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NATURE MATERIALS| VOL 8 | JUNE 2009 | www.nature.com/naturematerials 457

REVIEW ARTICLEPUBLISHED ONLINE: 21 MAY 2009 |DOI: 10.1038/NMAT2441

Ahuman embryo in its first eight weeks o lie undergoes anextraordinary transormation rom a single cell to a 3-cm-long etus with a beating heart, gut, nervous system, and

limbs with fingers and toes. Tis progression involves massive

growth, physical olds and twists, and myriad cellular and molecularevents o breathtaking complexity; yet it is the ultimate goal o tissueengineering (E) to recreate some o these processes in microcosm,to replace and regenerate lost tissue. At last the field has entered aperiod o ruition, and seems set to realize its potential to treat amultitude o debilitating and deadly conditions such as myocardialinarction, spinal injury, osteoarthritis, osteoporosis, diabetes, livercirrhosis and retinopathy. Te general strategy is usually to seed cellswithin a scaffold, a structural device that defines the geometry o thereplacement tissue and provides environmental cues that promotetissue regeneration. E skin equivalents have been in clinical usesince 1997 (re. 1) and a ast-growing arsenal o replacement devicesis in clinical trials or already approved as therapies or tissues includ-ing cartilage, bone, blood vessel and pancreas (able 1). In two

recent high-profile studies, seven patients benefited rom E blad-ders2, and a 30-year-old woman became the first person to receive aE tracheal segment, a procedure that saved her le lung3.

Aside rom the obvious human benefits, tissue engineering couldbring substantial financial rewards to those who succeed in trans-lating this new technology to the clinic. Sales o regenerative bio-materials already exceed US$240 million per annum4and the widermarkets that tissue engineering taps into are colossal: costs relatedto organ replacement account or 8% o global healthcare spending,and by 2040 as much as 25% o the US GDP is expected to be relatedto healthcare5. Nevertheless, i the short history o industrial tissueengineering has taught us anything, it is that the provision o effec-tive products is not in itsel sufficient to ensure commercial success(Fig. 1). Early E efforts were plagued by product issues related to

scale-up, shel-lie, quality control and distribution, and sufferedrom inappropriate business models and withdrawal o privatefinance in the early 2000s1,6. Since then the field has matured, evi-denced by the return o large-scale investment and the first regen-erative medicine companies becoming profitable4.

Alongside these positive developments, progress in biomaterialsdesign and engineering are converging to enable a new generation oinstructive materials to emerge as candidates or regenerative medi-cine. Which o these materials compete successully in the marketwill depend on a combination o clinical perormance, marketingand cost-effectiveness. A central dilemma is that to influence cellbehaviour, scaffolding materials must bear complex inormation,

Complexity in biomaterials for tissue engineeringElsie S. Place1,2, Nicholas D. Evans1,2and Molly M. Stevens1,2

The molecular and physical information coded within the extracellular milieu is informing the development of a new generationof biomaterials for tissue engineering. Several powerful extracellular influences have already found their way into cell-instructive scaffolds, while others remain largely unexplored. Yet for commercial success tissue engineering products must benot only efficacious but also cost-effective, introducing a potential dichotomy between the need for sophistication and ease ofproduction. This is spurring interest in recreating extracellular influences in simplified forms, from the reduction of biopolymersinto short functional domains, to the use of basic chemistries to manipulate cell fate. In the future these exciting developmentsare likely to help reconcile the clinical and commercial pressures on tissue engineering.

coded in their physical and chemical structures. On the other hand,financial considerations dictate that complexity must be kept to aminimum. Clearly there is a danger, by over-engineering devices,o making their translation to clinical use unlikely. Te solutions

to this challenge lie at every phase o product development, begin-ning with identiying the simplest unctional perormance requiredto resolve a defined clinical problem. Te ambitious early aimso reconstructing entire organs have largely given way to smaller,more attainable goals: or example, rather than trying to replace anentire heart, clinical advances in cardiac repair ocus on E coro-nary arteries, valves and myocardium. Organogenesis Inc. andAdvanced issue Sciences Inc. suffered heavily as a result o theiroverestimating the number o chronic wounds cases that were bestsolved by high-tech, E skin substitutes (respectively, Apligra andDermagra; Dermagra is now produced by Advanced Biohealing)as opposed to acellular products that aid ongoing repair 6(able 1,Fig. 1). Similarly, an emerging philosophy in tissue engineeringis that rather than attempting to recreate the complexity o living

tissues ex vivo, we should aim to develop synthetic materials thatestablish key interactions with cells in ways that unlock the bodysinnate powers o organization and sel-repair. In this review we willconsider how this can be achieved, emphasizing how even relativelysimple engineering solutions can deliver considerable unctionalbenefits. Along the way we will explore how some o these princi-ples have been applied to specific scientific and commercial tissue-engineering challenges.

Regenerative potential of tissuesEven without any therapeutic intervention, living tissues can havea staggering capacity or regeneration. For example, the humanliver will regrow to its original size even when more than 50% oits mass is excised7. Tis has been taken to the extreme in rats,

where one group has reported that a single rats liver was able toregenerate ully ollowing each o 12 sequential hepatectomies, afinding that can be explained by the high replicative potential othe cell types that make up the liver. Several other tissues boneand skin, or example also have an innate capacity to regener-ate to fill injuries below a critical size, helped by local or recruitedstem cells. Te clinical potential o stem cells has long been rec-ognized by haematologists, who in the 1960s showed that trans-planted haematopoietic (literally blood-making) stem cells romthe bone marrow o a healthy mouse could replace the destroyedimmune system o another mouse, paving the way or a cure orleukaemia8,9. Te discovery o other types o cell with multilineage

1Department of Materials; 2Institute for Biomedical Engineering, Imperial College London, London SW7 2AZ, UK. e-mail: [email protected]

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potential has since ollowed, including neural stem cells romthe brain, and mesenchymal stem cells, which can differentiateinto bone, at, cartilage and muscle cells10,11. Indeed, more recentevidence suggests that stem cells or progenitor cells can be isolatedrom almost every tissue o the body12,13. Under the correct condi-tions, these cells can be stimulated to orm new tissue, as we recentlydemonstrated using a simple biomaterials-based approach. Here,

either calcium-crosslinked alginate gels or modified hyaluronicacid gels were injected into an artificial space between the tibiaand the periosteum, the fibrous outer lining o bone. Tis stimu-lated bone and cartilage ormation rom resident progenitor cellsin the inner layer o the periosteum14, illustrating that complextissues can be generated rom relatively simple materials by usingthe body as a bioreactor.

Table 1 | Commercial tissue engineering products and biomaterials at various stages of development.

Tissue Product Regulatory

status

Description Material Cells Use Form

Synthetic

R

esorbable

A

nimalderived

P

lantorbacteria

d

erived

H

umanderived

G

rowthfactor

A

llogenic

A

utologous

Skin TransCyte, Advanced

Biohealing

1997 Nylon mesh coated with porcine

collagen, containing non-viable human

fibroblasts, with upper layer of silicon

Burns Sheet

Apligraf, Organogenesis 1998 Lower layer of human fibroblasts

and bovine collagen, upper layer of

keratinocytes

Leg ulcers Sheet

Dermagraft, Advanced

BioHealing

2001 Cryopreserved human fibroblasts on a

polyglactin 910 (2-hydroxy-propanoic

acid polymer with polymerized

hydroxyacetic acid) mesh

Diabetic foot

ulcers

Sheet

ICX-SKN, Intercytex Phase II Allogenic fibroblasts and human

collagen with additional layer of

keratinocytes

Burns and

acute wounds

Sheet

Integra Dermal

Regeneration Template,

Integra Lifesciences

1996 Porous bovine collagen crosslinked

with chondroitin-6-sulphate with

upper layer of silicon

Burns Sheet

Integra Flowable Wound

Matrix, Integra Lifesciences

2007 Granulated bovine

collagen crosslinked with

chondroitin-6-sulphate

Ulcers Gel

Oasis Wound Matrix,

Healthpoint

2006a Decellularized porcine small intestinal

submucosa

Burns, ulcers,

other wounds

Sheet

PriMatrix, TEI Biosciences 2008 Decellularized fetal bovine skin Wounds Sheet

Xelma, Molnlycke 2005 EU ECM protein (amelogenins) in

propylene glycol alginate carrier

Leg ulcers Gel

Bone INFUSE Bone Graft,

Medtronic

2002 Bovine type I collagen sponges soaked

in rhBMP-2 in LT-CAGE Lumbar

Tapered Fusion Device

Spinal fusion Solid

OP-1, Stryker 2001 Bovine type I collagen with rhBMP-7 Bone injury Paste

PuraMatrix, 3DM Preclinic Synthetic 16-amino-acid peptide,

forming nanofibres

Dental bone

defects

Gel

Vitoss Scaffold FOAM,

Orthovita

2004 Porous foam comprising -TCP and

bovine type I collagen

Bone injury Foam

Bioset IC, Pioneer surgical 2008 Human demineralized bone matrix

with bovine bone chips in type I

collagen carrier

Bone injury Paste

FortrOss, Pioneer Surgical 2008 Nanocrystalline hydroxyapatite

and E-matrix (porcine collagen

co-polymerized with dextran)

Bone injury Paste

Regenafil, Regeneration

Technologies/Exatech

2005 Human mineralized bone matrix in

porcine gelatin carrier

Bone injury Paste

GEM 21S, BioMimetic

Therapeutics

2005 -TCP particles and recombinant

human platelet-derived growth

factor-BB (PDGF-BB)

Dental bone/

gum defects

Paste

BCT001, Bioceramic

Therapeutics

Preclinic Strontium releasing bioactive glasses Bone defects Granules,

paste

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REVIEW ARTICLENATURE MATERIALS DOI: 10.1038/NMAT2441

Such simple strategies unortunately do not offer a universalregenerative solution. Healing may be restricted by an age-relateddecline in progenitor populations, by the intrinsically low regenera-tive potential o certain tissues, or by scarring or inflammation, such

as ollows myocardial inarction in the heart or stroke in the brain.In these cases, or where the original tissue has been completelydestroyed, biomaterials interventions that include an external sourceo cells may be required. Over 700 adult stem-cell therapies are

Table 1 (continued) | Commercial tissue engineering products and biomaterials at various stages of development.

Tissue Product Regulatory

status

Description Material Cells Use Form

Synthetic

Resorbable

A

nimalderived

Plantorbacteria

derived

H

umanderived

G

rowthfactor

A

llogenic

A

utologous

Cartilage Synvisc, Genzyme 1997 Hyaluronic acid (Hylan GF-20 and

Hylan B) from chicken combs

Synovial fluid

replacement

Gel

CaReS, Arthro Kinetics 2007

Germany

Rat-tail type I collagen matrix seeded

with chondrocytes

Articular

cartilage injury

3D disc

Bioseed-C, Biotissue

Technologies

Clinic 2001b Polyglycolic/polylactic acid and

polydioxane scaffold containing

chondrocytes

Articular

cartilage injury

3D disc

Menaflex, Regenbiologics 2008 Bovine type I collagen with hyaluronic

acid and glycosaminglycans, hydrated

Meniscus

cartilage injury

Mesh

Hyalograft C autograft, Fidia

advanced biopolymers

2008 EUc HYAFF (esterified derivative of

hyaluronic acid) scaffold with

autologous chondrocytes

Articular

cartilage injury

3D disc

MACI, Genzyme Unreg EUd

Type I collagen membrane seededwith chondrocytes Articularcartilage injury Sheet

T/L GraftJacket, Wright

Medical Technology

Unrege Decellularized human skin Tendon and

ligament repair

Sheet

X-Repair, Synthasome FDA reviewf Poly-L-lactic acid woven mesh Tendon and

ligament repair

Sheet

BV VascuGel, Pervasis Phase II Gelfoam porcine gelatin foam sponges

seeded with endothelial cells

Vessel

reconstruction

Tubular

Vascu-guard, Synovis 1994 Decellularized bovine pericardium Vessel

reconstruction

Sheet

HV Cryovalve SG pulmonary

human heart valve, Cryolife

2008 Decellularized human heart valves Heart valve

replacement

Valve

Retina NT-501, Neurotech Phase II/III Genetically modified cells secreting

ciliary neurotrophic factor in hollow

permeable polyethersulphone fibres

Retinitis

Pigmentosa

Tubular

Nerve NeuraGen, Integra 2001 Semipermeable bovine type I collagen

nerve conduits

Nerve injury Tubular

Pancreas Islet Sheet, Cerco Medical Preclinic 250-m-thick alginate sheet with

encapsulated pancreatic islets (pig

or human)

Diabetes

mellitus

Sheet

Amcyte/ReNeuron Phase II Alginate/poly--lysine encapsulated

human pancreatic islets

Diabetes

mellitus

Bead

Novocell Phase I/II Alginate/polyethylene glycol

encapsulated human pancreatic islets

Diabetes

mellitus

Bead

Bladder Neo-bladder, Tengion Phase II Poly(lactic-co-glycolic acid)

seeded with urothelial and smooth

muscle cells

Spina bifida,

bladder

dysfunction

Sheet

Dura DurADAPT, PegasusBiologicals

2005 Decellularized, crosslinked equinepericardium

Reconstructionof dura mater

Sheet

Various Extracel, Glycosan

Biosystems

Preclinic Crosslinkable bacterial hyaluronic

acid, bovine and porcine gelatin,

porcine heparin and PEG derivatives

g g g Examples:

cartilage, bone,

vocal fold TE

Gel,

sheet,

tubular

Tissue abbreviations: T/L, tendon and ligament; BV, blood vessel; HV, heart valve. Regulatory status: dates indicate year approved by regulatory body (FDA unless stated as European Union (EU) or Germany).

Preclinic indicates preclinical development, phase I and phase II indicate clinical trial stage. Date of regulatory approval does not necessarily coincide with market release. aReclassified in 2006 from an earlier

product. bFirst clinical trials were in 2001. cCommercial sale began in 2000. dUnregulated in EU, not available in the United States. eUnregulated product. fDecision expected 2009. gAddition of growth factors

and cells is dependent on application. A list of references is available online as Supplementary Information.



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currently in clinical studies in the US, covering a host o therapeuticapplications including ollowing myocardial inarction15. Celltherapy alone, however, may have as yet undetermined, unwantedand poorly controlled consequences. A recent study in mice hasrevealed that stem cells injected into heart muscle post-inarctionwent on to mineralize, possibly because the stiffer mechanical envi-ronment o the scar tissue was not conducive to myogenic differen-tiation16,17. Control o cell ate is perhaps the most limiting actor inthe translation o embryonic stem-cell therapy. When implanted in

an immunocompromised mouse they will orm teratomas benigntumours made up o a variety o adult tissues, or example teeth, hairand sections o gut epithelium18. On the other hand, a bank o only150 embryonic stem-cell lines could provide a good tissue matchin more than 80% o the UK population19and it is now possible tomake cells resembling embryonic stem cells (induced pluripotentcells) by artificially introducing up to our genes into adult cells2023.Te latest report achieved reprogramming in human cells with nopermanent genetic modification24, making translation to clinicaluse a tantalizing goal as tissue-specific, transplantable cells couldbe host-derived.

Another important challenge is that o how to replace wholetissues, which are made o many cell types whose organization iscrucial to unction. Cells, o course, have natural powers o sel-

organization, and under the correct conditions will spontaneouslyorm complex structures, such as the sprouting tubular networksormed by the endothelial cells that line blood vessels. ransmissionand receipt o complex molecular inormation involved in cellsorting, boundary ormation in tissues and cell movement can beeffected through direct cellcell interaction, largely through cadher-insa amily o transmembrane glycoproteins that regulate cellcelladhesion25. When two cell types expressing two different cadherinmolecules are mixed in suspension they will spontaneously sortthemselves on the basis o their cadherin expression26, an outcomeundamental or tissue development and healing. Many embryo-logical processes rely on cadherin communication. For instance, theormation o the central nervous system requires the neural tubeto bud off rom epithelial cells, a process that depends on a change

in the expression o cadherins rom E-cadherin to N-cadherin27

.Simpler, artificial cell adhesions have been engineered in a schemeinvolving periodate oxidation o cell suraces ollowed by biotinconjugation. Te subsequent addition o avidin triggered the assem-bly o multicellular aggregates through biotinavidin interaction28,which was intended to assist the development o more complexcellular interactions.

Extracellular matrix scaffoldsAs well as requiring inormation rom each other, cells derive a vastwealth o inormation rom their environments, including the mate-rial that surrounds and separates them within tissues, the extra-cellular matrix (ECM). A E material scaffold must take on thisinstructive role to some degree in order to maintain cell viability and

control cell behaviour. Clues or how to construct bioactive artifi-cial scaffolds come rom naturally bioactive scaffolds. For example,implantation o demineralized bone matrix (DBM, bone rom whichmineral and cells have been removed, leaving only proteinaceousmaterial) in muscle induces the ormation o bone in the surround-ing muscle tissue29. Tis remarkable observation led to the isola-tion o bone morphogenic protein (BMP) rom DBM, and severalcompanies currently produce DBM commercially rom cadavers orimplantation in bone deects30(able 1). As with DBM, many othercadaver- or animal-derived decellularized ECM products have aninherent bioactivity sufficient to induce regeneration and have oundclinical use: or instance, products derived rom the small intestinalsubmucosa o pigs (an example being Oasis Wound Matrix) are usedroutinely in reconstructive surgery, and ECM derived rom the peri-cardium o horses can be used as a reconstructive material in the dura

2009 President Obama lifts ban on federal funding of embryonic

stem-cell research

2008 Implantation of tracheal segment engineered from

decellularized tissue

2007 Creation of induced pluripotent stem cells from adult

human skin cells

Osiris named Biotech Company of the year

~170 companies offering TE products or services, sales

in excess of US$1.3 billion; >1 million patients treated;

aggregate economic activity fivefold higher than in 2002

Organogenesis breaking even, reinvesting profits;

Apligraf sales of US$60 milllion per year

2006 TE bladder appears in the Lancet

Launch of Proteus Venture Capital Fund, the first

dedicated regenerative medicine fund

2005 Carticel becomes profitable

2003 Organogenesis emerges from bankruptcy

2002 Integra Dermal Regeneration Template by Integra Life

Sciences approved for treatment of severe burns

FDA approval of Medtronics INFUSE Bone Graft

Organogenesis and ATS, both previously valued at

US$1 billion, file for bankruptcy

TE activity halved since 2000, loss of 800 full-

time employees, capital value of publicly-traded

TE corporations drops from US$2.5 billion to

US$300 million

Circe bioartificial liver completes Phase III clinical trial

with statistically significant benefit for a subset of

patients; FDA approval not granted as patient group

was not identified in advance

42% increase in stem-cell firms, coining of term

Regenerative medicine

2001 President Bush restricts federal funding for embryonic

stem-cell research

2000 Time magazine names Tissue Engineer as Hottest Job

for the future, 3,000 people pursue TE careers

US$580 million spent annually on TE R&D, public

TE companies valued at US$2.5 billion

1998 FDA approves Apligraf, first allogenic TE product

Human embryonic stem cells isolated

1997 TransCyte becomes first FDA-approved TE product

Carticel autologous cartilage implant approved by FDA

1996 The Tissue Engineering Society founded (now Tissue

Engineering Regenerative Medicine International

Society, TERMIS)

1990 US$3.5 billion invested worldwide in TE, 90% from

private finance

Late 1980s Early TE work in Massachusetts, term TE appearsin literature

Early 1970s Cells combined with biomaterials in research into artificial

skin and biohybrid pancreas

1968 First bone-marrow transplant

1950s Organ transplantation with identical twins

Figure 1 | Tissue engineering timeline. Tissue engineering gained

in profile through the nineties, hitting a peak around the turn of the

millennium, but several early commercial ventures failed and large-scale

private financing was withdrawn. Improved business planning and a

sounder scientific base have since propelled it towards a new

era of success14,22,23.



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mater layer o the brain meninges ollowing a craniotomy (able 1).In a urther development, last years transplanted E airway con-firmed this approach as being at the oreront o whole-organ tissueengineering3,31. Te scaffold in this case was a decellularized humandonor trachea that was repopulated with the patients own cellsexpanded rom biopsy. In contrast with traditional transplant surgery,the decellularization protocol solved the problem o tissue rejectionby removing virtually all traces o human leukocyte antigens (theproteins that to a large extent determine tissue compatibility), with

the consequence that the patient required no immunosuppressivedrugs. As well as immediately restoring airway patency, the deviceacilitated the rapid development o an internal cellular lining andblood vessel network. Although we ocus here on scaffolds designedand assembled in bottom-up mode in the laboratory, it is apparentthat both lab-built scaffolds and decellularized tissues offer distinctand important benefits or tissue engineering, and equally, that nei-ther approach represents a universal biomaterials solution.

Substituting physical aspects of the extracellular matrixypically, biomaterials-engineering approaches ocus on a ewmechanisms (chemical or physical) by which ECM influences cells,and attempt to present these influences effectively or a given tissue.Regardless o the chemistry that we apply within scaffolds, the con-

struct must usually also provide some level o physical support romthe moment o implantation, to assist tissue unction while newmatrix is being deposited3235. For example, the extreme sonesso the lamina propria o the human vocal old (elastic modulus

E= 1001,000 Pa) is essential or proper phonation, and its unctionis easily impaired by scarring. Tis has prompted the development oso (E 500 Pa), highly elastic gels o double-crosslinked hyaluronicacid microparticles or vocal-old tissue engineering36. Te par-ticles are synthesized by crosslinking with divinyl sulphone, thensurace-oxidized and lightly crosslinked together using hyaluronicacid modified with adipic dihydrazide. Te gels have controllableviscoelasticity, and a reduction in dynamic viscosity with requencyoccurs at a similar rate to that o human vocal-old mucosa.

In many cases, physical demands on scaffolding materials arecomplicated by the anisotropy inherent in most living tissues (con-sider the parallel arrangement o collagen fibres within tendonsand the concentrically layered sheets o the intervertebral disk).Nevertheless, engineering solutions need not be costly or comp-licated: substituting a rotating or a static collector yields orien-tated electrospun fibres37, and crosslinking hydrogels under straincan result in highly biomimetic anisotropic mechanical properties.For instance, thermal cycling o poly(vinyl alcohol) leads to thegrowth o crystallites that unction as physical crosslinks, leadingto gelation. Early in the crosslinking process, these crystallites canbe aligned by applying strain, the degree o which dictates the levelo anisotropy. Termal cycling is recommenced, with the numbero cycles determining the overall amount o crosslinking and thus

stiffness. By optimizing these two parameters, hydrogels with aniso-tropic stiffnesses closely resembling those o porcine aorta havebeen developed38. issues are also heterogeneously organized intomechanically distinct zones, or example the superficial, transitional,

Achieving a strong bond between mechanically dissimilar materialsis as much a challenge in tissue engineering as in other branches oengineering. Te morphological specialities and mechanical gra-dients seen at interaces between musculoskeletal tissues in vivoreduce impedance mismatch and minimize stress concentrationsas loads are redistributed, yet even with natures elegant solutions,

many chronic musculoskeletal injuries occur at tissue boundaries.Unsurprisingly, rupture at insertion sites is also the most commoncause o ailure o ligament and tendon gras131. Although aware-ness o this problem is growing, most orthopaedic E devicesdo not eature distinct transition zones to improve load transerbetween tissues. Tis includes most osteochondral plugs bilami-nar bone and cartilage E constructs that have been developed toimprove the assimilation o cartilage into joints. Here, the accu-mulation o matrix can effect good integration between the twophases132,133, but ew examples contain regions o calcified cartilagereminiscent o the tidemark seen adjacent to subchondral bonein vivo. An interesting exception is an osteochondral gra consist-ing o a bone component o hydroxyapatite populated with BMP-7transduced fibroblasts (connective tissue cells), and a poly(lactic

acid) sponge seeded with chondrocytes (cartilage cells). Pocketso mineralized cartilage were seen at the boundary between thetwo layers o this scaffold134. Conversely, trilaminar scaffolds bydesign possess a middle layer with a distinct composition135and/or seeded with different cell types. Any combination o cells can bestraightorwardly zoned within hydrogels at the point o abrica-tion by the layer-by-layer partial photo-polymerization o cell andmacromolecular precursor suspensions136. Constructs resemblingligament insertion sites, wherein bone and ligament are united bymeans o fibrocartilage (Fig. B1), have been produced by seedingfibroblasts, chondrocytes and osteoblasts (bone cells) separatelyinto the three layers o a preormed scaffold137. Another strategyuses just one cell type, namely fibroblasts, to produce scaffoldswith an internal gradient o mineralization. Retrovirus encoding

the bone-specific transcription actor Runx2 was immobilized ona layer o poly(-lysine). Te thickness o the poly(-lysine) layercould be graduated by dip-coating collagen scaffolds, leading inturn to a tapering o retrovirus concentration, osteoblastic geneexpression, mineralization and stiffness138. Although the ligamentcomponents in these examples were not optimized or immediatetensile load bearing, E ligaments with high tensile toughness (suchas braided polymers139) have been developed34. It will be interestingto observe how, in the uture, these two strands o ligament tissueengineering will be united.

Box 1 | Challenges in interface engineering for orthopaedics.

F

BV

Fb

Cc

Ob

a

CF

B

L

T

b

T

L

F

CF

B

Figure B1 | Histology of interface between bone and ligament.

a, Schematic; b, photomicrograph of tibial insertion of rabbit anterior

cruciate ligament. Adapted with permission from ref. 144. 1996 Wiley.

Ligament (L) insertions occur by means of fibrocartilage, which is divided

at the tidemark (T, black line in b) into non-calcified (F) and calcified (CF)

regions. The calcified fibrocartilage interdigitates with the underlyingsubchondral bone (B). Fb, fibroblast; Cc, chondrocyte; Ob, osteoblast;

BV, blood vessel.



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radial and tight zones o cartilage, some implications o which arediscussed in Box 1.

Beyond their structural and biomechanical roles, physical prop-erties also influence many aspects o cell behaviour. In one recentstudy, scaffold geometry was used to align cardiac muscle cells,to elicit directional contractions that are essential or efficientblood transer39. Te crosslinking o microstructured honeycombpoly(glycerol sebacate) sheets was optimized to mimic the aniso-tropic stiffness o rat ventricular myocardium. Tese sheets pos-

sessed microscale pores in the orm o two overlapping squarestilted at 45, which directed the alignment o neonatal rat heartcells. Te resulting constructs displayed anisotropic electrical exci-tation thresholds as a result o this long-range order. It is now wellknown that scaffolds that provide a particular physical environmentcan be cell-instructive as well as (potentially) contributing towardsthe physical unction o the tissue. Te stiffness o ECM alone canhave effects at a transcriptional level, determining whether stemcells make the decision to turn into cells as unctionally diverseas nerve and bone tissue40,41, and the importance o other physicaleatures such as topography and three-dimensionality o the matrixhas been reviewed or demonstrated elsewhere4244. Tese physicalactors continue to be investigated intensively and provide a com-plementary approach to the provision o molecular inormation to

cells inside scaffolds.

Engineering bioactive scaffoldsissue-engineering scaffolds can be designed to interact with cellsby emulating key molecular eatures o the ECM. ECM containsmany macromolecules such as proteoglycans, collagens, laminins,fibronectin and sequestered growth actors, and it is primarily thismolecular inormation that coners its bioactivity. For example,the sequences o many ECM proteins are recognized by dimericcell-surace receptors known as integrins. Binding o integrins toECM molecules can trigger a cascade o signalling events lead-ing ultimately to gene expression. Te gallery o ECM proteinspresented to cells in a given tissue is likely to be critical in deter-mining how cells behave within that tissue.As integrins are dim-

ers o alpha and beta subunits, they can associate in a variety ocombinations, and thus bind to a diverse range o ligands. Teindispensable nature o integrins and the ECM is demonstrated bygenetic knock-outs: the absence o the integrin 1subunit

45or ofibronectin46is lethal at the early embryonic stage. One way to pro-vide sites or integrin attachment in scaffolds is to include purifiedECM proteins such as collagen or fibrin. able 1 demonstrates thesuccess o this strategy, particularly regarding the use o collagen(and gelatin, a low-cost denaturation product o collagen), whichis present in commercial products or most o those tissues whereE replacements are available. Another widely incorporated ECMmolecule is hyaluronic acid, a polysaccharide that is deposited atsites o wound repair and also during morphogenesispreciselythe processes that we wish in some way to emulate. Hyaluronic

acid is used therapeutically as a putative lubricating actor inarthritic joints (or example Synvisc; able 1), although hereand in tissue engineering it may also interact directly with cellsthrough CD44 and other cell-surace receptors47. Te ubiquity othese ECM molecules has prompted the development o Extracel(able 1)48, a modular ECM system based on derivatives o gela-tin and hyaluronic acid. Te components are combined in varyingproportions to suit many different cell-culture and in vivoappli-cations, crosslinked with poly(ethylene glycol) (PEG) diacrylateand supplemented with additives such as heparin-bound growthactors as needed. For example, whereas equal quantities (weightor weight) o gelatin and hyaluronic acid are suitable or most tis-sue applications, a specialized composition enriched in hyaluronicacid (95:5 w/w hyaluronic acid/gelatin) is required or vocal-oldrepair. Tis adaptability is especially valuable or niche markets,

where the costs o development and manuacturing can be highrelative to volume o sales.

Matrix constituents have disadvantages associated with purifica-tion and processing, and coupled with the desire or greater controlover material properties, this has led to the investigation o ullysynthetic bioactive systems. Te ability to unctionalize bioinert sub-stances could improve the suitability or tissue engineering o a host omaterials with remarkable properties, such as high-strength syntheticpolymers and (nano)composites or bone tissue engineering49,50. O

particular interest are systems amenable to minimally invasive deliv-ery, including injectable or shape-memory materials that gel or regaintheir original orm in response to stimuli such as ultraviolet illumi-nation or physiological conditions (temperature, pH or solvent)5154.Materials that do not adsorb protein, such as PEG gels, can effectivelybe used as a blank template on which to coner bioactivity with theminimum amount o modification. In some cases specific unctionso biopolymers can be attributed to small unctional domains whichmay be included in place o the ull protein55,56. Te best known othese is the integrin-binding arginineglycineaspartic acid (RGD)sequence ound in many ECM proteins, including fibronectin,laminin, collagen type IV, tenascin and thrombospondin5759. RGDmodification alone is sufficient to transorm alginate rom a relativelyinert polysaccharide into a substance that supports the ormation o

convincing growth-plate-like structures when mouse osteoblasts andchondrocytes are co-cultured within it60. Adhesive and inormationalpeptides ound in ECM are reviewed elsewhere61,62.

In addition to providing cell-directing elements, ECM is itselhighly responsive to the actions o cells. It is requently remodelledby cells during development, homeostasis and healing, a processthat involves digestion by a variety o proteases (such as cathep-sins and matrix metalloproteases) ollowed by deposition o reshmatrix. Many scaffolds undergo a hydrolysis route to degradationwhich can lead to sudden loss o mechanical strength and struc-ture. Cell-mediated scaffold degradation is more likely to generate amaterial temporal profile in tune with the generation o new tissue.In this approach, pioneered by the Hubbell group, the materials arecrosslinked by enzyme-degradable peptide sequences, and a combi-

nation o cell-mediated degradation and integrin binding allows thecells to migrate through the gel in a process reminiscent o tissueremodelling63. Cleavage sequences can also be incorporated intomultidomain peptides. One example is a recombinant, crosslink-able elastin-like protein which harbours an adhesion moti (REDV)and an elastase-sensitive sequence. Cleavage o the latter yields abioactive Val-Gly-Val-Ala-Pro-Gly (VGVAPG) ragment intendedto stimulate cell prolieration and improve tissue repair64. Tis unc-tionality mimics the multilayered bioactivity o the ECM, wherebyenzymatic remodelling can liberate cryptic sites contained withinthe amino acid sequences o ECM proteins. In act it is becomingincreasingly clear that ragments o many ECM proteins possessbioactivity with effects ranging rom cell migration to differentia-tion, prolieration and angiogenesis which only becomes

recognizable to the cells when the ECM is modified in some way65

.Physical manipulation by cells can exert effects on the ECM: theinteraction o integrins with fibronectin induce conormationalchanges o this large molecule, exposing cryptic sites which allowit to sel-assemble66,67. In another example, myofibroblasts, cellsimplicated in wound healing, can release sequestered transorm-ing growth actor beta (GF-) rom its latent binding protein bypulling on the ECM, a process thought to be integrin-dependent 68.Once relinquished, GF- is a powerul regulator o cell unction,even to the extent o determining whether a cell lives or dies.

Soluble signalling moleculesGrowth actors, such as GF- and BMPs, are important signallingmolecules in both tissue healing and development. Some o theseproteins act as morphogens, determining the spatial arrangement



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o cell types within the developing embryo. Tey may also haveproound effects in the control o tissue regeneration. For example,injured muscle tissue secretes several Wnt proteins, stimulating aresident population o cellsto divide and differentiate to orm newmuscle tissue69, and as we have already seen, BMPs can induce theormation o bone ectopically in muscle tissue29. Tese observa-tions have ound resonance in stem-cell research with the result thatmany growth actors are important components in the differentia-tion regimes or both adult and embryonic stem cells 7072. In tissue

engineering, the application o growth actors within biomaterialsalso represents a powerul tool or controlling cell differentiationand unction. For instance, when murine muscle lacerations weretreated by transplantation o muscle precursor cells within RGD-coupled alginate gels, recovery was greatly improved by the additiono hepatocyte growth actor (HGF) and fibroblast growth actor-2(FGF-2)73. Already, growth actors eature in a handul o commer-cially available E products (able 1), one o which MedtronicsINFUSE represents the fields biggest financial success yet4.INFUSE is supplied with powdered recombinant human BMP-2,which is reconstituted in water and added to a collagen spongeimmediately beore use.

Controlled-release strategies are requently adopted to overcomethe short hal-lie and residence o ree growth actors in solution.

For example, microspheres abricated by double emulsion canrelease protein payloads rom aqueous pockets within the particles,and can now be made to nanoscale dimensions using a single sur-actant74. Furthermore, in developmental pathways, different actorsbecome active at different times, and growth actor release profilesthat recapitulate these dynamics are likely to provide more lever-age over cell behaviour than those that apply growth actors indis-criminately. Materials schemes based on different degradation ratesor diffusive properties o polymers have been designed with this inmind75,76(Fig. 2).

Although most efforts so ar have concentrated on evaluatingthe effects o reely diffusible orms o growth actors in solution,most in act unction at interaces in vivo, bound to ECM com-ponents or as part o membrane complexes 77. Although concern

undoubtedly arises over the cellular accessibility and activity osurace-immobilized proteins, even relatively simple tethering o agrowth actor to a biomaterial matrix can elicit desired biologicalresponses78,79. For example GF-1 covalently tethered to PEG notonly retained its ability to stimulate matrix production in vascu-lar smooth muscle cells, but also did so significantly more thana comparable concentration o the soluble orm o the protein 80.Fixinggrowth actors covalently in place carries the added benefito preventing internalization o growth-actorreceptor complexesby cells. More precise, site-specific couplings can be engineeredthrough the use o recombinant proteins into which additionalamino acids are introduced at the termini, or example Cys-tagsor enzyme substrate sequences that lead to proteolytic release81,82.Systems or controlling the kinetics o growth actor release and

presentation have shown potential or aiding blood vessel growthinto scaffolds (Box 2, Fig. 3).More natural mechanisms o growth actor binding and release

are also being pursued. In vivo, glycosaminoglycans (GAGs), mostlyas components o proteoglycans, have key parts in growth actoractivity, including sequestering them within the matrix, prevent-ing their degradation and presenting them to cell-surace recep-tors. GAGs are complex molecules with tissue-specific distributionand multiple physiological unctions, but they share characteristiclinear structures o repeating hexosamine-uronic acid disaccha-ride units83. Heparin, and heparan-, chondroitin-, keratin- anddermatan-sulphate GAGs (HS, CS, KS, DS, respectively) also havetightly regulated regiospecific sulphation patterns, which determinetheir specific interactions with proteins84,85. Tese interactions canbe essential or the physiological effects o growth actors. FGF-1,

or example, requires HS binding or dimerization and receptoractivation86. Heparin has been widely incorporated into E scaffoldsto offer a slow release mechanism87,88(Fig. 2), and CS in commer-cial products (able 1) may perorm a similar unction, includingmodulating the activity o cell-derived signalling actors.

Simplifying biomolecules for use in biomaterialsFew approved products include recombinant growth actors, butthe enormous success o INFUSE shows the potential commercialviability o these material/growth actor combinations (able 1): itattracted nearly US$700 million o sales in 2007 (re. 4), an order omagnitude more than any o its competitor products. Furthermore,the sophisticated use o growth actors is likely to be important in

advanced E applications. For example, the patterning o growthactors within preabricated scaffolds could aid the generation oheterogeneous tissues89. As already discussed or integrin-bindingand protease-digestible proteins, growth-actor-mimicking thera-peutics where some o the growth actor unction is condensedinto relatively short peptide ragments, typically o 3040 aminoacids90,91, hold promise. Some o these peptides bind their respec-tive receptors with comparable affinities to recombinant growth ac-tors, and can trigger signal transduction and lead to appropriate cellresponses. Although the concentrations required to elicit biologicaleffects are variable, and in some cases exceed those o the nativeproteins by orders o magnitude, angiogenesis has been inducedby one FGF-2 mimetic peptide at similar concentrations to recom-binant FGF-2 (re. 91). Tis molecule contains two 15-amino-acidreceptor-binding domains and a 9-amino-acid heparin-binding

Rele

aseof

>1growthfactor

Mode

sofspa

tia

lpre

sen

ta

tion

Scaffoldsurface

Differentrates

of diffusion

Different ratesof polymerbreakdown

Loaded polymerand microspheres

Loaded polymercoatings

Rele

asestrateg

ies

Protease Cleavablepeptide

Cell-demandedrelease

GAGsequestered

Enhancedbinding

Free in solution

Tethered:random

orientation

Tethered:specific

orientation

Use of spacersuch as PEG

Figure 2 | Presentation and release of growth factors from TE scaffolds.

Anticlockwise, from top: growth factors within TE scaffolds may be loaded

into polymers whose rate of degradation or diffusive properties can be

modulated to tailor release rate, and which may be combined into systems

releasing multiple factors with distinct kinetics75,76. The exposure of cells

to different growth factors with time may therefore imitate developmental

pathways and healing responses. An alternative to presenting growth factors

in soluble form is to bind them to a surface in either random or specific

orientations, with the possible use of a spacer molecule7880. Non-covalent

associations with matrix components, particularly glycosaminoglycans

(GAGs), can effect slow release and in some cases may potentiate binding

to membrane receptors87,88. Cell-demanded release is based on the presence

of protease-sensitive peptide sequences within the growth factor protein81,82.



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sequence. Furthermore, such mimetics can have demonstrableeffects at the whole-organism level: a 15-amino-acid peptide, basedon the neural cell adhesion molecule (NCAM) binding site or FGF-receptor-1, imparted to rats a long-lasting improvement in memoryupon intracerebroventricular administration92. A urther advantageto these mimetics is their relatively high stability relative to nativegrowth actors, such as BMP-2, which are necessarily used in supra-physiological doses93.

Even active compounds bearing no relation to the primarystructures o growth actors can be identified rom peptide 94 orsmall-molecule95 libraries. As many cytokines and growth actorsand their receptors are arranged in dimers, bi- or oligovalency canincrease the activity o these compounds. Te covalent dimeriza-tion o a 20-amino-acid erythropoietin (EPO) mimetic peptide

increased its affinity or the EPO receptor 100-old94

. Mice erythro-poiesis assays, which measure the incorporation o radioactive59Fe into the blood, revealed a similar increase in in vivopotencyo this peptide, although its activity still remained orders o mag-nitude short o native EPO. More widely, an expanding palette osmall molecules and ions such as retinoic acid, dexamethasoneand thyroid hormones are known to influence differentiation95,96.Bioactive glasses such as PerioGlas can be made to release variousions including calcium and silicon, which can effect upregulationo genetic pathways relevant to bone differentiation97,98. Te bioac-tive glass BC001 additionally releases strontium to help combatosteoporosis (able 1).

Te ability to bind growth actors and thus modulate cellularunctions can be recreated in synthetic GAG mimetics84,85. Synthetic,sulphated di- and tetra-saccharides in side-chain positions along

a polymer backbone can successully compete with neural CS orgrowth actor binding, despite non-native molecular architectures84.One o these glycopolymers, a polymerized CS tetrasaccharide, hadsimilar potency to neural CS. Interestingly, non-specific chemicalsulphation o hyaluronic acid, a GAG occurring naturally in a non-sulphated orm, induces less extensive structural rearrangements oadsorbed and covalently bound fibronectin, which translates intoa higher level o cell attachment99. Alginate can also be chemicallysulphated to yield a substance with binding affinities to growthactors (or example VEGF, PDGF-BB and HGF) comparable to orhigher than heparin and with the ability to enhance FGF-inducedblood-vessel ormation100. A sulphated and carboxylated dextranderivative potentiated VEGF binding to its receptors, resulting inangiogenic effects101. Even incorporating sulphonated monomers

(sulphopropyl acrylate potassium) into poly(acrylamide) gelsincreases the uptake o serum proteins102. Tese materials carry theadvantages o scalable chemical synthesis and more closely definedmaterial properties, and are gaining interest as replacements orheparin and CS as modulators o growth actor release and activity.

In addition to binding a broad spectrum o proteins, small oligo-saccharide domains present within larger GAG sequences canalso regulate cellular unction through their involvement in spe-cific structural interactions with their binding partners85,86,103,104.etrasaccharides, or example, represent the minimal CS epitopenecessary to stimulate neuronal growth105, and the anticoagulantactivity o heparin has been localized to a pentasaccharide moti thatinteracts selectively with antithrombin106. Tus, in the same way thatshort peptide sequences have been used to isolate specific proteinunctions, oligosaccharide ragments can emulate the unction o

Creating a unctional vasculature represents one o the mostundamental challenges in tissue engineering, and most notablesuccesses so ar have been in thin or avascular structures suchas skin, bladder and cartilage. Surgical approaches wherebyimplants are sited alongside a rich external blood supply2 arelikely to complement materials strategies that attempt to induce

or organize vessel ormation, either de novo (vasculogenesis)or by sprouting o existing vessels (angiogenesis). Endothelialcells have an inherent ability to orm tubular structures, but itis essential that these are stabilized i regression is not to occur.Permanent vasculature possesses smooth muscle cells and peri-cytes as well as an endothelial component, and several studieshave shown the potential o co-culture with various cell typesto improve the longevity o vascular networks127,140,141. Pericytesand endothelial cells in co-culture produce tissue inhibitor ometalloproteinase (IMP) -3 and -2, respectively, which stabi-lizes vessels by arresting the matrix breakdown that is associatedinitially with vascular invasion and lumen ormation, but alsoultimately with regression127. Several materials-based approacheshave used vascular endothelial growth actor (VEGF), a potent

angiogenic actor involved in the early stages o blood vessel or-mation. VEGF has a narrow therapeutic concentration range,above which capillary ormation is prolific but aberrant: theresulting structures are malormed, leaky and unstable, regress-ing quickly on withdrawal o VEGF124. On top o this, VEGF hasa hal-lie o under 90 minutes in the circulation142, hence theneed or it to be delivered through biomaterials that can releaseit in low concentrations on a timescale o weeks. An interestingscheme devised to this end involved the production o a recom-binant VEGF variant, which was enzymatically incorporatedinto fibrin matrices, and released upon matrix breakdown bycell-produced enzymes. Te release rate was accelerated by the

selection o VEGF isoorms with a plasmin-cleavable sequencenear the conjugation site. Interestingly, durable vessel ormationand in vivovascularization was higher with this VEGF molecularvariant than or native VEGF in a mouse model despite a lowerupregulation o VEGF receptor 2 (the receptor through whichVEGF exerts its effects on endothelial cells)143.

Other groups have taken a combinatorial approach to growthactor delivery whereby VEGF is released in tandem or sequentiallywith other growth actors involved in the orchestration o angio-genesis, such as platelet-derived growth actor-BB (PDGF-BB),FGF-2 and angiopoietin-1 and -2 (res 140, 142). PDGF-BB isimportant in recruiting smooth muscle cells to stabilize nascentvessel walls; when packaged with VEGF-A inside alginate hydro-gels, the two growth actors show distinct release kinetics becauseo their different affinities or alginate. Used therapeutically, thismaterial stimulated the ormation o mature blood vessels withassociated smooth muscle cells, and improved cardiac unctionin a rat model o myocardial inarction125. Yet another possibleavenue is to usegene transer such that cells constitutively producelow levels o VEGF or other desired proteins124. One target is the

transcription actor hypoxia-inducible actor 1 (HIF-1), whichholds the key to intracellular detection o hypoxia and subsequentupregulation o VEGF and other proteins involved in the cascadeo angiogenesis126. A gene was delivered that encoded a stabilizedorm o HIF-1, which lacked the oxygen-sensitive degradationdomain present in the native orm and could thereore initiateangiogenic events under normoxic conditions. Te plasmid waspackaged within designer peptides, one o which incorporateda actor XIIIa substrate sequence as a means to immobilize theDNA within a fibrin gel (Fig. 3). Clearly, although vascularizationremains an inadequately resolved challenge, encouraging develop-ments are being made in this area.

Box 2 | Challenges in vascularization.



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proteoglycans while keeping structural complexity to a minimum107.Understanding o the relationships between structure and unctionwithin polysaccharides lags well behind that o proteins and nucleicacids, owing to the difficulty o obtaining structurally defined oligo-saccharides in bulk rom natural polysaccharides, and to complicatedchemical synthesis and inadequate sequencing methods108. However,recent developments herald a new era o understanding in this field,and with it the potential or therapeutic application, including in

tissue engineering. New synthetic routes, including engineered celllines, chemoenzymatic routes, solid-phase automated chemicalsynthesis and one-pot methods, allow more efficient production ooligosaccharides o ever increasing complexity109112. Molecules thatmight once have taken months to produce can now be created in aday108. Once these techniques are readily available, array technolo-gies such as carbohydrate chips promise to shed light on proteinoligosaccharide interactions113, and lead to the discovery o relativelysimple and synthetically tractable structures or use in tissue engi-neering and elsewhere. For now the ull potential o these advancesremains unrealized, and developments are eagerly anticipated.

Non-specific cellbiomaterial interactionsAs well as introducing defined molecular recognition elementsinto biomaterials, recent studies have ocused on using more basic

chemistry to influence cell ate, potentially paving the way orstructurally simple, yet cell-instructive, biomaterials. In the absenceo adhesion peptides, cells interact with scaffolds by means oadsorbed protein, and in this regard topography and hydrophilicityare key considerations. For example, fibrous meshes with nanoscalefibre diameters have shown selective take-up o proteins relevant orcell attachment, such as fibronectin and vitronectin114. And whereashydrophobic scaffolds tend to adsorb protein in sub-optimal con-

figurations (with hydrophobic residues displaced towards the sca-old surace), hydrophilic polymers adsorb protein in a hydratedinteracial phase wherein the proteins are more likely to retain theirnative conormation115. In one study116, introducing different unc-tional groups to suraces changed the conormation o adsorbedfibronectin leading to altered integrin binding, such that indices oosteoblastic differentiation (expression o bone markers, alkalinephosphatase activity and mineralization) were significantly upregu-lated on OH- and NH2-unctionalized suraces compared with CH3and COOH. Tere is considerable interest in using such simplechemistry to improve cell responses in avour o particular applica-tions. Recent studies have described high-throughput arrays or thesystematic evaluation o large numbers o biomaterials-cell interac-tions. Tese investigations typically involve robotic spotting o poly-mer islands (1,152 in one example117) onto a glass slide coated with

Constitutive VEGF

expressionGF upregulation

Stabilized

HIF-1

To nucleus

Dimerization,

transcription

Sequential release

VEGFPDGF-BB

Slow, low-level release

Therapeutic

window

Time

GFrelease

Co-culture:

Paracrine

signals

Cadherins

VEGF

PDGF-BB

Endothelial

cells

Smooth musclecells, pericytes

Endothelial tube

Stabilized vessel

VEGF

rapid burst

Leaky vessels

Regression

PericyteEC

TIMP-3

TIMP-2

Genetransfer

Cellc

ellinteractions

Grow

thfacto

rreleasekinetics

Grow

thfactordelivery

Figure 3 | Vascularization of tissue-engineering scaffolds. Centre/bottom: new blood vessels form initially by endothelial cell organization into tubes,

which are later stabilized by smooth muscle cells and pericytes. From left: many attempts to induce this process within TE scaffolds have involved the use

of VEGF. A low level of sustained release is demanded as burst-release results in the formation of unstable, leaky vessels124. Subsequent release of platelet-

derived growth factor BB (PDGF-BB) helps to recruit smooth muscle cells, which favours vessel maturation125. Materials strategies for releasing growth

factors are indicated in Fig. 2. Gene transfer has been used to generate populations of cells that constitutively express either VEGF or a stabilized variant ofhypoxia-inducible factor 1(HIF-1). In the latter case, the protein translocates to the nucleus where it initiates a hypoxic response involving the activation

of a host of genes and upregulation of angiogenic growth factors126. Co-culture of different cell types leads to paracrine and membrane interactions that

can enhance angiogenesis. Endothelial cells (EC) and pericytes in co-culture increase the production of tissue inhibitor of metalloproteases TIMP-2 (EC)

and TIMP-3 (pericytes), which inhibits tube regression127.



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High LowMedium

Structural synthetic mimics

Anionic and

phosphate groups

Multi-domain

peptide

Sulphated and

carboxylated dextran

Enzyme-sensitive

peptide crosslinkers

Cryptic site peptides

Sulphonated

synthetic

polymers

Poly(glutamic

acid) peptide

Domain III

Integrin binding

sequence

Fibronectin

Structure and function of ECM molecules

Protease

Glycosylated

synthetic

polymers

Proteoglycan aggregate

Chondroitin

sulphate,

a GAGSulphated groups

Proteoglycan

Enzymatic cleavage

sequences

Amino acids

Bone sialoprotein interaction

R

GD

Cell behaviour Functional synthetic mimicsCellcell adhesion

Global response: including viability, adhesion and differentiation

Biotinavidin

crosslinking

Polymer microarray

Bone sialoprotein-

derived peptideP

CO O

O

OO

O

Synthetic polymer matrix

Dimerized affinity

peptides

Receptor binding

Heparin

binding

Biotin

Avidin

Cell surface

Protein complex

Cell membrane

E-cadherin

Cytoskeletal actin

Cell

junctions

Fibroblast

growth factor 1

(FGF-1)

bound as dimer

FGF receptor 2Bound

heparan sulphate

Collagen

triple helices

Hydroxyapatite

crystals

S O

O

O

-

SO O

O-Oligosaccharides

such as heparin oligomer

- Poly(glutamic acid)

sequences

Integrin

recognition sequences

RGD

IKVAV

PHSRNYIGSR

PDSGR

EEEEEEEE

E

E

EEEEEEEE

...GPQGIWQG...

...GPQ

G IWQG...

e

f

Active

fragment

released

Integrin binding, protease sensitivity

Mineralization mediators

Growth factors

Protein binding

a

b

d

Carbohydrates

c

Proteins

Figure 4 | Synthetic mimics of biological structures. Many characteristics of ECM macromolecules have been reproduced in simpler compounds with

biologically inspired structures. a, Certain protein functions, including integrin binding for cell attachment and protease degradability, can be isolated to

short amino-acid sequences. These sequences can be combined with synthetic polymers or incorporated into complex peptides to enable cells to attach

to or break down the material, respectively55,56,5860,63,64. b, Some glycoproteins involved in bone mineralization, such as bone sialoprotein, possess runs of

negatively charged amino acids. Peptides that incorporate these sequences, and synthetic polymers with negatively charged chemical groups, can display

improved mineral-nucleating activity120,121,128,129. c, Growth factor action has been demonstrated in peptides possessing receptor-binding domains; heparin-

binding sequences may also be included to aid growth factor sequestration90,91. Random peptide libraries have allowed the identification of peptides with

affinity to particular receptors, and dimerization of these molecules in some cases can improve receptor binding and physiological response94. Growth

factor action is sometimes potentiated through the actions of glycosaminoglycans (GAGs) such as the binding of heparan sulphate to the FGF-1/FGF

receptor 2 complex. This specific interaction can be achieved using short heparin oligosaccharides86. d, Furthermore, the protein-binding function of ECM

GAGs such as chondroitin sulphate can be mimicked by grouping sulphated oligosaccharides by polymerization, by sulphating natural carbohydrates such

as dextran, or by sulphonating synthetic polymers84,99102,130. Additionally, certain biological functions can even be supported by chemistries with no relation

to biological structures. e, Whereas cellcell adhesion occurs predominantly through the complex binding of cell surface cadherin proteins, biotinavidin

interactions have been used to artificially aggregate cells28. f, A range of responses, such as cell adhesion, viability and differentiation, can be differentially

affected by particular synthetic substrates. These cellmaterial interactions can be assayed using high-throughput screening of cells on polymer

microarrays 117119. (Depictions of protein and peptides do not represent structures accurately.)



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a non-adhesive polymer such as poly(hydroxyethyl methacrylate),which prevents cell migration between spots. Following cell culture,standard immunohistochemical techniques and microarray scan-ning can be perormed. Tis provides a way o identiying poly-mers that support desired responses rom specified cell types, orexample those that promote differentiation o human embryonicstem cells117119.

Another approach has been to select chemical unctionalitiesbased on their resemblance to characteristic chemical eatures

o particular ECMs. Earlier we described the bio-inspired use osulphonate groups within hydrogels, mimicking their presence inGAG chains, which increased protein uptake102. Tis approach iswell established in bone tissue engineering, where there exist manyexamples o materials incorporating anionic chemical moieties thatimprove mineral deposition, or instance by NaOH treatment oscaffold suraces or by the incorporation o unctionalized mono-mers such as methacrylated amino acids (GlyMA, SerMA, AspMA,GluMA)120,121. Tis practice stems rom the observation that manyglycoproteins involved in bone mineralization display a high pro-portion o negatively charged amino acids: or example, bonesialoprotein (BSP-II) possesses two polyglutamic acid sequences,and osteopontin (BSP-I) contains a run o 1012 aspartic acid resi-dues122. Phosphate groups also nucleate mineral, and although oen

delivered in soluble orm in vitro (as -glycerophosphate) can beincorporated covalently into scaffolds.

Moreover, these chemical groups may also be instructive tocells. In a recent study, small defined chemical groups were incor-porated into PEG gels, and encapsulated human mesenchymal stemcells differentiated towards cells o those tissues that the unctionalgroups chemically resembled54. Tus, those cells cultured in gelswith charged phosphate groups increased expression o RUNX2(CBFA1; an early bone transcription actor), produced a collagen-rich pericellular matrix, and synthesized osteopontin. Hydrophobict-butyl groups pushed cells towards an adipocytic (at cell) lineage,demonstrated by upregulation o the transcription actor PPARand the deposition o intracellular lipid deposits. It is unknown (andor practical purposes, arguably irrelevant) whether the role o the

chemical modifications was to act directly on the cells, or to cause thepreerential accumulation o particular cell-derived molecules, thesemolecules in turn providing behavioural signals to cells. An exampleo the latter mechanism in action is the ability o mineral deposits tosequester osteopontin, which improves cell adhesion and viabilitywithin phosphate-containing PEG gels123. Whatever the modes oaction, the complexity o biomaterials could be massively reduced ithe essential chemical character o ECM influences could be distilledinto simple chemical unctionalities. A summary o the various waysin which relatively simple molecules can mimic the molecular inor-mation within the ECM is given schematically in Fig. 4.

Concluding remarks and perspectivesTe examples discussed herein demonstrate the importance o

the extracellular environment in determining cell behaviour, andhighlight the need or regenerative materials to provide cells withbiological cues. Much is still unknown about the mechanisms bywhich tissues orm and heal, yet already insights rom developmen-tal biology and other biological disciplines are actively guiding thedevelopment o intelligent materials that work with natures ownmechanisms o repair. Tese expanding possibilities raise the ques-tion o how much extrinsic physiochemical inormation is requiredto mobilize endogenous or transplanted cells into producing acomplex tissue, and specifically, what minimum level o materialscomplexity is required or a given task. Evidently, a careul appraisalo the job in hand will reveal that the broader cost and treatmentimplications or any biomaterials approach vary with severalinterrelated actors including the orm o the device, the mode odelivery, the nature o the cellular component and any regulatory

implications. o elaborate briefly, injectable matrices help to tackleproblems o surgical invasiveness whereas tissue engineering prod-ucts in sheet orm (able 1) conront problems related to nutrientsupply by limiting diffusional distances. Moreover, materials thatcan recruit endogenous cells into scaffolds avoid the expense anddifficulties associated with culture, storage and distribution o cells,not to mention immune considerations. Encouragingly, however,it is clear that comparatively simple materials in combination withan appropriate cellular component can support a high level o tis-

sue organization14,60. Te optimization o mechanical and struc-tural eatures o scaffolds and their potential to direct aspects o cellbehaviour illustrates that unctional sophistication is not necessarilysynonymous with high manuacturing costs.

A large number o commercially viable products or connectivetissues are based on purified ECM components such as collagensand hyaluronic acid (able 1), representing a relatively generic ECMbackdrop conducive to the activities o differentiated cells. Imposing atissue-specific identity on stem cells in many cases is likely to requiremore specific influences, within materials i not during cell culture.Tese more advanced biomaterial approaches are just beginning totrickle through product-development pathways, but the runaway suc-cess o INFUSE4demonstrates the potential impact o schemes thatmake use o growth actor activity. It is promising that the outcome

o growth actor administration can be improved enormously withthe use o technically simple slow-release schemes, such as deliveryusing polymers. Such considerations may prove critical or the resolu-tion o complex tissue engineering challenges such as that o vascu-larization (Box 2). However, the generation o thick or heterogeneousconstructs, and even complex organs, will require urther innovationsin biomaterials research. Interest is also growing in the exciting pos-sibility o using simple chemistries to influence cell behaviour, and inthe development o a range o therapeutics with intrinsic or modulat-ing growth actor activity, including designer carbohydrates. Severallaboratories in their own ways are actively pursuing simple but effec-tive solutions to tissue engineering problems, such that the ideal ostructurally simple, yet unctionally complex, biomaterials is becom-ing a plausible possibility or the near uture. More widely, there is

evidence in the resurgence o tissue engineering since the gloomydays o the early millennium that companies offering these productshave become wise to the demands and realities o the marketplace.Te industry has benefited rom a heavy dose o reality and, lessonslearned, is ready to prosper.

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engineered autologous bladders or patients needing cystoplasty. Lancet367,12411246 (2006).

3. Macchiarini, P.et al.Clinical transplantation o a tissue-engineered airway.Lancet372,20232030 (2008).

4. Lysaght, M. J., Jaklenec, A. & Deweerd, E. Great expectations: Private

sector activity in tissue engineering, regenerative medicine, and stem celltherapeutics. issue Eng. Part A14,305315 (2008).

5. US Department o Health and Human Services. 2020: A New Vision A Future for Regenerative Medicine (2006); available at.

6. Bouchie, A. issue engineering firms go under. Nature Biotechnol.20,11781179 (2002).

7. Michalopoulos, G. K. & DeFrances, M. C. Liver regeneration. Science276,6066 (1997).

8. Ford, C. E., Hamerton, J. L., Barnes, D. W. & Loutit, J. F. Cytologicalidentification o radiation-chimaeras. Nature177,452454 (1956).

9. Mathe, G., Amiel, J. L., Schwarzenberg, L., Cattan, A. & Schneider, M.Haematopoietic chimera in man aer allogenic (homologous) bone-marrowtransplantation. (Control o the secondary syndrome. Specific tolerance due tothe chimerism). Br. Med. J.5373,16331635 (1963).

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http://www.nature.com/doifinder/10.1038/nmat2441http://www.nsf.gov/pubs/2004/nsf0450/start.htmhttp://www.hhs.gov/reference/newfuture.shtmlhttp://www.hhs.gov/reference/newfuture.shtmlhttp://www.nsf.gov/pubs/2004/nsf0450/start.htmhttp://www.nature.com/doifinder/10.1038/nmat2441


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AcknowledgementsWe thank R. Langer, K. Godula and A. Ratcliffe or eedback on themanuscript. M.M.S. acknowledges an ERC Individual Investigator Grant orunding, and EPSRC or the unding o E.S.P. and N.D.E.

Additional informationSupplementary inormation accompanies this paper on www.nature.com/naturematerials.
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