Hydrogels for Tissue Engineering [602217]

Hydrogels for Tissue Engineering
Kuen Yong Lee and David J. Mooney*
Departments of Biologic & Materials Sciences, Chemical Engineering, and Biomedical Engineering, University of Michigan,
Ann Arbor, Michigan 48109
Received November 15, 2000
Contents
I. Introduction 1869
II. Design Parameters for Hydrogels in Tissue
Engineering1871
III. Hydrogels from Natural Polymers 1872
A. Collagen and Gelatin 1872B. Hyaluronate 1872
C. Fibrin 1872D. Alginate 1873
E. Agarose 1873
F. Chitosan 1873
IV. Hydrogels from Synthetic Polymers 1874
A. Poly(acrylic acid) and Its Derivatives 1874B. Poly(ethylene oxide) and Its Copolymers 1874
C. Poly(vinyl alcohol) 1875D. Polyphosphazene 1875
E. Polypeptides 1876
V. Future Perspectives 1876
VI. Acknowledgments 1877
VII. References 1877
I. Introduction
Every year, millions of patients suffer the loss or
failure of an organ or tissue as a result of accidentsor disease. Over 8 million surgical procedures areperformed to treat these patients in the U.S. eachyear, and the overall cost of these problems to theU.S. economy is estimated to exceed $400 billion peryear.
1Tissue or organ transplantation is a generally
accepted therapy to treat these patients. However,this approach is extremely limited by a donor short-age. For example, according to the American HeartAssociation, only 2300 people received a heart trans-
plant in 1997, while approximately 40 000 patientsin the U.S. alone could benefit from this therapy.Similarly, over 10 000 patients require skin grafts totreat severe burns or skin cancers in the U.S. eachyear.
2
An exciting and revolutionary strategy to treat
patients who need a new organ or tissue is theengineering of man-made organs or tissues (Figure1). Tissues or organs can be potentially engineeredwith a number of different strategies, but a particu-larly appealing approach utilizes a combination of apatient’s own cells combined with polymer scaffolds.In this approach, tissue-specific cells are isolatedfrom a small tissue biopsy from the patient andharvested in vitro. The cells are subsequently incor-porated into three-dimensional polymer scaffolds thatact as analogues to the natural extracellular matricesfound in tissues. These scaffolds deliver the cells tothe desired site in the patient’s body, provide a spacefor new tissue formation, and potentially control thestructure and function of the engineered tissue.
3,4A
variety of tissues are being engineered using thisapproach including fabricated artery, bladder, skin,cartilage, bone, ligament, and tendon. Several ofthese tissues are now at or near clinical uses.
5-10In
addition, various approaches have been introducedto coax differentiated or undifferentiated cells (i.e.,stem cells) into the desired cell phenotype.
11
A critical element in virtually all tissue engineering
approaches is the polymer scaffold. The polymerpotentially mimics many roles of extracellular ma-trixes found in tissues. Extracellular matrices, com-prised of various amino acids and sugar-based mac-romolecules, bring cells together and control thetissue structure, regulate the function of the cells,and allow the diffusion of nutrients, metabolites, andgrowth factors.
12Various types of polymers have been
studied and utilized to date in tissue engineering.13
Aliphatic polyesters including poly(glycolic acid) (PGA),* To whom correspondence should be addressed. David J. Mooney,
Department of Chemical Engineering, University of Michigan, AnnArbor, MI 48109-2136. Tel: 734-763-4816. Fax: 734-763-0459.E-mail: mooneyd@umich.edu.Volume 101, Number 7 July 2001
10.1021/cr000108x CCC: $36.00 © 2001 American Chemical Society
Published on Web 05/31/2001

poly(lactic acid) (PLA), and copolymers (PLGA) of
these materials are the most widely used syntheticpolymers (Figure 2).
14,15These polymers have a long
history of use in medical applications and are con-sidered safe in many situations by the FDA. How-ever, the use of these types of polymer scaffoldsrequires the surgeon to make incisions (cuts) suf-ficiently large to enable placement of the polymer/cell constructs.
An exciting alternative approach to cell delivery for
tissue engineering is the use of polymers (i.e., hy-drogels) that can be injected into the body. Thisapproach enables the clinician to transplant the celland polymer combination in a minimally invasivemanner. Hydrogels have structural similarity to the
macromolecular-based components in the body andare considered biocompatible.
16Hydrogels have found
numerous applications in tissue engineering as wellas in drug delivery. Tissue engineering is a mostrecent application of hydrogels, in which they areused as scaffolds to engineer new tissues (Figure 1).
17
David Mooney was born in Madison, WI in 1964. He received a B.S. at
the University of Wisconsin (1987) and a Ph.D. from MIT (1992), both inChemical Engineering. He served as a postdoctoral fellow in the SurgeryDepartment at Harvard Medical School, before moving to the Universityof Michigan. He is currently an Associate Professor in ChemicalEngineering, Biomedical Engineering, and Biologic & Materials Sciences.Research in his laboratories is focused on elucidating the mechanismsby which cells receive information from materials, and utilizing thisinformation to design new biomaterials that precisely regulate cellular geneexpression. The resultant biomaterials are currently being tested in a varietyof drug delivery and tissue engineering applications.
Kuen Yong Lee was born in Seoul, Korea in 1968. He received his B.S.degree in Fiber and Polymer Science from Seoul National University(1992), and his M.S. (1994) and Ph.D, (1998) degrees from Seoul NationalUniversity in Polymer Chemistry. He held postdoctoral positions at theKorea Institute of Science and Technology (1998) and at the Universityof Michigan (1998 −2001). He is currently an Assistant Research Scientist
of Biologic & Materials Sciences at the University of Michigan. His researchinterest has included design, modification, and characterization ofbiomaterials for drug delivery and tissue engineering applications. Hiscurrent research activities are focused on elucidating interactions betweenpolymers and cells, degradable polymeric scaffolds, and delivery of growthfactors for tissue engineering.
Figure 1. Schematic illustration of typical tissue engi-
neering approaches. Cells are obtained from a small biopsyfrom a patient, expanded in vitro, and transplanted intothe patient either by injection using a needle or otherminimally invasive delivery approach, or by implantationat the site following an incision (cut) by the surgeon to allowplacement.
Figure 2. Chemical structure of (a) poly(glycolic acid), (b)
poly(lactic acid), and typical structures of (c) porous scaf-folds of poly(lactide- co-glycolide) and (d) nonwoven fabrics
of poly(glycolic acid). These latter materials have beenwidely used for tissue engineering applications. (Reprintedfrom ref 14 with permission. Copyright 1998 John Wiley& Sons, Inc.)1870 Chemical Reviews, 2001, Vol. 101, No. 7 Lee and Mooney

In this review, we discuss the critical design
parameters of hydrogels to be used in tissue engi-neering. Hydrogels, currently used or with potentialapplications in tissue engineering, are divided intotwo categories, according to their natural or syntheticorigin. Hydrogels from natural polymers have beenwidely used for tissue engineering approaches. How-ever, limitations of gels from natural polymers havemotivated approaches to modify these polymers aswell as to use various synthetic polymers. A widerange of synthetic polymers may potentially havesuitable chemical and physical properties for theseapplications. In addition, incorporation of growthfactors and the role of mechanical signals to enhancetissue development will be discussed as future direc-tions.
II. Design Parameters for Hydrogels in Tissue
Engineering
Hydrogels in tissue engineering must meet a
number of design criteria to function appropriatelyand promote new tissue formation. These criteriainclude both classical physical parameters (e.g.,degradation and mechanics) as well as biologicalperformance parameters (e.g., cell adhesion). Anabsolutely critical parameter is the biocompatibilityof hydrogels. Biocompatibility relates to the materi-al’s ability to exist within the body without damagingadjacent cells or lead to significant scarring orotherwise elicit a response that detracts from itsdesired function. This may be especially problematicas the inflammatory response to a hydrogel can affectthe immune response toward the transplanted cellsand vice versa.
18,19Naturally derived polymers fre-
quently demonstrate adequate biocompatibility, whilesynthetic polymers may elicit significant negativeresponses from the body. Therefore, one may havesome restrictions when preparing hydrogels fromsynthetic polymers for these applications.
The mechanism of gelling, which may include ionic
or covalent cross-linking and inherent phase transi-tion behavior, should be considered next. Ionic cross-linking with multivalent counterions is a simple wayto form hydrogels. However, those ions could be
exchanged with other ionic molecules in aqueousenvironments, resulting in an uncontrolled deteriora-tion of the original properties of hydrogels.
20Covalent
cross-linking is a common method to precisely controlthe cross-linking density of hydrogels. However, thetoxicity of cross-linking molecules must be consid-ered, and nondegradable cross-link formation may bedisadvantageous in most tissue engineering applica-tions. One recent approach to form hydrogels is theutilization of the phase transition behavior of certainpolymers.
21For example, a very small change of
temperature near the lower critical solution temper-ature (LCST) can trigger the phase transition of apolymer solution to a gel, and significant researchhas been performed to control the LCST (e.g., designit to be close to body temperature).
22Hydrogels, cross-
linked in situ with minimal temperature rise duringpolymerization, have also been reported and utilizedfor orthopedic tissue engineering.
23The mechanical properties of hydrogels are impor-
tant design parameters in tissue engineering, as thegel must create and maintain a space for tissuedevelopment. In addition, the adhesion and geneexpression of cells are tightly related to the mechan-ical properties of the polymer scaffold.
24The mechan-
ical properties of hydrogels mainly depend on theoriginal rigidity of polymer chains, types of cross-linking molecules and the cross-linking density, andswelling as a result of hydrophilic/hydrophobic bal-ance.
25
The controlled degradation of hydrogels is also
critical in tissue engineering, whether the gels areoriginated from natural resources or are syntheticallycreated. Typically, one desires to coordinate thedegradation rate of a scaffold to tissue development,and this time will be dependent on the tissue type tobe engineered.
26Degradation of hydrogels can be due
to hydrolysis, the action of enzymes, and/or dissolu-tion. The degradation rate and mechanical propertiesof cross-linked gels are typically coupled to eachother. However, sometimes those properties can bedecoupled by intentionally introducing network de-fects, resulting in the formation of soft hydrogels withlonger degradation times than stiffer, more cross-linked gels (Figure 3).
27
The interactions of cells with hydrogels signifi-
cantly affects their adhesion as well as migration and
Figure 3. (a) Decoupled degradation behavior of poly-
(aldehyde guluronate) (PAG) hydrogels cross-linked withadipic acid dihydrazide. t
1/2indicates the time when gels
lose 50% of their initial modulus.27(b) Photomicrograph of
representative tissue section following osteoblast/PAG gelconstructs transplantation into mice. The tissue sectionwas taken after 9 weeks and stained with hematoxylin andeosin. The original picture was taken at 100 magnifica-
tion, and the photomicrograph has labels for remaininghydrogel (H) and newly formed bone tissue (B) (unpub-lished data).Hydrogels for Tissue Engineering Chemical Reviews, 2001, Vol. 101, No. 7 1871

differentiation. The adhesion may be cell-type specific
and is dependent on the interaction of specific cellreceptors with ligands that are a component oradsorbed onto the materials.
28Inappropriate interac-
tions could cause undesirable tissue formation.
III. Hydrogels from Natural Polymers
A. Collagen and Gelatin
Collagen is the most widely used tissue-derived
natural polymer, and it is a main component ofextracellular matrices of mammalian tissues includ-ing skin, bone, cartilage, tendon, and ligament.Physically formed collagen gels are thermally revers-ible and offer a limited range of mechanical proper-ties. Chemical cross-linking of collagen using glut-araldehyde
29or diphenylphosphoryl azide30can
improve the physical properties. However, these gelsare still short of physical strength, potentially im-munogenic, and can be expensive.
31Furthermore,
there can be big variations between produced col-lagen batches. However, collagen meets many of thebiological design parameters, as it is composed ofspecific combinations of amino acid sequences thatare recognized by cells and degraded by enzymessecreted from the cells (i.e., collagenase). Collagenhas been used as a tissue culture scaffold or artificialskin due to the ready attachment of many differentcell types and its cell-based degradation. The attach-ment of cells to collagen can be altered by chemicalmodification, including the incorporation of fibronec-tin, chondroitin sulfate, or low levels of hyaluronicacid into the collagen matrix.
32Collagen gels have
been utilized for reconstruction of liver,33skin,34blood
vessel,35and small intestine.36
Gelatin is a derivative of collagen, formed by
breaking the natural triple-helix structure of collageninto single-strand molecules. There are two types ofgelatin, gelatin A and gelatin B. Gelatin A is pre-pared by acidic treatment before thermal denatur-ation, while gelatin B is processed by alkaline treat-ment that leads to a high carboxylic content.
37
Gelatin easily forms gels by changing the tempera-ture of its solution. It has been used in many tissueengineering applications due to its biocompatibilityand ease of gelation. Gelatin gels have also beenutilized for delivery of growth factors to promotevascularization of engineered new tissue.
38However,
the weakness of the gels has been a problem, and anumber of chemical modification methods have beeninvestigated to improve the mechanical properties ofgelatin gels.
39,40
B. Hyaluronate
Hyaluronate is one of the glycosaminoglycan com-
ponents in natural extracellular matrices and playsa significant role in wound healing. Hyaluronate canbe formed into hydrogels by covalent cross-linkingwith various kinds of hydrazide derivatives (Figure4)
41,42and radical polymerization of glycidyl meth-
acrylate.43Hyaluronate is degraded by hyaluronidase,
which exists in cells and serum.44Hyaluronate has
shown excellent potential for tissue engineeringapplications such as artificial skin,45facial intra-
dermal implants,46would healing,47and soft tissue
augmentation.48However, hyaluronate requires thor-
ough purification to remove impurities and endotox-ins that may potentially transmit disease or act asan adjuvant in eliciting an immune response.
49In
addition, hyaluronate gels typically possess low me-chanical properties. These issues have limited theapplications of hyaluronate.
C. Fibrin
Fibrin has been used as a sealant and an adhesive
in surgery as it plays an important role in naturalwound healing. Fibrin gels can be produced from thepatient’s own blood and can be used as an autologousscaffold for tissue engineering. No toxic degradationor inflammatory reactions are expected from thisnatural component of the body. Fibrin forms gels bythe enzymatic polymerization of fibrinogen at roomtemperature in the presence of thrombin.
50An in-
teresting feature of fibrin is the degradation andremodeling by cell-associated enzymatic activity dur-ing cell migration and wound healing, and its deg-radation rate can be controlled by apronitin, aproteinase inhibitor.
51
Fibrin gels might promote cell migration, prolifera-
tion, and matrix synthesis through the incorporationof platelet-derived growth factors and transforminggrowth factor â.
52Bidomain peptides with a factor
XIIIa substrate in one domain and a bioactive peptidecontaining RGD sequence in another domain havebeen covalently incorporated into fibrin gels duringcoagulation through the action of the transglutami-nase factor XIIIa, resulting in gels with potentialneurological applications.
53Fibrin gels have also been
utilized to engineer tissues with skeletal musclecells,
51smooth muscle cells,54and chondrocytes.55
However, fibrin gels are limited in mechanicalstrength, and this prevents their use in certainapplications.
Figure 4. Chemical structure of (a) hyaluronic acid and
various cross-linking molecules that can be used to formhydrogels, including (b) 3,3 ′-dithiobis(propanoic dihy-
drazide), (c) 1,3,5-benzene(tricarboxylic trihydrazide), and(d) poly(ethylene glycol)-diamine tetrapropanoic tetra-hydrazide.1872 Chemical Reviews, 2001, Vol. 101, No. 7 Lee and Mooney

D. Alginate
Alginate is a well-known biomaterial obtained from
brown algae and is widely used for drug delivery andin tissue engineering due to its biocompatibility, lowtoxicity, relatively low cost, and simple gelation withdivalent cations such as Ca
2+,M g2+,B a2+, and Sr2+
(Figure 5a).56Alginate has found uses to date as an
injectable cell delivery vehicle57as well as wound
dressing, dental impression, and immobilization ma-trix.
58,59Alginate gel beads have also been prepared
and used for transplantation of chondrocytes,60hepa-
tocytes,61and islets of Langerhans to treat diabetes.62
Despite its advantageous features, alginate itself
may not be an ideal material because it degrades viaa process involving loss of divalent ions into thesurrounding medium, and subsequent dissolution.This process is generally uncontrollable and unpre-dictable. Therefore, covalent cross-linking with vari-ous types of molecules and different cross-linkingdensities has been attempted to precisely control themechanical and/or swelling properties of alginate gels(Figure 5b -d).
25In addition, the molecular weights
of many alginates are typically above the renalclearance threshold of the kidney.
63Recently, hydro-
lytically degradable and covalently cross-linked hy-drogels derived from alginate were reported.
64An
attractive approach to control the degradation ofalginate involves the isolation of polyguluronateblocks with molecular mass of 6000 Da from alginate,and the subsequent oxidation and covalent cross-linking of these derivatives with adipic acid dihy-drazide. The gelling of these polymers could bereadily controlled, and their mechanical and degra-dation properties were also regulated depending onthe cross-linking density.
27In addition, slightly oxi-
dized alginate itself was found to be degradable inaqueous media depending on the pH and tempera-ture of the solution (Figure 5e).
65
Another potential limitation in using alginate gels
in tissue engineering is the lack of cellular inter-action. Alginate is known to discourage proteinadsorption due to its hydrophilic character, and it is
unable to specifically interact with mammalian cells.
66
Therefore, alginate has been modified with lectin, acarbohydrate specific binding protein, to enhanceligand-specific binding properties.
67An RGD-contain-
ing cell adhesion ligand has also been covalentlycoupled to alginate gels to enhance cell adhesion.These modified alginate gels have been demonstratedto provide for the adhesion, proliferation, and expres-sion of differentiated phenotype of skeletal musclecells (Figure 6).
68
E. Agarose
Agarose is another type of marine algal polysac-
charide, but unlike alginate it forms thermallyreversible gels.
69The proposed gel structure is bundles
of associated double helices, and the junction zonesconsist of multiple chain aggregation.
70The physical
structure of the gels can be mainly controlled byusing a range of agarose concentrations, whichresults in various pore sizes. The large pores and lowmechanical stiffness of the gels at low concentrationsof agarose may enable the migration and proliferationof cells, and these factors have been found to affectneurite growth in vitro.
71Chitosan has been co-
valently bound to agarose gels to incorporate chargeinto the gels, and this significantly contributed toneurite growth as well.
3Cell adhesion peptides
(CDPGYIGSR) have also been covalently coupled toenhance the interaction with cells.
72
F. Chitosan
Chitosan is prepared by N-deacetylation of chitin
and usually contains less than 40% of N-acetyl- D-
Figure 5. Chemical structure of (a) sodium alginate and
various cross-linking molecules used in covalent cross-linking reactions, including (b) adipic acid dihydrazide, (c)
L-lysine, and (d) poly(ethylene glycol)-diamine. Alginate can
be oxidized with sodium periodate under mild reactionconditions to infer main chain lability to hydrolysis as well(e).
Figure 6. Myoblast adhesion onto (a) unmodified and (b)
GRGDY-modified alginate hydrogels. Very few cells adhereto unmodified alginate gels, while cells readily adhere,spread, and function on the modified gels. (Reprinted fromref 68 with permission. Copyright 1999 Elsevier Science.)Hydrogels for Tissue Engineering Chemical Reviews, 2001, Vol. 101, No. 7 1873

glucosamine residues (Figure 7). Chitosan has found
many biomedical applications, including tissue en-gineering approaches, due to its biocompatibility, lowtoxicity, structural similarity to natural glycosami-noglycans, and degradation by enzymes such aschitosanase and lysozyme.
73However, chitosan is
easily soluble in the presence of acid, and generallyinsoluble in neutral conditions as well as in mostorganic solvents due to the existence of amino groupsand the high crystallinity. Therefore, many deriva-tives have been reported to enhance the solubilityand processibility of this polymer.
74,75Chitosan forms
hydrogels by ionic76or chemical cross-linking with
glutaraldehyde.77Azide-derivatized chitosan was also
reported to form gels by UV irradiation.78
Numerous derivatives have been developed to alter
the biological functions of chitosan, including en-hancement of cellular interactions for tissue engi-neering approaches. Chitosan has been modified withsugar residues such as fructose or galactose forculture of hepatocytes,
79,80and with proteins such as
collagen, gelatin, and albumin for neural tissueengineering.
81In addition, methylpyrrolidinone-de-
rivatized chitosan has been reported to promote boneformation.
82
IV. Hydrogels from Synthetic Polymers
A. Poly(acrylic acid) and Its Derivatives
One of the most studied synthetic hydrogels is
hydrolytically stable cross-linked poly(2-hydroxyethylmethacrylate) (HEMA). The permeability and hydro-philicity of these gels are dependent on the cross-linking agents.
83Poly(HEMA) has been used for
ophthalmic uses including contact lens,84as well as
in many drug delivery applications.85Macroporous
poly(HEMA) gels have been prepared by freeze/thaw,or particulate leaching techniques for cartilage re-placement.
86Many different types of molecules and
cells have also been encapsulated into poly(HEMA)gels, and this approach has been reported to besuccessful for delivery of insulin or other proteins intothe body.
87Poly(HEMA) gels are not degradable in
physiological conditions. Therefore, dextran-modifiedpoly(HEMA) gels have been synthesized, and re-ported to be degradable by enzymes.
88In addition,
enantiomeric oligo( L-lactide) and oligo( D-lactide) were
grafted to poly(HEMA) to induce stereocomplexation,resulting in the formation of poly(HEMA) gels with-out using any toxic chemical reagents.
89
Poly( N-isopropylacrylamide) (PNIPAAm) is poten-
tially very attractive for tissue engineering applica-tions as it exhibits phase transition behavior abovethe lower critical solution temperature (LCST). TheLCST of PNIPAAm in water is approximately 32 °Cand can be matched to body temperature by copo-
lymerization.
90Therefore, the use of PNIPAAm and
its copolymers in tissue engineering would be verybeneficial as one can easily prepare a mixed solutionof cells and the polymer at room temperature or evenat a lower temperature and inject it into the desiredsite. This will result in the formation of a solid cell/polymer construct as the gel warms to body temper-ature. NIPAAm has been copolymerized with acrylicacid, methacrylic acid, or butylmethacrylic acid,depending on the desired final applications.
91-93
Acrylamide derivatives have also been cross-linked
with native proteins,94oligodeoxyribonucleotides,95or
engineered coiled-coil proteins96to form temperature-
responsive gels. In this situation, conventional poly-mers could potentially be modified to exhibit thermaltransition behavior by utilizing a variety of cross-linking molecules that can induce phase separationin response to temperature changes.
The unique temperature-responsive nature of these
polymers is leading to a variety of biological applica-tions. In standard cell culture, cells are recoveredfrom the dishes by treatment with proteases (e.g.,trypsin). However, the culture of cells on PNIPAAmenables one to easily recover intact cell sheetswithout damage by simply decreasing the tempera-ture and modulating the hydrophilicity of the gel.
97
These polymers are also being investigated as aninjectable delivery vehicle for cartilage and pancreasengineering.
91,98This mechanism of phase transition
may be ideal for delivery of cells, as unlike chemicallycontrolled cross-linking (e.g., alginate), the timing ofphase transition is not set, but simply depends onthe temperature change upon introduction to thebody. However, limitations of these gels are thenondegradable cross-links, and the vinyl monomersand cross-linking molecules are toxic, carcinogenic,or teratogenic.
21In an effort to obviate these issues,
dextran-grafted PNIPAAm copolymers have beensynthesized, and these may modulate degradation insynchronization with temperature (Figure 8).
99
B. Poly(ethylene oxide) and Its Copolymers
Poly(ethylene oxide) (PEO) has been approved by
the FDA for several medical applications due to its
Figure 7. Chemical structure of chitosan.
Figure 8. Synthesis of dextran-grafted poly( N-isoprop-
ylacrylamide- co-N,N-dimethylacrylamide) (NIPAAm- co-
DMAAm).991874 Chemical Reviews, 2001, Vol. 101, No. 7 Lee and Mooney

biocompatibility and low toxicity. It has been exten-
sively studied for uses including preparation ofbiologically relevant conjugates,
100surface modifica-
tion of biomaterials,101and induction of cell mem-
brane fusion.102PEO itself is very hydrophilic and
can be synthesized by anionic or cationic polymeri-zation of ethylene oxide. PEO gels can be preparedby UV photopolymerization of the precursor thatconsists of PEO with acrylate termini at each end inthe presence of R-hydroxy acid.
103The peptide se-
quence of Ala -Pro-Gly-Leu has also been intro-
duced into these gels, to make the gels susceptibleto enzymes existing in the body, and these gels maybe useful for tissue engineering applications.
104
Branched PEO having a cinnamylidene acetyl moietyas a pendant group has been synthesized and photo-polymerized to form gels, and these gels demon-strated antithrombogenic properties.
105Star-shaped
PEO was cross-linked by irradiation to form hydro-gels and also modified with galactose moieties toenhance the interaction with liver cells.
106
Various PEO-based copolymers have been reported
and utilized, especially in drug delivery applica-tions.
107-109One interesting copolymer is a triblock
copolymer of PEO and poly(propylene oxide) (PEO-b-PPO- b-PEO), which is known by the trade name
Pluronics or Poloxamers, and is commercially avail-able in various lengths and compositions. Thesepolymers form thermally reversible gels without anypermanent cross-links, unlike PNIPAAm and itscopolymer gels. In addition, it was reported thatPEO -PPO -PEO triblock copolymers could be de-
signed to form gels at body temperature by forminga liquid crystalline phase.
110PEO -PPO triblock
copolymers have been mainly used for drug deliveryapplications, as they are known to enhance drugpenetration, and also enhance the activity of antine-oplastic agents against tumors.
111There have been
few reports on the utilization of these gels in tissueengineering to date,
112but they may find multiple
applications in this field.
Although PEO -PPO -PEO triblock copolymers
form hydrogels in aqueous solutions in response totemperature changes, they have limitations for bio-medical uses. These limitations include the lack ofbiodegradation. Therefore, a variety of biodegradabledi- or triblock copolymers of PEO and poly(lactic acid)(PLA) have been synthesized, as PLA is degradableand has been already proved to be safe in manymedical applications (Figure 9).
113Alternating multi-
block copolymers of PEO and PLA were also synthe-sized by the condensation reaction of
L-lactic acid in
the presence of succinic acid. These gels exhibitedtemperature-dependent reversible gel -sol transitions
near body temperature.114These gels may be useful
in tissue engineering as they can be easily formulatedwith protein drugs or cells at room temperature orlower, and subsequently delivered to the desired sitein a minimally invasive manner.
C. Poly(vinyl alcohol)
Poly(vinyl alcohol) (PVA) is generally obtained from
poly(vinyl acetate) by alcoholysis, hydrolysis, or ami-nolysis.
115The hydrophilicity and solubility of PVA
can be readily controlled by the extent of hydrolysisand molecular weight. PVA forms hydrogels bychemical cross-linking with glutaraldehyde
116or ep-
ichlorohydrin.117To avoid the toxicity and leaching
problems of chemical cross-linking agents, a repeatedfreezing/thawing method,
118or electron beam119has
been applied to form PVA hydrogels. The gels formedby the repeated freezing/thawing method were re-ported to be stable at room temperature, and highlyelastic.
118However, these gelling methods are not
appropriate inside the body in situ, and PVA is notdegradable in most physiological situations. There-fore, these gels are mostly likely to be useful as along-term or permanent scaffold. PVA hydrogels havebeen utilized in tissue engineering for regenerationof artificial articular cartilage,
120hybrid-type artificial
pancreas,121and bone-like apatite formation.122
Oligopeptide sequences have been introduced onto
the surface of PVA gels to enhance cellular inter-action. For example, a Gly -His-Lys sequence re-
sponsible for hepatocyte attachment,
123and an RGDS
sequence for the adhesion of corneal epithelial cells124
have been investigated.
D. Polyphosphazene
Polyphosphazenes have been attractive in many
biomedical applications, as they are degradable inphysiological situations. The degradation kinetics canbe controlled by changes in the side-chain structurerather than the polymer backbone, unlike aliphaticpolyesters, polyanhydrides, or poly(ortho esters).
125
Polyphosphazene, an organometallic polymer, con-tains alternating phosphorus and nitrogen atomswith two side groups attached to each phosphorusatom. Poly(dichlorophophazene) has been used as anintermediate to synthesize stable polyphosphazenes(Figure 10). Poly(dichlorophosphazene) is usuallysynthesized by thermal ring-opening polymerizationof hexachlorocyclotriphosphazene,
126solution con-
densation of p-trichloro- N-(dichlorophosphoryl)mono-
phosphazene,127or living cationic polymerization of
phosphoamines to control molecular weight distribu-tion.
128A hydrophilic backbone, as well as the struc-
tural versatility as a result of various substitutionreactions, offers possibilities in designing new classes
Figure 9. Synthetic scheme of poly(ethylene oxide)-poly-
(lactic acid) (PEO -PLA) block copolymers.
Figure 10. Synthetic scheme of polyphosphazene using
poly(dichlorophosphazene) as an intermediate.Hydrogels for Tissue Engineering Chemical Reviews, 2001, Vol. 101, No. 7 1875

of polyphosphazene gels. Various modifications of
polyphosphazenes have been reported including poly-(aryl/alkyl) phosphazenes,
129poly[(amino acid-ester)
phosphazenes],130and methoxy-poly(ethylene glycol)-
substituted polyphosphazenes with temperature-responsive features.
131Two types of hydrogels, non-
ionic and ionic, can be prepared from polyphospha-zenes. Nonionic polyphosphazene gels are based onwater-soluble polyphosphazenes containing glucosylor glyceryl side groups.
132Ionic polyphosphazene
hydrogels, formed with divalent ions or60Co gamma
irradiation, have been extensively studied in con-trolled delivery of protein drug due to their abilityto respond to environmental changes such as pH orionic strength.
133,134These polymers might be useful
for skeletal tissue regeneration135or encapsulation
of hybridoma cells.136
E. Polypeptides
Proteins are a major component of the natural
matrixes of tissues, and there is wide interest insynthesizing polypeptides to mimic natural proteins.Polypeptides are usually prepared by using N-car-
boxyanhydride as a starting monomer, and a largenumber of polypeptides and copolypeptides can besynthesized from various combination of amino acids.However, it is very difficult to precisely control thesequence of amino acids as desired and thus veryexpensive. In addition, most polypeptides are in-soluble in common organic solvents.
31Recently, a
polymerization strategy to synthesize polypeptideswith well-defined amino acid sequences and a widerange of molecular weights was reported by usingorganonickel initiators that suppress chain-transferand termination side reactions.
137One striking technique to bypass the above-
mentioned problems is the synthesis of geneticallyengineered polypeptides. In brief, one may insertDNA templates of predetermined sequences into thegenome of bacteria and produce polypeptides withpredetermined structure and controlled proper-ties.
138,139This method enables one to design and
engineer various sequences of polypeptides withknown functions, including elasticity, stiffness, deg-radation, and cellular interactions. Silk-like polypep-tides have been prepared by this technique,
140and a
Gly-Ala-rich sequence has been introduced into
these artificial proteins to form reversible hydrogelsin response to environmental changes of pH ortemperature.
141Elastin-mimetic polypeptides, com-
prised of a Gly -Val-Pro-Gly-any amino acid se-
quence, have also been studied and considered tohave potential for artificial extracellular matrices intissue engineering.
142-144However, this technique is
not appropriate to economically produce biomaterialsin large scale at the current time, and one is alsounable to easily modify the polymer product as anychange requires re-engineering of the entire system.
V. Future Perspectives
We have summarized a wide range of hydrogels
that are frequently used to date, or will potentiallybe useful in tissue engineering. Hydrogels shouldmeet certain design parameters to be useful for thisarea, regardless of whether they originate fromnatural resources or are synthetically created. Hy-drogels comprised of naturally derived macro-molecules have potential advantages of biocompat-ibility, cell-controlled degradability, and intrinsiccellular interaction. However, they may exhibit batch
Figure 11. Schematic illustration of blood vessel formation promoted by including growth factors (a) or by seeding
endothelial cells (d) into the polymer scaffold. Growth factors encourage existing blood vessels in the surrounding hosttissue to grow into the scaffold (b), and the transplanted endothelial cells will form new blood vessels within the scaffoldand grow outward toward the host tissue (e). Ultimately, new vessels combine with existing blood vessels to create functionalblood vessels capable of blood flow (c,f).1876 Chemical Reviews, 2001, Vol. 101, No. 7 Lee and Mooney

variations and generally exhibit a narrow and limited
range of mechanical properties. In contrast, syntheticpolymers can be prepared with precisely controlledstructures and functions. However, many syntheticpolymers do not degrade in physiological conditions,and the use of toxic chemicals in their synthesis orprocessing may require extensive purification steps.We believe no one material will satisfy all designparameters in all applications, but a wide range ofmaterials will find uses in various tissue engineeringapplications.
A critical future challenge facing this field is how
polymers may be used to promote blood vesselnetwork formation in the tissue. This is critical toprovide nutrient transport to the engineered tissueand integrate it with the rest of the body.
145One
important approach to actively modulate the vascu-larization process is the local delivery of eitherangiogenic factors or blood vessel forming cells (en-dothelial cells) to the engineered site using hydrogels(Figure 11).
2Controlled and sustained release of
angiogenic factors from hydrogels may optimizelocalized vessel formation. Various growth factorsincluding vascular endothelial growth factor(VEGF),
146basic fibroblast growth factor (bFGF),147
epidermal growth factor (EGF),148and bone morpho-
genetic protein (BMP)149could be incorporated into
hydrogels depending on the desired tissue type.Alternatively, delivery of plasmid DNA containinggenes encoding the angiogenic proteins may be
another approach to enhance vascular network for-mation in engineered tissues.
150Co-transplantation
of endothelial cells, which comprise blood vessels,along with the primary cell type of interest may allowone to rapidly form blood vessels in an engineeredtissue. This approach is based on the observation thatendothelial cells spontaneously form capillary-likestructures in vitro if cultured in an appropriateenvironment.
151
Another critical issue in the design of hydrogels for
tissue engineering is that many tissues (e.g., bone,muscle, and blood vessels) exist in a mechanicallydynamic environment. Many current hydrogels donot possess appropriate mechanical properties forthese mechanically dynamic environments. In addi-tion, it has been previously demonstrated that me-chanical signals result in alterations of cellularstructure, metabolism, and transcription and/or trans-lation of various genes.
152-154So the gels must ap-
propriately convey the mechanical signals to theseincorporated cells. We have recently reported thatmechanical signals may be exploited to control growthfactor release from hydrogels, and this could providea novel approach to guide tissue formation in me-chanically stressed environments (Figure 12).
146
VI. Acknowledgments
The National Institute of Dental and Craniofacial
Research (NIH) supports the authors’ research in thisarea. We thank Martin Peters for assistance withillustrations.
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