building bone tissue: matrices and scaffolds in physiology and … · 2003. 7. 24. · in...

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Braz J Med Biol Res 36(8) 2003

Building bone tissueBrazilian Journal of Medical and Biological Research (2003) 36: 1027-1036ISSN 0100-879X

Building bone tissue: matricesand scaffolds in physiology andbiotechnology

1Dipartimento di Medicina Sperimentale,Universita’ dell’Aquila, Rome, Italy2Parco Scientifico Biomedico San Raffaele, Rome, Italy3Dipartimento di Medicina Sperimentale e Patologia,Universita’ La Sapienza, Rome, Italy

M. Riminucci1,2

and P. Bianco2,3

Abstract

Deposition of bone in physiology involves timed secretion, depositionand removal of a complex array of extracellular matrix proteins whichappear in a defined temporal and spatial sequence. Mineralizationitself plays a role in dictating and spatially orienting the deposition ofmatrix. Many aspects of the physiological process are recapitulated insystems of autologous or xenogeneic transplantation of osteogenicprecursor cells developed for tissue engineering or modeling. Forexample, deposition of bone sialoprotein, a member of the smallintegrin-binding ligand, N-linked glycoprotein family, represents thefirst step of bone formation in ectopic transplantation systems in vivo.The use of mineralized scaffolds for guiding bone tissue engineeringhas revealed unexpected manners in which the scaffold and cellsinteract with each other, so that a complex interplay of integration anddisintegration of the scaffold ultimately results in efficient and desir-able, although unpredictable, effects. Likewise, the manner in whichbiomaterial scaffolds are “resorbed” by osteoclasts in vitro and in vivohighlights more complex scenarios than predicted from knowledge ofphysiological bone resorption per se. Investigation of novel biomaterialsfor bone engineering represents an essential area for the design oftissue engineering strategies.

CorrespondenceP. Bianco

Dipartimento di Medicina

Sperimentale e Patologia

Universita’ La Sapienza

Viale Regina Elena 324

00161 Rome

Italy

E-mail: [email protected]

Presented at SIMEC 2002

(International Symposiumon Extracellular Matrix),Angra dos Reis, RJ, Brazil,

October 7-10, 2002.

Unpublished research included in

this article was supported by

grants from Telethon (E.1029),

AIRC, ASI, and MIUR.

Received February 5, 2003

Accepted June 3, 2003

Key words• Extracellular matrix• Bone matrix• Bone formation• Biomaterials• Tissue engineering

The extracellular matrix of bone

For decades, attention devoted to the bi-ology of the extracellular matrix (ECM) ofbone has been centered on the quest forunique properties thereof, which would ex-plain its unique ability to mineralize in ahighly controlled fashion. Originally, the driv-ing idea was that one or more protein speciesproduced in bone but not in other connectivetissues would act as tissue-specific inducers,

nucleators and regulators of mineral deposi-tion. Given the ubiquitous nature of the ma-jor collagen species in bone, collagen type I,the search for noncollagenous componentsendowed with the “King Mida’s touch” whichwould turn tissue into stone, focused mostlyon noncollagenous components. This lead-ing idea fueled a large volume of studies,which originally capitalized on the identifi-cation of biochemical procedures for extrac-tion of proteins buried in the mineral com-

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M. Riminucci and P. Bianco

partment of bone (1,2), and thus not ame-nable to extraction by usual denaturingagents. Identification of a number of bone-enriched noncollagenous proteins and cog-nate genes ensued, which was later followedby the development of a number of trans-genic and knockout models developed withthe aim of identifying the specific biologicalfunction of individual proteins.

Today, the original idea of the mechanis-tic relationship between bone proteins andmineralization is in need of significant revi-sion. Most of the proteins originally thoughtto be unique of the bone ECM have beenproven to be expressed in other tissues aswell, whereas, ironically enough, a role forosteocalcin (the only protein still reasonablyassumed to be bone specific) in bone miner-alization has been dispelled by the regularoccurrence of bone mineralization in osteo-calcin-deficient mice (3). A single well-de-fined function has not been identified con-clusively for any of the bone-enriched non-collagenous proteins. Nonetheless, a num-ber of unexpected biological events in whichbone noncollagenous proteins are involvedhave emerged and are currently the focus ofintensive investigation. Many of these eventshave revealed complexities of bone physiol-ogy which were not accounted for by theoriginal, simplistic idea of a bone-specificmineral nucleator and also exceed the limitsof biomineralization to extend into cell mi-gration, angiogenesis, assembly of the ma-trix, maintenance of its mechanical proper-ties, cell-matrix-interaction, and more.

A detailed discussion of the biochemis-try of bone matrix can be found elsewhere(4). Ninety percent of the organic matrix ofbone is made up by collagen type I, and 10%by a variety of noncollagenous proteins. Theratio of collagen to noncollagen protein inbone is highly variable throughout the dif-ferent physiological scenarios of bone for-mation (from embryonic development toadult remodeling to aging, Ref. 5) and re-flects specific changes in the secretory func-

tion of the bone-producing cells, the osteo-blasts. Primary embryonic bone has a muchlower collagen-to-noncollagen ratio com-pared to postnatal secondary bone (reviewedin Ref. 5), and this results in different typesof internal organization of the matrix (wovenvs lamellar bone). Thus, the composition andstructure of the bone matrix is not one and thesame under all physiological circumstances,and insights have started to accumulate onthe identity of proteins secreted in associa-tion with specific physiological settings.

Gene targeting experiments have revealedsignificant specific roles for individual pro-teins or families of proteins. Small proteo-glycans found in bone (6,7), for example,play a role in regulating the assembly andstructure of the collagen fibrils in bone, andtheir deficiency induced by gene targetingresults in reduced bone mass and strength,and abnormalities in collagen fibrils similarto those found in Ehlers-Danlos syndromesin a variety of connective tissues (8). Smallintegrin-binding ligand, N-linked glycopro-teins include osteopontin and bone sialopro-tein (BSP), which are closely related to eachother in terms of structure and post-transla-tional modifications (4,9-11). Both featurean integrin-binding RGD motif and are in-volved in cell-matrix interactions. Osteo-pontin is widely distributed in different tis-sues, whereas BSP is highly enriched inbone and skeletal cartilages, is expressed inthe human trophoblast (12), and is ectopicallyexpressed in a variety of malignant epithelialcancers (13-15), but not in the cognate nor-mal tissues. In bone, BSP is expressed byfully mature osteogenic cells (Figure 1A)competent to deposit a mineralizing matrix(12), but only during early phases of de novobone formation (16). Extracellular BSP lo-calizes to newly formed mineralizing bonematrix, and at the electron microscope level,its distribution spatially and temporally co-incides with nascent mineral deposits (17),with some of which bone cells establishclose contacts (Figure 1B). In systems in

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Building bone tissue

which mineralization is impaired, such aschick embryos incubated in vitro in the ab-sence of the shell (which naturally suppliesthe calcium necessary for bone mineraliza-tion), BSP is abundantly expressed in com-petent cells, but not deposited in the ECM(Riminucci M, Gentili C, Cancedda R andBianco P, unpublished results). In vitro, BSPsecreted by osteoblast-like cells is localizedto focal adhesions and possibly other adhe-sion structures (Figure 1C) (Riminucci Mand Bianco P, unpublished data).

Scaffolds in physiological boneformation

Deposition of bone is a highly oriented,polarized phenomenon. Osteoblasts seem toknow where to deposit bone and where notto. For example, this allows the new bonebuilt by osteoblasts during bone remodelingto go and fill the space generated by theprevious resorption phase, and not elsewhere.This ensures not only a homeostatic balancebetween resorption and formation, but alsothe restoration and preservation of the struc-tural and morphological layout of existingbone tissue. The high degree of polarity ofbone deposition clashes with the notion thatosteoblasts, like all connective tissue cells,have not evolved efficient mechanisms formaintaining cell polarity and polarized se-cretion. In epithelial cells, polarized secre-tion is made possible by the existence ofpolarized domains of the plasma membrane,which in turn are established and stabilizedthrough the interaction with a basement mem-brane and the segregation of membrane do-mains by systems of tight junctions. Osteo-blasts do not reside upon a basement mem-brane and do not establish systems of tightjunctions. How then do bone cells depositbone in a polarized fashion without beingpolarized cells? They do so by using scaf-folds and templates which direct depositionof matrix proteins that are secreted in anonpolarized fashion (18).

Creation of a scaffold in de novobone formation

In physiology, bone formation occurs a)on preexisting bone surfaces, or b) on pre-existing nonosseous but mineralized surfaces,or c) de novo in the context of unmineralizedtissue. Instances a) and b) represent the for-mation of bone tissue on a preexisting min-eralized scaffold, which accounts for mostof the total bone formation occurring duringgrowth, and for all bone formation occurringafter skeletal growth has ceased with sexualmaturity. De novo bone formation has uniquebiochemical features which distinguish itfrom bone formation occurring under differ-ent circumstances. BSP is specifically ex-pressed by osteoblasts depositing bone de

A

B

C

Figure 1. Expression and depo-sition of bone sialoprotein (BSP)by bone cells in vivo and in vitro.A, De novo bone formation in a17-day rat embryo, with BSP im-munolocalization. BSP is ob-served in the Golgi apparatus ofmature osteoblasts and in thenascent bone matrix. B, Elec-tron microscopy immunolocal-ization of BSP. BSP is localizedto discrete structures (double ar-rows) in the nascent bone ma-trix, corresponding to early min-eral deposits. The single arrowpoints to one such structure inclose contact with an osteo-blast. C, Primary culture of new-born rat calvarial cells, with BSPimmunofluorescence. Note BSPlocalization to intracellular secre-tory granules and to adhesionstructures (arrows). Bars = 8 µm(A); 0.1 µm (B); 10 µm (C).

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novo (Figure 2A), and much less or not at allby osteoblasts adding bone to preexistingbone (16). De novo bone formation is theonly scenario in which no preexisting scaf-fold is available to guide the deposition ofnew bone matrix synthesized and depositedby osteoblasts. Interestingly, the generationof a scaffold is the first event in de novo boneformation. Deposition of the first bone ma-trix at any site of de novo bone formationtakes place between rows of cells facingeach other (vis-à-vis cells) (19). This allows

the space between facing rows of cells tobecome highly enriched in proteins other-wise secreted in a nonpolarized fashion.Mineral is deposited at the same time and indirect physical association with the deposi-tion of certain bone proteins, first and fore-most BSP. Electron microscopy analysis re-veals that early mineral clusters cannot bephysically distinguished by early spots ofnewly deposited BSP (17,19). The creationof mineral clusters at nascent osteogenicsites further promotes the adsorption of os-teoblast-secreted proteins, thus establishinga preferential direction for bone depositionin spite of the lack of determinants for polar-ized secretion of matrix proteins. Subsequentdirectional accretion of matrix onto the nas-cent sites then becomes polarized by defaultonce a scaffold has been created (Figure2B).

Deposition of bone on a preexistingscaffold in physiology

Endochondral bone formation providesthe easiest scenario in which to seek a sim-plified representation of the nature and tem-poral sequence of events characterizing theinteraction of osteogenic cells with a preex-isting mineralized scaffold. The scaffold isprovided by mineralized cartilage coreswhich have formed at the climax of growthplate maturation. On these cores, newly dif-ferentiated osteoblasts deposit temporarybone, fated to be remodeled into secondarytrabecular bone. A specific histological struc-ture - the cement line - separates the cartilagecore from the primary bone which is depos-ited onto it. The cement line found in thetrabeculae of the endochondrally formed pri-mary spongiosa is structurally and chemi-cally identical to cement lines found in sec-ondary and even postnatal bone, where theyphysically mark the reversal of a resorptionphase to a new formation phase. Cementlines do not contain collagen type I or type II,mineralize to a higher extent than bone, and

Figure 2. A, Deposition of a bone sialoprotein (BSP)-enriched matrix at the sites of nascentbone formation in rat embryonic ribs. Rings of BSP-rich matrix surround the cartilageanlagen of the ribs. At these sites, BSP immunoreactivity and early mineral deposits co-distribute in space and time. Both osteoblasts located on the outer surface of the cartilageanlage and peripheral hypertrophic chondrocytes located at the edge of the anlage (“bor-derline” chondrocytes) contribute to the deposition of the nascent mineralizing matrix. ABSP-rich material forms the interface between bone and cartilage. B, Diagram illustratingthe generation of a scaffold at sites of de novo bone formation. Rows of cells competent todeposit a mineralizing matrix are arranged vis-à-vis. Secretion of a noncollagenous matrixenriched in BSP results in a local enrichment in mineral nucleators in the space betweenthe facing rows of cells. Mineral deposits form rapidly in this milieu and adsorb osteoblast-produced matrix proteins, thus determining the polarized deposition of the products ofnonpolarized secretion. These events “prime” the subsequent deposition of collagen-richbone matrix. Once a template (scaffold) is generated in this way, subsequent deposition ofbone remains spatially oriented. C, Electron microscopy immunolocalization of BSP in anendochondrally formed bone trabecula in the primary spongiosa of growing bone. Here,bone is formed on the surface of a preexisting core of mineralized cartilage (cartilage). Notethat BSP immunolabeling marks the cement line at the interface of bone and cartilage, andalso material (called cement) found between collagen fibrils. In this Lowicryl K4M section,collagen fibrils appear as electron-lucent space. Bars = 20 µm (A); 0.2 µm (C).

A

B

Bone

Cartilage

Osteoblasts

Nascent boneVis-à-vis

cells

C

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are highly enriched in BSP (20). Since ce-ment lines are formed by the first matrixwhich is laid down during bone formation,their enrichment in BSP is a further instanceof the role of this protein in de novo boneformation. As bone formation progresses,increasing amounts of collagen are producedby osteoblasts and layered onto the BSP-richcement line. Among collagen fibrils, a struc-tural phase with electron microscopic, cyto-chemical and immunocytochemical charac-teristics identical to those of the cement lineis recognizable (Figure 2C). To emphasizethis similarity in structure and nature, thisphase (a true structurally defined noncollag-enous phase in bone) has been named ce-ment (20).

Scaffolds in bone tissue engineering -evolution of a concept

Attempts at tissue engineering of bonelong precede the current implementation ofstem cell-based technologies. Before meth-ods became available for isolating, expand-ing and culturing osteogenic progenitor cellsto be used for bone reconstruction, scaffoldswere used alone to guide and promote regen-eration of bone tissue after trauma or extir-pative surgery (21-26). Most of the materialsused under these circumstances were hy-droxyapatite-based (27-33), and a crucialrequirement of their design and use was thattheir internal structure should be such as toaccommodate the ingrowth of blood vessels,and to guide the subsequent deposition ofbone in a way that would ultimately mimicthe haversian structure of normal compactbone. (Macro)porosity of the material wasthus an important characteristic. Hydroxy-apatite-based materials are osteoconductive,meaning that they lead to bone deposition,provided that fully differentiated and com-petent osteogenic cells are available at thesite of implantation. It is important to distin-guish osteoconductivity from osteoinductiv-ity, which means the ability of a given chem-

ical species to induce de novo differentiationof competent osteogenic cells from non-osteogenic and uncommitted cells - a prop-erty only ascribed in biology to members ofthe bone morphogenetic protein family. Hy-droxyapatite is osteoconductive because itadsorbs osteogenic growth factors from thelocal milieu and from the circulation, thuscreating suitable local conditions for boneformation when implanted in an osseousenvironment. It is not osteoinductive be-cause it is not sufficient to induce differen-tiation of osteogenic cells when implanted ina nonosseous environment such as skin ormuscle. In addition, rigid hydroxyapatitematerials fulfill the requirement of bone cellsfor a template onto which to deposit bone,and provide some mechanical resilience atthe site of implantation, even though thefragility and inelasticity of hydroxyapatiteceramic (HAC)-based carriers warrants theuse of internal fixation whenever a HACdevice is used for restoration of a load-bearing skeletal segment.

Integration, disintegration, andresorption of HAC scaffolds

Ideally, a HAC carrier, whether used aloneor in combination with cells, should be eas-ily integrated in bone, and also resorbableand amenable to gradual replacement bynewly formed bone. Curiously, a rigorousand consistent definition of “integration” isnot easily identified in the literature. On theother hand, common concepts of resorbabilityof HAC carriers are not based on rigorousexperimental assessment. Integration of aHAC-based material used for bone graftingimplies the establishment of a structural andfunctional continuity between the implantedmaterial and the bone surfaces available atthe implantation site (34). It also implies,however, the establishment of the same con-tinuity between HAC surfaces available atthe implantation site and the bone which isnewly deposited after implantation, either

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by resident osteogenic cells, or by osteo-genic cells included in a “biologized” im-plant (that is, a HAC/cell construct).

Integration of bone and HAC at the mi-croscopic level reveals unexpected complexi-ties. Boyde et al. (35) investigated the long-term fate of large porous HAC cylinderswhich were implanted in a weight-bearingbone of large mammals in order to promoterepair of an experimental critical size defect.This study revealed that integration of HACinto bone occurs at multiple dimensionallevels, reflecting different orders of porosityof the material employed. Bone fills macro-pores (size of the order of hundreds of mi-crons) as well as micropores down to the sizeof 2 µm. Thus, bone cells are able to fill gapsand spaces in the HAC structure that are oneorder of magnitude smaller than their ownsize, as if bone matrix was deposited in afluid state before solidifying into a mineral-ized phase. In addition, continuing mechan-ical effects on the implanted material gener-ate breaks and clefts within the material.This generates additional HAC surfaces avail-able for osteoconduction, and progressivelysmaller portions of HAC become incorpo-rated into bone. A disintegration mechanismthus boosts integration of the HAC.

Resorbability of HAC-based materials iscommonly thought to reflect chemical com-position (relative content of hydroxyapatitevs tricalcium phosphate) and hydroxyapatite

crystal size, which in turn is a function of thesintering temperature. However, adequateinvestigations of how osteoclasts (the cellsinvolved in resorption of bone and thus envi-sioned as the HAC-resorbing cells) behavewith respect to HAC-based biomaterials invitro and in vivo are largely missing. Theideal approach involves the use of isolatedosteoclasts in a pit resorption assay (36),followed by adequate assessment not only ofpit number and diameter but also of pit depth,which significantly affects the actual vol-ume of material which is resorbed (36). Dataobtained using this approach (Boyde A andBianco P, unpublished results) reveal specif-ic manners of HAC-osteoclast interaction,which are not accounted for by the simplisticassumption that osteoclasts would deal withHAC in the same manner as they deal withbone. Thus, the manner in which bone cellsat large (i.e., both bone-forming and bone-resorbing cells) handle HAC-based materi-als represents an important area for futureinvestigation, at the cross-road of bone cellbiology and material science.

Scaffolds and skeletal stem cells -mimicking physiology in tissueengineering

The recognition of the existence of skel-etal stem/progenitor cells in the postnatalbone marrow of humans and other mammalshas opened important perspectives in bonetissue engineering (37). Skeletal progenitorcells are found among the adherent, clono-genic and fibroblastic cells associated withthe bone marrow stroma (38). These cellscan be significantly expanded ex vivo (Fig-ure 3) in order to generate sufficient num-bers of cells competent to differentiate intomature osteoblasts and form new bone uponin vivo transplantation (39-42). Transplanta-tion of about 106 such cells in conjunctionwith suitable carriers in the subcutaneoustissue of immunocompromised mice gener-ates several mm3 of new bone and associated

Figure 3. The principle of bone reconstruction using stromal stem cells and biomaterialscaffolds. Stromal stem cells (skeletal stem/progenitor cells) are isolated from the bonemarrow as adherent clonogenic cells. Strains obtained from ex vivo expansion are thenloaded onto suitable carriers (most of which are hydroxyapatite ceramic (HAC)-basedmaterials available either as solid structures or granular powders) and transplanted intoexperimental animals. Ectopic transplantation (e.g., in the subcutaneous tissue of immuno-compromised mice) is used to assay the differentiation potential of the cell lines; orthotopictransplantation in bone (e.g., at the site of an experimentally created bone gap) of similarHAC/cell constructs prepared with autologous cells is used in preclinical models of bonetissue engineering.

HAC carrierStromal cell culture

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tissues (bone marrow) organized in a struc-tural layout closely resembling natural bonehistology (38,43,44).

Under these experimental circumstances,transplanted skeletal stem cells recapitulatethe formation of bone and marrow in a pre-cisely defined temporal sequence. The depo-sition of bone precedes the establishment ofa suitable hematopoietic stroma and its colo-nization by blood-borne hematopoietic stemcells. In the resulting “ossicle”, a bony andstromal framework of donor origin (i.e.,formed by the transplanted cells) harbors acomplete hematopoietic tissue formed byhost hematopoietic progenitors (45). In theearly stages of bone deposition which ini-tiate and trigger the whole process, deposi-tion of BSP is the first recognizable step(Figure 4), much like in normal bone forma-tion (Riminucci M, Corsi A, Quarto R,Cancedda R and Bianco P, unpublished re-sults). The surface of the carrier is paintedwith a BSP-enriched layer (cement) whichremains detectable upon the subsequent ad-dition of a collagenous phase. An additionalcommon feature of the HAC-bone interfacegenerated upon transplantation of HAC/pro-genitor cell constructs is the presence of amore or less regular row of cells adjacent tothe scaffold surface and embedded in thenewly deposited matrix (Figure 4). Giventhe nature of BSP as an RGD motif-contain-ing, cell-binding protein, this finding maysignify a role of BSP in promoting (andmediating) cell-scaffold interactions in theearly phases of deposition of new bone onthe HAC material.

Thus, the essential microevents of initialbone formation under natural conditions areduplicated when bone is engineered. Both innatural settings and in bone tissue engineering,bone cells handle a scaffold surface in a simi-lar manner. They deposit a layer of cement, anoncollagenous matrix enriched in BSP (andother cell-binding proteins) as a preliminarystep in the deposition of the collagenous ma-trix. The high affinity of BSP for mineral

surfaces probably plays a role in promotingthe adsorption of a cell-derived protein phaseonto a preexisting mineral surface, and mayplay a role in creating a mineral surface bynucleating mineral deposition. The resultingorganic-inorganic structure provides a tem-plate for polarized bone deposition. Thus,mineral itself plays a biological role in guid-ing bone formation via interactions with cellproducts and cells themselves.

HAC space

Bone

ft

D

C

B

A

HAC space

HAC space

Figure 4. A, Bone formation onthe surface of a hydroxyapatiteceramic (HAC)/skeletal stem cellconstruct which was trans-planted into the subcutaneoustissue of an immunocompro-mised mouse. The tissueformed at the site of transplan-tation was harvested 2 weekspost-transplant, demineralized,processed for histology, andused for immunolocalization ofbone sialoprotein (BSP). Disso-lution of the HAC material dur-ing decalcification generatesempty space (HAC space) whichcorresponds to the space previ-ously occupied by the material.Bone forms on the surface ofthe HAC material, and a distinctlayer of BSP-enriched matrixmarks the HAC/bone interface(B, D, arrowheads). On thegrowing surface of the newlyformed bone, individual osteo-blasts are BSP-immunoreactive(B, C, arrows). In D, note severalosteocytes (arrows) residingnext to the BSP-rich layer (ar-rowheads). ft = fibrous tissue.Bars = 200 µm (A); 60 µm (B);100 µm (C); 10 µm (D).

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The need for a template is retained inattempts to reconstruct bone tissue usingskeletal stem cells. Local transplantation ofskeletal stem cells in conjunction with asuitable physical carrier (Figure 5) repre-sents a major promise for bone repair oraugmentation in orthopedics and dentistry(37,38,43). To date, a substantial amount ofpreclinical investigation indicates the feasi-bility of the approach. Animal models havebeen developed in which autologous skel-etal stem cells isolated from bone marrowcan generate significant amounts of boneorthotopically (i.e., in critical size defectscreated in bone) (46-49). In these systems,the use of skeletal progenitor cells conveysdistinct advantages over the use of the scaf-fold alone. Bone formation occurs at a fasterrate and results in the formation of a com-

posite bone/HAC phase which is effective intemporary bone repair (46-49). Preliminaryclinical observations confirm the significantpotential of the approach (50).

The inclusion of stem cell technologiesin the current approaches to bone recon-struction brings about novel challenges inthe design and conception of scaffolds andcarriers. Porosity of the material, tradition-ally seen as a necessary inherent characteris-tic, assumes a novel significance in this con-text, namely as the property whereby a uni-form access is granted to cells employed togenerate a biotechnological construct. Littlework has been done to determine the actualpermeability of materials in use with respectto cells suspended in a fluid phase permeat-ing the micropores of the scaffold. Moreimportant, materials that are efficient in pro-viding physical support and appropriate ge-ometry for bone formation may not neces-sarily be endowed with the properties re-quired to ensure stem cell survival. This isparticularly relevant when materials are con-sidered for the production of devices to beused in clinical procedures that have to last avery long time, such as those performed ininfants or adolescents with skeletal disor-ders. Experience from separate fields, suchas skin reconstruction using epidermal stemcells, has clearly indicated the critical role,in this specific context, of the supportingmaterials that are used (reviewed in Ref. 37).While the new perspectives opened by stemcell technologies have essentially employedmaterials conceived for a previous era oftissue engineering, in which autochthonouscells residing at the site of implantation wouldmerely use a biomaterial scaffold, signifi-cant innovations have been introduced in thefield of bone biomaterial proper. These nota-bly include the design of injectable mineralphases capable of crystallization in vivo, orthe design of biological matrices suited todirect cell growth and differentiation locally(reviewed in Ref. 37). Directing efforts to-wards the development of novel materials in

Figure 5. A, Low power scan-ning electron microscopic (SEM)view of a hydroxyapatite ceramic(HAC) sample used for studiesin bone tissue engineering. B,SEM view of a HAC sample re-trieved from an intraosseous site5 months after implantation.Bone has filled HAC pores. Bars= 250 µm (A); 50 µm (B).

B

A

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view of the specific challenges posed byrapid advances in the field of skeletal stemand progenitor cell biology will undoubtedlybe rewarding.

Acknowledgments

We gratefully acknowledge Alessandro

Corsi, University of L’Aquila, Italy; RanieriCancedda and Rodolfo Quarto, Centro Bio-tecnologie Avanzate, Genoa, Italy; PamelaGehron Robey, National Institute of Dentaland Craniofacial Research, National Insti-tutes of Health, Bethesda, MD, USA, andAlan Boyde, University College London,UK, for valuable collaboration.

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