
Vascularizationcould be a naturalhost response to the implantor
could be artificiallyinduced by slow release of angiogenic factors. The polymer could be
degradable or nondegradable. Materialsthat disappear from the body after they performtheir
functionobviate concerns about long-termbiocompatibility.
SCIENCE * VOL.260 * 14 MAY1993

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ARTICLES
graftnecrosis,inadequatebloodsupply,and
other complications.Copolymertubesconsistingof lactic and glycolicacid have been
sutured into dogs after removal of 5-cm
esophagealsegments. Over time, connective tissueandepitheliumcoveredthe polymergraft(whichhad begundegrading)and
the dogs were able to drink freelyand eat
semisolidfood (31). In a similarapproach,
fetal intestinalcells have been placedonto
these copolymer tubes and implanted in
rats. Histologicexaminationseveralweeks
later revealedthat some animalshad welldifferentiatedintestinal epithelium lining
the tubes, and this epitheliumappearedto
secrete mucous (32). Tubular structures
have also been usedin kidneyreplacement
studies. Current treatmentsfor end-stage
renal failureare basedsolely on nonphysiological drivingforcesand are not able to
mimic active moleculartransportaccomplishedby renaltubularcells. As a firststep
towardcreatinga bioartificialkidney, renal
tubularcells have been grownon acrylonitrile-vinyl chloride copolymersor microporouscellulose nitrate membranes.In vitro, these cells transportedinsulin, glucose
andtetraethylammonium
in the presenceof
a hemofiltratefrom a uremicpatient (33).
This approachhas not yet, to our knowledge, been exploredin vivo.

tion of artificialpolymeror metal prostheses. Problemsarise,however,in that donor

tissuefortransplantation
is limited,andit is
extremelydifficultto form delicate threedimensionalimplantsfrom host cartilage.
Artificialprosthesesmayresultin infection
and adhesivebreakdownat the host-prosthesis interface. A prosthesisalso cannot
adaptin responseto environmentalstresses
as does cartilage(34).
The need for improvedtreatmentshas
motivatedresearchaimed at creatingnew
cartilagethat is based on collagen-glycosaminoglycantemplates(35), isolatedchondrocytes (36), and chondrocytesattached
to natural(37) or synthetic (38, 39) polymers (Fig. 3C). In mice chondrocytes
grownfor 1 to 6 months on highly porous
scaffoldsof polyglycolicor polylactic acid
maintained the scaffold'soriginal threedimensional shape, appeared glistening
white macroscopically,contained sulfated
glycosaminoglycansand type II collagen,
andcloselyresembledcartilagehistologically (39). It is critical that cartilagetransplantsof appropriatethicknessbe mechanically functional. Recently, chondrocytes
grown in agarose gel culture have been
shown to producetissue with stiffnessand
compressibilitysimilarto articularcartilage
(40). The use of well-stirredbioreactorsfor
cultivatingchondrocyteson polymerscaffolds in vitro may enable nutrientsto penMesoderm
etrate the center of this nonvascularized
Cartilage,bone,andmuscle.Over 1 million tissue,leadingto relativelystrongand thick
surgical proceduresin the United States (up to 0.5 cm) implants(41).
each year involve cartilage replacement.
Over 1 million operationsannuallyinCurrent therapies include transplantation volve bone repair. Conventionally, bone
(removinghealthycartilage,carvingit into ingrowthis acceleratedthroughthe use of
desiredshapes, and reimplantingit where autogenousbone graftsor allogenic bone.
needed in the same patient) and implanta- The formercan be a successfulprocedure
Fig. 3. Histologicsections of engineered tissues. (A) Dopamine-producing cells in a polymercapsule
inthe guinea pig striatum12 weeks
aftertransplant.Cells were visualized (orange color) by immunostaining for tyrosine hydroxylase
[reprinted with permission from
(9)]. (B) New dermis produced
fromneonatalfibroblaststhatwere
placed on degradable polymers
witha thinoverlyingskin graftand
transplanted into a mouse. Photo
was taken 10 days afterthe transplant. (C) New cartilage produced
from chondrocytes seeded onto
degradable polymers 7 weeks after transplantinto a nude mouse.
(B) and (C) are stained withhematoxylinand eosin.

but is often material limited and causes

donorsite morbidityand contourirregularities (42). The lattercan alsobe a successful
procedure,but cell-mediatedimmune responsesto transplantationalloantigensand
pathogenscan be problematic.
Metals and ceramics are also used in
severalforms:bioinert(forexample,alumina), resorbable(for example, tricalcium
phosphate),porous(forexample,hydroxyapatite-coatedmetals), and bioactive (for
example,hydroxyapatites
and certainglasses). Bioactive materialsform a bond with
the surroundingtissue upon implantation.
Such materialsare currentlybeing used in
middle ear surgery,vertebralsurgery,and
other applications. Alumina prostheses
have been used in a varietyof dental and
orthopedicproceduresbecauseof theirminimal interaction with surroundingtissue
and low coefficientsof friction and wear
rates.Porousmaterials(poresize > 100 ,um)
allow bone to grow into the pores, which
strengthensthe union betweenthe implant
and bone. In practice,it may be desirable
for these materialsto degradeover time
because they lose strengthas they age. A
critical aspect of ceramic design is determining the appropriatecomposition, microstructure,poresize, porosity,andsurface
chemistryto matchthe specificbiologicand
metabolicrequirementsof tissues and disease states (43).
Synthetic and natural polymers have
been exploredfor bone repair, but it has
been difficultto createa polymerdisplaying
optimal strength and degradationproperties. Another approachinvolves implantation of demineralizedbone powder(DBP),
which is effective in stimulating bone
growthin animalsand humans;however,
DBP cannot induce sufficientbone formation in most non-bonysites. Bone morphogenic proteins (BMP), now producedby
genetic engineeringbut originallyderived
fromDBP, or growthfactorssuch as transforminggrowthfactor-3 (TGF-P), areother promisingstrategies.The formerinduce
formationof both cartilageand bone (including marrow)and the latter augment
bone growth (44). Such molecules have
theoretically unlimited availability;effective deliverysystemsfor such agentswill be
important. Bone growth can also be induced when cells are grown on synthetic
polymersor ceramics.For example, when
marrowcells are grownon porouscalcium
phosphatesin mice, bone formsinside the
poreswithin 3 weeks (45).
The abilityto generatemusclefibersmay
be usefulin the treatmentof muscleinjury,
cardiacdisease,disordersinvolving smooth
muscleof the intestineor urinarytract,and
in patients with muscular dystrophy. It has
been difficult to find drug therapies to treat
such diseases; however, gene therapy or

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923

cell-basedtherapiesmayprovidea meansof
treatingdiseasessuch as Duchennemuscular dystrophy(DMD) (46). Normal myoblastsfrom unaffectedrelativeshave been
transplantedinto patientswith DMD and
shown to produce dystrophinfor 1 to 6
months after transplant,althoughthe efficiency of myoblasttransferwas low. Myoblastscan migratefromone healthymuscle
fiberto another (47); thus, if the efficiency
of transfercan be increased,cell-basedtherapies may be useful in treatingDMD and
other muscleatrophies.
Heartdiseaseis the singlegreatestkiller
in the United States. Once patients become symptomatic,their life expectancyis
usuallymarkedlyshortened.This decline is
generallyattributedto the inabilityof cardiac cells to regenerateafter injury. In
contrast, skeletal muscle has the capacity
for tissue repair, presumablybecause of
satellite cells that have regenerativecapability. Recently, the feasibility of using
autotransplantedskeletal muscle satellite
cells multiplied in vitro and placed into
damagedheart muscle was explored in a
canine model. Preliminaryresultsshowed
that muscleformationoccurredat 8 weeks,
but not at 14 weeksaftertransplant(48).
Blood vesselsand cells. The design of
artificialblood vesselsand vasculargraftsis
an active researcharea. Although largediameter[>5 mm internaldiameter(i.d.)]
vasculargraftshave been successfullydeveloped with polymers such as Dacron or
expanded polytetrafluoroethylene,it has
been difficultto develop vasculargraftsof
<5 mm i.d. becauseof biologicalreactions
at the blood-materialand tissue-material
interface.These reactionslead to stricturing and total occlusion from clotting and
scarring.To circumvent these problems,
graftshave been madefromrelativelyinert
materials(with either heparincoatings or
polyethyleneoxide surfaces)or frommaterials that interact in a desirableway with
blood cells (49). One idea has been to line
polymersin vitro with endothelialcells to
promote hemocompatibility (50). Such
graftshave allowed these blood vessels to
stay open in short-termclinical studies.
Endothelializationin vivo can be induced
by the healing responsesof host tissue,
whichleadsto coverageof graftswith endothelial and smoothmusclecells (51). Polymersurfacemodificationby chemicalmeans
(for example,plasmadischarge)or protein
adsorptionmayalsobe desirable.The latter
approachmaybe usefulin designingmaterials that interactappropriately
with cells, but
it may be difficultto design materialsthat
selectively supportendothelial cell adhesion. Materialsthatpromoteendothelialcell
attachmentunfortunatelyoften simultaneously promoteattachmentof plateletsand
smooth muscle cells, with the attendant
924

adverseeffectsof clottingandpseudointimal
thickening. Polymershave recently been
designedthat containa cell adhesionligand
specificfor endothelialcells (52).
Thereare 18 millionhumanbloodtransfusionsin the United States annually.Becausedonorblood suffersfromproblemsof
limited storage time, donor shortage, requirementsfor typing and cross-matching,
and infectiousdiseasetransmission,thereis
a critical need for blood cell substitutes.
Red blood cells providea numberof functions, one of which is oxygen transport.
Oxygen-containingfluidsor materialsoffer
enormousapplicationsforuse in emergency
resuscitation, angioplasty, shock, tumor
therapy, exchange transfusion,and organ
preservation.Several oxygen transporters
are underdevelopment.A primarycandidate is hemoglobin,which not only serves
as the naturaloxygen transporterin blood
but also functionsin carbondioxide transport, as a buffer,and in regulatingosmotic
pressure.Early clinical trials of cell-free
hemoglobinwerecomplicatedby its lack of
purity,instability,andhigh oxygenaffinity,
but these problemshave subsequentlybeen
addressedby variouschemicalmodifications
(53). One remainingproblemis the limited
source of hemoglobin. It is unlikely that
there will be sufficient outdated human
blood to preparepracticalquantitiesof hemoglobinfor widespreadclinical use. Geneticallyengineeredhumanhemoglobinor
hemoglobinfrom bovine sourcescould be
an alternativeto humanhemoglobinif no
toxic effectsare associatedwith their use.
Solutions of perfluorocarbons(PFCs;
largeorganicmoleculesin which hydrogens
are replaced by fluorines)dissolve 40 to
70%oxygen per unit volume, nearlythree
times the oxygen-carryingcapacity of
blood. PFCs are not metabolizedand are
immisciblewith blood. They mustbe emulsified with dispersing agents such as
nonionic detergentsor phospholipids.Critical factorsin the choice of PFCs include
theiremulsifyingability,emulsionstability,
tissueretention time, vaporpressure,safety, and the effectivenessand safetyof the
emulsifyingagentrequired.AlthoughPFCs
have advantagesin termsof unlimitedsupply and oxygen-carrying
capacity,they also
have a numberof disadvantages,including
complement activation, toxicity, and retention by the reticuloendothelialsystem
(RES), which then reducesthe body'sability to clearwaste products(53).
Clinicaltrialsof both modifiedhemoglobins and PFCshave been conducted.The
resultsof the hemoglobintrialshave varied
dependingon the protocoland the specific
hemoglobinpreparationtested. Some studies have shown allergicor toxic responses,
and othershave showngood toleranceand
clinical improvement(for example, in a

studyof patientswith sickle cell anemia).

No single preparationhas demonstrated
clear superiority,and furtherstudy is required. Clinical trials with PFCs have
shown efficacy,but toxic effectshave limited the currentallowabledose in humans.
Researchto createfunctionalsubstitutes
for platelets(by encapsulatingplatelet proteins in lipid vesicles) has also been conducted (54). The success of this research
will depend on the identificationof the
membraneproteinscriticalto plateletfunction andthe abilityto minimizeRESuptake
and toxicity.
Finally, it is possiblethat bone marrow
stem cells could be maintainedin culture
and induced to multiply and differentiate
into the variouscellularelementsof blood.
Severalculturesystemsare underdevelopment to foster cell division (55-57). For
example, a combinationof rapidmedium
exchange and use of appropriategrowth
factorshas allowed up to a sixfold expansion of stem cells in 2 weeks (55). Maintenance of low oxygen concentrations(56)
and the additionof stemcell factormayalso
facilitatein vitro cell expansion(58).

Future Research
Numerousresearchareasarecriticalfor the
successof tissueengineering.Muchmustbe
learned from cell biology, such as what
controlscellulardifferentiationand growth
and how extracellularmatrix components
affectcell function (59). Immunologyand
moleculargenetics will contribute to the
designof cells (for example, by gene therapy) or cell transplantsystemsthat are not
rejectedby the immunesystem(60).
Cell source and cell preservationare
other importantissues. The transplanted
cells may come from cell lines or primary
tissues-from the patientsthemselves,other human donors, animal sources,or fetal
tissue. In choosing the cell source, a balance mustbe struckbetweenethical issues,
safetyissues,and efficacy.Cryopreservation
has been used successfullyfor certain cells
(61), but proceduresneed to be broadened
so that cell bankscan be createdfor many
different tissues. Large-scalecell culture
systemsare also importantto ensureproliferation of needed cells in vitro prior to
transplantationand to solve nutrienttransport issues (62). Sterilizationof the transplantsis also critical.
The materialsusedin tissueengineering
representa major area of study. Natural
materials are advantageousin that they
contain information(for example,particular amino acid sequences) that facilitates
cell attachmentor maintenanceof differentiated function. Counteringthis advantage
is the fact that manynaturalmaterialssuffer
batch-to-batchvariationsor scale-updiffi-

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-m-

___

culties. Synthetic polymers,on the other

hand, allow precisecontrolover molecular
weight, degradationtime, hydrophobicity,
andotherattributes,yet they maynot interact with cellsin a desiredmanner.Recently,
the advantagesof both naturalandsynthetic
polymershave been combinedin strategies
wherebycriticalaminoacid sequencesfrom
naturalpolymersare graftedonto synthetic
polymers(63). Polymerprocessingis another key issue. Many implantsare made of
compositematerialsor highly porousstructures;methods of manufacturingsuch immay be crucialto their
plantsreproducibly
success(64). The developmentof controlled
release systems, which deliver molecules
over long time periods,will be importantin
numeroustissue-inducing
facadministering
tors, growthfactors,and angiogenesisstimulators (65). Finally, it will be useful to
develop methods of surface analysis for
studyinginterfacesbetweencell andmaterials (66) and mathematicalmodels (67) and
in vitrosystems(68) that can predictin vivo
cellularevents.
and
Currentmethodsof transplantation
reconstructionare among the most costly
therapiesavailabletoday.Tissueengineering
futuresavoffersthe possibilityof substantial
ings by providingsubstitutesthat are less
expensivethandonororgansandbyproviding
a meansof interventionbeforepatientsare
criticallyill. In addition,cell transplantsystems may complementgene therapy approachesin facilitatingtransferof largepopua desiredphenotype.
lationsof cellsexpressing
Few areasof technologywill requiremore
researchthan tissue engiinterdisciplinary
neeringor have the potentialto affectmore
positivelythe qualityandlengthof life.
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understudy: (i) purifyingalginates [A. M. Sun, I.

Vacek, I.Tai,in Microcapsuiesand Nanoparticles
in Medicine and Pharmacy, M. Donbrow, Ed.
(CRC Press, Boca Raton, FL, 1992), pp. 315322]; (ii)synthesizinghydrogelswithpolyethylene
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(1992)]; (iii) encapsulating cells with synthetic
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as polyacrylates-interestingly,procedures for
using polyacrylatesexpose cells to organic solvents, yet viabilityand functionof a numberof
mammaliancell types are retained[M.V. Sefton,
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Release 19, 289 (1992); H. Uludag and M. V.
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72. We thank J. Stoudemireand L. Blanksteinfor
assistance, and J. Hubbell,G. Naughton,S. Weinstock, H. Blau, R. Tompkins,R. Pops, and C.
Mullonfor reviewingthe manuscript.Supported
by AdvancedTissue Sciences, Inc.,LaJolla,CA,
NSF, NIH,the ThomasAnthonyPappas Charitable Foundation,Inc., and the HollyAnn Soulard
Research Fund.

Development of Methods for

Gene Delivery

The transductionof appropriatetargetcells

representsthe critical first step in gene
therapy;consequently,the developmentof
methodsof gene transfersuitablefor different formsof therapyhas been a majorfocus
64.
of research(Table 1). The single common
65.
feature of these methods is the efficient
66.
deliveryof genes into cells. In the case of
retroviralvectors and adeno-associatedvi67.
rusvectors, the transferred
DNA sequences
68.
are stablyintegratedinto the chromosomal
DNA of the targetcell. These vectorshave
been consideredmostoften forex vivo gene
therapy, which involves removal of the
69.
relevant targetcells from the body, transductionof the cells in vitro, andsubsequent
reintroductionof the modifiedcells into the
70.
patient. All of the other methodsof gene
transferresultprimarilyin the introduction
71.
of DNA sequencesinto the nucleus in an
unintegratedform. Those methods, which
result in high, but transient,gene expression, have predominantlybeen considered
for use in in vivo gene therapies,in which
genetic materialis directlytransferredinto
cells and tissuesof the patient. As discussed
below, little is known about the fate and
propertiesof DNA delivered to cells by
these other methods.
RichardC. Mulligan
Retroviral
vectors.Interestin retrovirusThe developmentoverthe past decade of methodsfordeliveringgenes to mammaliancells
mediatedgene transferstemsprimarilyfrom
has stimulatedgreat interestin the possibilityof treatinghumandisease by gene-based
the abilityof some vectors to stably transtherapies. However,despite substantialprogress, a numberof key technicalissues need
duce close to 100%of targetcells.
to be resolvedbeforegene therapycan be safely and effectivelyappliedinthe clinic.Future
Although retrovirally mediated gene
technologicaldevelopments,particularly
in the areas of gene deliveryand cell transplan- transferis ideal for many ex vivo applicatation,willbe criticalfor the successful practiceof gene therapy.
tions of gene therapy,severalfeaturesof the
gene transfermethodmay limit its applicability, particularlywith regardto in vivo
therapies.First,retrovirusentryinto cells is
Ever since the developmentof recombi- by transferof genetic materialinto specific absolutelydependent on the existence of
nant DNA technology, the promiseof the cells of a patient, ratherthan by conven- the appropriateviral receptoron the target
technologyfor dramaticallyimprovingthe
tional drugs-has yet to make its markin
cell. To providea means of infectingmost
practice of medicine has been vigorously medicine. Although the concept may ap- cells of interest,researchershave developed
championed.Most of the advancesaffect- pearto be elegantlystraightforward
and the packagingcell lines capableof generating
ing the clinical managementof patients most direct application of recombinant viruses of a variety of host ranges (2).
have involved either the developmentof
DNA technology, researchhas indicated Because the identities of most retroviral
new moleculartechniquesfor the diagnosis that successful implementation of gene receptorsareunknown,however,it has not
of specificinheritedandacquireddiseasesor transferin the clinic will requirethe coor- been possibleto determinethe distribution
the developmentof new therapeuticprod- dinated developmentof a variety of new of receptorsin differentcell types.Problems
uctsmadepossibleby the abilityto engineer technologies and the establishment of encountered in transducingspecific cell
the overexpressionof specific genes. Re- unique interactionsbetween investigators types (such as hematopoietic stem cells)
combinantDNA technologyhas also pro- from divergentmedical and basic science may be due, in part, to the lack of expresduced the means for definingthe roles of disciplines.
sion of appropriateviralreceptors.Second,
specificgene productsin the pathogenesis
Although severalreviewsof gene thera- replicationof the targetcells is necessaryfor
of humandisease.The abilityto character- py researchhave been published (1), few proviral integration to occur. Although
ize disease in such molecular terms has have focused on the technical issues that previouslyit had been assumedthat this
alreadyled to more precise and effective continue to impede the translationof pre- requirement reflected the necessity for
clinical interventions.
clinical studiesof gene therapyinto effec- DNA synthesis,recent studiessuggestthat
However,the idea underlyinggene ther- tive clinical protocols. In this review and viral integration may depend on mitosis
apy-that humandiseasemight be treated commentary,I have attempted to define (3). Thus, successfulgene transferdepends
those issues and to suggest new areas of on the abilityto induceproliferationof the
Theauthoris at the WhiteheadInstituteforBiomedical
investigation that may help to resolve targetcell, at least forshortperiodsof time.
Researchand Departmentof Biology,Massachusetts
Instituteof Technology,Cambridge,MA02142.
them.
Another problemis that the retroviral

The

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Gene

Science
Therapy

of

SCIENCE * VOL. 260 * 14 MAY1993

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