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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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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
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derived peptideP
CO O
O
OO
O
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Dimerized affinity
peptides
Receptor binding
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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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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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