Upload File

sintese do complexo

13
Development of bone substitute materials: from ‘biocompatible’ to ‘instructive’† Matilde Bongio, Jeroen J. J. P. van den Beucken, Sander C. G. Leeuwenburgh and John A. Jansen * Received 22nd March 2010, Accepted 21st July 2010 DOI: 10.1039/c0jm00795a Progress made in basic research in the last decades led to a tremendous increase in quality of clinically applied bone substitute materials (polymers, ceramics and composites). The desired biological performance of these materials has consequently shifted from a passive role where materials were merely accepted by the body to an active role in which materials instruct their biological surroundings. Bone substitute materials were traditionally based on bioceramics, that can be optimized in terms of composition, structure and porosity. Now, polymers are increasingly gaining importance for use in medical applications due to their high versatility. This review provides an overview of the evolution from 1st generation biotolerant and bioinert materials via 2nd generation bioresponsive bone substitutes towards 3rd generation bioinstructive bone substitute materials that possess inherent biological cues for bone regeneration. 1 General background The change in living conditions during the twentieth century, compared to the centuries that preceded it, has brought major benefits to the welfare and health of mankind. However, the increased life-expectancy, the dynamism of activities (e.g. trans- portation methods and sport activities), and the growing world population lead to a substantial increase in patients who suffer from damaged, malfunctioning or diseased tissues or body parts. In view of bone tissue, these patients require safe and reliable bone substitute materials, which are available in sufficient quantities and possess the capacity to rapidly regenerate bone defects. The conventional treatment approach for bone defects involves bone grafting, which is a surgical procedure that utilizes a patient’s own bone (autograft), removed from another site (e.g. hip or ribs) or from a human donor (allograft) to fill up the defect. However, the scarce amount of bone that can be safely harvested and the risk of donor site morbidity limit the applicability of this approach. 1 Consequently, an alternative strategy for bone grafting is required in the form of synthetic materials that can promote bone regen- eration. A large variety of synthetic materials for bone substitute applications, (so-called biomaterials) have already been investi- gated for the reconstruction of bone defects. Generally, these materials can be categorized into three groups: ceramics, polymers and composites. Ceramic materials have a long history as bone implant coatings and bone substitute materials owing to their unique properties, including biocompatibility, bioactivity and osteoconductivity. Major drawbacks for the application of ceramics are the relatively slow degradation and poor mechanical properties. Although ceramics are still emerging as bone substi- tute materials, an increasing trend is seen towards the fabrication of polymer-based materials to obtain biodegradable and more versatile bone substitute materials that can be adjusted to specific requirements. Additionally, combinations of ceramics and poly- mers (i.e. ceramic/polymer composites) have been developed to combine the advantageous properties of both materials within a single bone substitute material. The aim of the present review is to provide an overview of the past and present bone substitute materials, and to highlight current developments in this multidisciplinary field. For reasons of convenience, a list of the abbreviations used throughout the paper is presented in Table 1. 2 Bone substitute materials 2.1 Past and present bone substitute materials: evolution and current commercially available products. Evolution of bone substitute materials. Reviewing the classical concepts and considering the evolution of materials science until today, the definition of a biomaterial has evolved from Table 1 List of abbreviations BMP Bone morphogenetic protein hrBMP Human recombinant bone morphogenetic protein BCP Biphasic calcium phosphate BMA Bone marrow aspirate hBMSCs Human bone marrow stromal cells CPC Calcium phosphate cement DBM Demineralized bone matrix ECM Extracellular matrix FGF Fibroblast growth factor HA Hydroxyapatite NF Nanofibrous OPF Oligo (poly (ethylene glycol) fumarate) PCL Poly-e-caprolactone PEG Poly(ethylene glycol) PGA Poly(glycolic acid) PLA Poly(lactic acid) PLEOF Poly(lactide-co-ethylene oxide- co-fumarate) PLGA Poly(lactic-co-glycolide acid) PPF Poly(propylene fumarate)(PPF) TIPS Thermally induced phase preparation SMP Shape memory material SME Shape memory effect VEGF Vascular endothelial growth factor Department of Biomaterials (309), Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: [email protected]; Fax: +31-24-3614657; Tel: +31-24-3614006 † This paper is part of a joint Journal of Materials Chemistry and Soft Matter themed issue on Tissue Engineering. Guest editors: Molly Stevens and Ali Khademhosseini. This journal is ª The Royal Society of Chemistry 2010 J. Mater. Chem., 2010, 20, 8747–8759 | 8747 FEATURE ARTICLE www.rsc.org/materials | Journal of Materials Chemistry Published on 23 August 2010. Downloaded by UNIVERSIDAD SAO PAULO on 09/05/2015 16:57:23. View Article Online / Journal Homepage / Table of Contents for this issue

Upload: marcos-antonio

Post on 07-Nov-2015

14 views

Category:

Documents


1 download

Report

DESCRIPTION

Sintese de um complexo de europio

TRANSCRIPT

  • Development of bone substitute materials: from biocompatible toinstructive

    Matilde Bongio, Jeroen J. J. P. van den Beucken, Sander C. G. Leeuwenburgh and John A. Jansen*

    ous increase in quality of clinically

    s). The desired biological performance

    materials were merely accepted by the

    rroundings. Bone substitute materials

    rms of composition, structure and

    e in medical applications due to their

    rom 1st generation biotolerant and

    towards 3rd generation bioinstructive

    one regeneration.

    in the form of synthetic materials that can promote bone regen-

    tute materials, an increasing trend is seen towards the fabrication

    requirements. Additionally, combinations of ceramics and poly-

    paper is presented in Table 1.

    Table 1 List of abbreviations

    enetic proteinbinant bone morphogenetic proteinum phosphateaspiratearrow stromal cellshate cementbone matrixmatrixwth factorte

    thylene glycol) fumarate)PCL Poly-e-caprolactone

    FEATURE ARTICLE www.rsc.org/materials | Journal of Materials Chemistry

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Online / Journal Homepage / Table of Contents for this issueStevens and Ali Khademhosseini.of polymer-based materials to obtain biodegradable and more

    versatile bone substitute materials that can be adjusted to specific

    PEG Poly(ethylene glycol)PGA Poly(glycolic acid)PLA Poly(lactic acid)PLEOF Poly(lactide-co-ethylene oxide- co-fumarate)PLGA Poly(lactic-co-glycolide acid)PPF Poly(propylene fumarate) (PPF)TIPS Thermally induced phase preparationSMP Shape memory materialSME Shape memory effectVEGF Vascular endothelial growth factor

    Department of Biomaterials (309), Radboud University Nijmegen MedicalCenter, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail:[email protected]; Fax: +31-24-3614657; Tel: +31-24-3614006

    This paper is part of a joint Journal of Materials Chemistry and SoftMatter themed issue on Tissue Engineering. Guest editors: Mollyeration. A large variety of synthetic materials for bone substitute

    applications, (so-called biomaterials) have already been investi-

    gated for the reconstruction of bone defects. Generally, these

    materials can be categorized into three groups: ceramics, polymers

    and composites. Ceramic materials have a long history as bone

    implant coatings and bone substitute materials owing to their

    unique properties, including biocompatibility, bioactivity and

    osteoconductivity. Major drawbacks for the application of

    ceramics are the relatively slow degradation and poor mechanical

    properties. Although ceramics are still emerging as bone substi-

    BMP Bone morphoghrBMP Human recomBCP Biphasic calciBMA Bone marrowhBMSCs Human bone mCPC Calcium phospDBM DemineralizedECM ExtracellularFGF Fibroblast groHA HydroxyapatiNF NanofibrousOPF Oligo (poly (eReceived 22nd March 2010, Accepted 21st July 2010

    DOI: 10.1039/c0jm00795a

    Progress made in basic research in the last decades led to a tremend

    applied bone substitute materials (polymers, ceramics and composite

    of these materials has consequently shifted from a passive role where

    body to an active role in which materials instruct their biological su

    were traditionally based on bioceramics, that can be optimized in te

    porosity. Now, polymers are increasingly gaining importance for us

    high versatility. This review provides an overview of the evolution f

    bioinert materials via 2nd generation bioresponsive bone substitutes

    bone substitute materials that possess inherent biological cues for b

    1 General background

    The change in living conditions during the twentieth century,

    compared to the centuries that preceded it, has brought major

    benefits to the welfare and health of mankind. However, the

    increased life-expectancy, the dynamism of activities (e.g. trans-

    portation methods and sport activities), and the growing world

    population lead to a substantial increase in patients who suffer

    from damaged, malfunctioning or diseased tissues or body parts.

    In view of bone tissue, these patients require safe and reliable bone

    substitute materials, which are available in sufficient quantities

    and possess the capacity to rapidly regenerate bone defects.

    The conventional treatment approach for bone defects involves

    bone grafting, which is a surgical procedure that utilizes a patients

    own bone (autograft), removed from another site (e.g. hip or ribs)

    or from a human donor (allograft) to fill up the defect. However,

    the scarce amount of bone that can be safely harvested and the risk

    of donor site morbidity limit the applicability of this approach.1

    Consequently, an alternative strategy for bone grafting is requiredThis journal is The Royal Society of Chemistry 20102 Bone substitute materials

    2.1 Past and present bone substitute materials: evolution and

    current commercially available products.

    Evolution of bone substitute materials. Reviewing the classical

    concepts and considering the evolution of materials science until

    today, the definition of a biomaterial has evolved frommers (i.e. ceramic/polymer composites) have been developed to

    combine the advantageous properties of both materials within

    a single bone substitute material.

    The aim of the present review is to provide an overview of the

    past and present bone substitute materials, and to highlight

    current developments in this multidisciplinary field. For reasons

    of convenience, a list of the abbreviations used throughout theJ. Mater. Chem., 2010, 20, 87478759 | 8747

  • a non viable material used in a medical device, intended to interact

    with biological systems to a substance that has been engineered

    to take a form which, alone or as part of a complex system, is used

    to direct, by control of interactions with components of living

    systems, the course of any therapeutic or diagnostic procedure, in

    human or veterinary medicine.2 These alterations in definition

    are accompanied by a shift in conceptual ideas on biomaterials

    and the expectations of their biological performance, which both

    have changed in time. In view of bone substitute materials, the

    shift in expectations on biomaterial performance is chronologi-

    material by bone tissue without limiting the regenerative

    process.11

    These advanced properties of synthetic bone substitute mate-

    rials provided the basis for a progression towards the demand for

    more powerful biomaterials to be used for the reconstruction of

    bone defects, and more specifically those for application in

    compromised clinical situations (e.g. critical-sized defect

    scenarios, osteoporotic patients, patients with impaired wound

    healing due to diabetes, etc.). Hence, these advances have

    resulted in the onset of 3rd generation biomaterial development,

    in which biomaterials can instruct the biological healing

    process. The more recent developments and the novel require-

    ments of 3rd generation bone substitute materials will be out-

    lined in more detail below.

    The main properties and the respective definitions for a bone

    substitute material are listed in Table 3.

    Current commercially available bone substitute materials. In

    modern orthopedic and dental applications, several inorganic

    and organic bone substitute materials, including ceramics,

    demineralized allografts, polymers and composites have gained

    popularity thanks to their availability, cost-effectiveness and

    biological performance. Since the past decade, a growing range

    of synthetic scaffolds for bone regeneration has become

    commercially available with widespread usage.12 Currently, the

    major segments in bone substitute market belong to the 2nd

    generation bone substitute materials (Table 4). These bone

    substitute materials are prepared in various physical forms, such

    ng m

    n, dn/ineliv

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlinecally described below.

    The search for appropriate materials as bone substitute

    materials to repair and reconstruct bone defects due to trauma

    or disease began in the early part of the 20th century. At that

    time, selection was restricted to materials that seemed (i) suit-

    able to fill bone defects, and (ii) tolerated by the biological

    environment. However, before the advent of modern immu-

    nology, many of these early attempts were pathogenic or toxic

    and triggered rejection, adsorption, or inflammatory responses.

    These first insights established the basis for the development of

    a continuing progress within the field of biomaterials science.

    Concurrently, other ongoing developments in several disci-

    plines, from molecular biology and chemistry to computer

    science and engineering, have contributed to a significant

    acceleration towards understanding of biological processes and

    biomaterials.3 In this review, the historical development of bone

    substitute materials is divided in three consecutive generations,

    the characteristics of which are not always confined to one

    specific generation but represent the optimization of require-

    ments during this evolution (Table 2).

    The 1st generation biomaterials, the so-called traditional

    materials, included inert and tolerant biomaterials, which were

    designed to withstand physiological stress without, however,

    stimulating any specific cellular responses.4,5 Despite the long-

    term integrity, the inability of the body to adapt to these mate-

    rials determined high probability of failure.

    Consequently, attention shifted from tolerant and inert

    1st generation biomaterials towards responsive 2nd genera-

    tion biomaterials that were able to elicit biological responses.6

    In particular, huge emphasis has been placed on synthetic

    biomaterials that encompassed bioactive properties (i.e.

    actively interacting with surrounding bone tissue to establish

    a direct bond between the synthetic material and bone),7

    osteointegration (i.e. healing process of implant placed within

    the bone)8 and osteoconductive properties (i.e. the capacity to

    guide bone-forming tissue on a surface or down into

    pores).9,10 Another revolutionary step for these 2nd generation

    biomaterials was the development of biodegradable materials,

    which allows the gradual replacement of a bone substitute

    Table 2 Evolution of three biomaterial generations and the correspondi

    Generation Material characteristics Tissue responses

    1st Tolerant, inert Fibrous tissue encapsulation

    2nd Responsive Bone bonding, osteoconductio3rd Instructive Bone bonding, osteoconductio

    angiogenesis, biomolecules d8748 | J. Mater. Chem., 2010, 20, 87478759as powders, granules, dense blocks, putties, pastes, gels and

    porous scaffolds, depending on the components and treatment.

    In 2008, the fastest growing bone graft substitute material was

    aterial characteristics and biological performances

    Examples

    Titanium and its alloys, alluminia, zirconia, porousceramics, thermoplastic polymers

    egradation Bioglassduction,ery, degradation

    Tissue engineered biomaterials, smart biomaterials,biomimetic biomaterials

    Table 3 Definitions of properties relevant for a bone substitute material

    Requirement Definition

    Biocompatibility The ability to perform with an appropriate hostresponse in a specific situation131

    Bioactivity The ability to form a direct chemical bond withbone and thus a uniquely strong biomaterial-strong interface132

    Osteointegration The healing process of implant placed within thebone8

    Osteoconduction The capacity to guide bone-forming tissue ona surface or down into pores9

    Biodegradability The ability to gradually vanish and leave space fornew tissue growth without hindering theregeneration process11

    Osteoinduction The ability to induce bone formation byinfluencing the differentiation or maturation ofstem cells into bone-forming cells9This journal is The Royal Society of Chemistry 2010

  • Ta

    ble

    4S

    um

    ma

    ryo

    fty

    pic

    al

    com

    mer

    cia

    lly

    av

    ail

    ab

    leb

    on

    esu

    bst

    itu

    tem

    ate

    ria

    lsu

    nti

    l2

    00

    9133

    Pro

    du

    ctC

    om

    po

    siti

    on

    Fo

    rmO

    steo

    con

    du

    ctio

    nR

    eso

    rbab

    ilit

    yO

    steo

    ind

    uct

    ion

    Ino

    rgan

    icm

    atr

    ix-b

    ase

    db

    on

    esu

    bst

    itu

    tesa

    Pro

    Ost

    eon

    500R

    Co

    rall

    ine-

    der

    ived

    HA

    /CC

    com

    po

    site

    Gra

    nu

    lar

    or

    blo

    ckX

    X

    CO

    ND

    UIT

    TC

    PG

    ran

    ule

    s1

    00

    %b

    -TC

    PG

    ran

    ule

    sX

    X

    Op

    teM

    x

    HA

    /TC

    Pb

    iph

    asi

    cco

    mb

    inati

    on

    Gra

    nu

    les,

    stic

    ks,

    rou

    nd

    edw

    edg

    es,

    wed

    ges

    an

    dcy

    lin

    der

    sin

    sev

    era

    lsi

    zes

    XX

    Ma

    ster

    Gra

    ft

    Gra

    nu

    les

    Bip

    ha

    sic

    calc

    ium

    ph

    osp

    ha

    teG

    ran

    ule

    sX

    X

    No

    ria

    n

    SR

    S

    Calc

    ium

    ph

    osp

    hate

    Inje

    ctab

    lep

    ast

    eX

    X

    No

    ria

    n

    SR

    S

    Fa

    stS

    etP

    utt

    yC

    alc

    ium

    ph

    osp

    ha

    teM

    old

    ab

    lep

    utt

    yX

    X

    chro

    nO

    S

    b-T

    rica

    lciu

    mp

    ho

    sph

    ate

    Gra

    nu

    les,

    blo

    cks

    an

    dw

    edges

    XX

    C

    EL

    LP

    LE

    X

    Tri

    calc

    ium

    ph

    osp

    ha

    teV

    ari

    ou

    ssi

    zed

    gra

    nu

    les

    XX

    O

    rgan

    icm

    atr

    ix-b

    ase

    db

    on

    esu

    bst

    itu

    tesb

    All

    oF

    use

    Hea

    tse

    nsi

    tive

    cop

    oly

    mer

    wit

    hD

    BM

    Inje

    ctab

    legel

    an

    dp

    utt

    yX

    XX

    Inte

    rGro

    DB

    Min

    ale

    cith

    inca

    rrie

    rP

    ast

    e,p

    utt

    ya

    nd

    mix

    wit

    hH

    A/C

    Cco

    mp

    osi

    tegra

    nu

    les

    XX

    X

    Op

    tefo

    rm

    DB

    Ma

    nd

    cort

    ica

    lca

    nce

    llo

    us

    chip

    ssu

    spen

    ded

    ing

    elati

    nca

    rrie

    rF

    orm

    ab

    lep

    utt

    yo

    rd

    ryp

    ow

    der

    rea

    dy

    tob

    eh

    yd

    rate

    dw

    ith

    au

    tolo

    go

    us

    dil

    uen

    tso

    rsa

    lin

    e

    XX

    X

    Op

    tefi

    lD

    BM

    susp

    end

    edin

    gel

    ati

    nca

    rrie

    rIn

    ject

    ab

    leb

    on

    ep

    ast

    e,d

    ryp

    ow

    der

    rea

    dy

    tob

    eh

    yd

    rate

    dw

    ith

    blo

    od

    or

    sali

    ne

    XX

    X

    Op

    tecu

    re

    DB

    Msu

    spen

    ded

    ina

    hyd

    rogel

    carr

    ier

    Dry

    mix

    kit

    del

    iver

    edw

    ith

    or

    bu

    ffer

    edsa

    lin

    em

    ixw

    ith

    pa

    tien

    ts

    au

    tog

    eno

    us

    bo

    ne

    gra

    fto

    ra

    uto

    log

    ou

    sd

    ilu

    ents

    XX

    X

    Op

    tecu

    re

    +C

    CC

    DB

    Ma

    nd

    CC

    Csu

    spen

    ded

    ina

    hy

    dro

    gel

    carr

    ier

    Dry

    mix

    kit

    del

    iver

    edw

    ith

    bu

    ffer

    edsa

    lin

    eo

    rm

    ixw

    ith

    pa

    tien

    ts

    au

    tog

    eno

    us

    bo

    ne

    gra

    fto

    ra

    uto

    log

    ou

    sd

    ilu

    ents

    XX

    X

    Acc

    ell

    10

    0

    DB

    Mp

    utt

    yIn

    ject

    ab

    lep

    utt

    yX

    XX

    Acc

    ell

    Co

    nn

    exu

    sD

    BM

    plu

    sre

    ver

    sep

    hase

    med

    ium

    Inje

    ctab

    lep

    utt

    yX

    XX

    Acc

    ell

    TB

    M

    To

    tal

    bo

    ne

    ma

    trix

    ,1

    00

    %p

    refo

    rmed

    Va

    rio

    us

    size

    dst

    rip

    sX

    XX

    Op

    tiu

    mD

    BM

    DB

    Mco

    mb

    ined

    wit

    hg

    lyce

    rol

    carr

    ier

    Fo

    rma

    ble

    pu

    tty

    (bo

    ne

    fib

    ers)

    an

    din

    ject

    ab

    leg

    el(b

    on

    ep

    art

    icle

    s)X

    XX

    ICG

    raft

    Ch

    am

    ber

    DB

    Mp

    art

    icle

    san

    dca

    nce

    llo

    us

    chip

    sL

    yo

    ph

    iliz

    edan

    dp

    ack

    aged

    invari

    ou

    ssi

    zes

    wit

    hin

    ad

    eliv

    ery

    cham

    ber

    XX

    X

    Cel

    lect

    DB

    M

    DB

    Mfi

    ber

    san

    dca

    nce

    llo

    us

    chip

    sP

    rovid

    edin

    asp

    ecia

    lize

    dca

    rtri

    dge

    XX

    XIN

    FU

    SE

    Bo

    ne

    Gra

    ftrh

    BM

    P-2

    pro

    tein

    on

    an

    ab

    sorb

    ab

    leco

    lla

    gen

    spo

    ng

    eM

    ult

    iple

    kit

    size

    sX

    XX

    Pro

    gen

    ix

    DB

    MP

    utt

    yD

    BM

    inT

    yp

    e-1

    bo

    vin

    eco

    lla

    gen

    an

    dso

    diu

    ma

    lgin

    ate

    Rea

    dy

    tou

    sein

    ject

    ab

    lep

    utt

    yX

    XX

    Ost

    eofi

    lD

    BM

    DB

    Min

    po

    rcin

    egel

    ati

    nIn

    ject

    ab

    lep

    ast

    ean

    dm

    old

    ab

    lest

    rip

    sX

    XX

    DB

    X

    DB

    Min

    sod

    ium

    hy

    alu

    ron

    ate

    carr

    ier

    Pa

    ste,

    pu

    tty

    mix

    an

    dst

    rip

    XX

    XG

    raft

    on

    DB

    Mco

    mb

    ined

    wit

    hG

    lyce

    rol

    Fo

    rmab

    lep

    utt

    y,

    inje

    ctab

    legel

    ,p

    utt

    ym

    ixed

    wit

    hch

    ips,

    flex

    ible

    shee

    tsa

    nd

    ma

    trix

    XX

    X

    Gra

    fto

    nP

    lus

    DB

    Mco

    mb

    ined

    wit

    ha

    sta

    rch

    carr

    ier

    Pa

    ste

    XX

    XB

    ioS

    et

    DB

    Mco

    mb

    ined

    wit

    hn

    atu

    ral

    gel

    ati

    nca

    rrie

    rIn

    ject

    ab

    lep

    ast

    e,in

    ject

    ab

    lep

    utt

    y,

    stri

    ps

    an

    db

    lock

    sw

    ith

    cort

    ica

    lca

    nce

    llo

    us

    chip

    s

    XX

    X

    VIA

    GR

    AF

    DB

    Mco

    mb

    ined

    wit

    hg

    lyce

    rol

    Pu

    tty

    ,p

    ast

    e,g

    el,

    cru

    nch

    an

    dfl

    exX

    XX

    OP

    -1

    Imp

    lan

    trh

    BM

    P-7

    wit

    hty

    pe

    1b

    on

    eco

    llagen

    Lyo

    ph

    iliz

    edp

    ow

    der

    reco

    nst

    itu

    ted

    tofo

    rmw

    etsa

    nd

    X

    X

    This journal is The Royal Society of Chemistry 2010 J. Mater. Chem., 2010, 20, 87478759 | 8749

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Online

  • it into a putty or paste without reducing the osteoconductive

    Ta

    ble

    4(Contd.)

    Pro

    du

    ctC

    om

    po

    siti

    on

    Fo

    rmO

    steo

    con

    du

    ctio

    nR

    eso

    rbab

    ilit

    yO

    steo

    ind

    uct

    ion

    OP

    -1

    Pu

    tty

    rhB

    MP

    -7w

    ith

    typ

    e1

    bo

    ne

    coll

    agen

    Lyo

    ph

    iliz

    edp

    ow

    der

    reco

    nst

    itu

    ted

    tofo

    rmp

    utt

    y

    XX

    Co

    piO

    sC

    an

    cell

    ou

    sB

    on

    eG

    raft

    Bo

    vin

    eb

    on

    eC

    an

    cell

    ou

    sch

    ips,

    can

    cell

    ou

    scu

    bes

    an

    dw

    edg

    esX

    Pu

    ros

    Dem

    iner

    ali

    zed

    Bo

    ne

    Matr

    ixD

    BM

    pu

    tty

    Pu

    tty

    XX

    XIn

    org

    an

    ic/O

    rga

    nic

    ma

    trix

    -ba

    sed

    bo

    ne

    sub

    stit

    ute

    sH

    EA

    LO

    S

    Bo

    ne

    Gra

    ftR

    epla

    cem

    ent

    Min

    era

    lize

    dco

    lla

    gen

    ma

    trix

    Va

    riet

    yo

    fst

    rip

    size

    sX

    X

    Inte

    gra

    Mo

    zaik

    80

    %h

    igh

    lyp

    uri

    fied

    B-T

    CP

    Str

    ipa

    nd

    pu

    tty

    XX

    -2

    0%

    hig

    hly

    pu

    rifi

    edty

    pe-

    1co

    lla

    gen

    Ma

    ster

    Gra

    ft

    Ma

    trix

    Ca

    lciu

    mp

    ho

    sph

    ate

    an

    dco

    lla

    gen

    Co

    mp

    ress

    ion

    resi

    sta

    nt

    blo

    ckX

    X

    Ma

    ster

    Gra

    ft

    Pu

    tty

    Ca

    lciu

    mp

    ho

    sph

    ate

    an

    dco

    lla

    gen

    Mo

    lda

    ble

    pu

    tty

    XX

    V

    ito

    ss

    10

    0%b

    -TC

    PP

    utt

    y,

    stri

    p,

    flo

    w,

    mo

    rsel

    sa

    nd

    shap

    esX

    X-

    80

    %b

    -TC

    P/2

    0%

    coll

    ag

    enC

    als

    tru

    x

    Tri

    calc

    ium

    ph

    osp

    ha

    tew

    ith

    carb

    ox

    y-

    met

    hel

    cell

    ulo

    seM

    old

    ab

    lep

    utt

    yX

    X

    Co

    piO

    sB

    on

    eV

    oid

    Fil

    ler

    Dib

    asi

    cca

    lciu

    mp

    ho

    sph

    ate

    an

    dT

    yp

    eI

    coll

    ag

    enS

    po

    ng

    ea

    nd

    pa

    ste

    XX

    ab

    -TC

    P-b

    -tri

    calc

    ium

    ph

    osp

    hate

    ,H

    A/C

    C-

    Hyd

    roxyap

    ati

    te/c

    am

    ciu

    mca

    rbo

    nate

    .b

    CC

    C-

    Co

    rtic

    al

    can

    cell

    ou

    sch

    ips,

    DB

    M-

    Dem

    iner

    ali

    zed

    bo

    ne

    matr

    ix.

    8750 | J. Mater. Chem., 2010, 20, 87478759

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlineproperties. The success of DBM is mainly due to cost-effective-

    ness and availability from human tissue banks.13

    Within the bone substitute material market, ceramics also hold

    a prominent position. Biocompatibility and osteoconductive

    properties provide ceramics with desirable qualities as implant

    materials for different applications throughout the body,

    covering all areas of the skeleton, such as bone fillers, treatment

    of bone defects, fracture treatment, orthopedics, and cranio-

    maxillofacial reconstruction.16,17

    Although limited in extent, few current commercially available

    bone substitute materials belong to 3rd generation biomaterials.

    The limited number of currently commercially available 3rd

    generation biomaterials for bone substitute include materials

    endowed with high porosity and interconnectivity to allow

    attachment of bone-forming cells, nutrient/oxygen infiltration

    and vascularization, and high resorbability that permits better

    imaging and visualization of the healing process (e.g. CopiOs

    Bone Void Filler). In addition to osteoconductive properties, the

    supplementation of bone substitute materials with an autologous

    bone marrow aspirate (BMA) can provide osteoinductive prop-

    erties necessary for bone ingrowth. Finally, also growth factors,

    such as recombinant human bone morphogenetic protein 2

    (rhBMP-2) and rhBMP-7 (or Osteogenic protein 1, (OP-1)), have

    been incorporated into scaffolds (INFUSE Bone Graft, OP-1

    Implant and OP-1 Putty) to be released in a temporally and

    spatially controlled manner and to confer osteoinductive prop-

    erties by actively recruiting stem cells from surrounding tissue

    and blood, initiating the bone formation process.

    2.2 Current development for 3rd generation bone substitute

    materials

    Despite recent achievements, the majority of 3rd generation

    synthetic bone substitute materials is at research or test level and

    many improvements are still necessary before application in

    a clinical setting becomes feasible. Biomaterials science is an

    interdisciplinary field, in which many experts (e.g. mechanical

    engineers, material scientists, chemists, biologists, medical

    doctors, and surgeons) collaborate, expanding knowledge about

    composition, microstructure, properties, biological performance

    and ability to control these characteristics. Material researchers

    can now analyze and manipulate material properties in ways that

    were hard to imagine just a few years ago.demineralized bone matrix (DBM) allograft (data from Euro-

    pean Markets for Dental Bone Graft Substitutes and Other

    Biomaterials 2009, Published Sep 2009 by iData Research, Inc.:

    www.mindbranch.com). DBM possesses acknowledged osteo-

    conductive and osteoinductive properties.13 Nevertheless,

    different methods of preparation, carrier, sterilization tech-

    niques, storage, and donor specifications determine significant

    differences in osteogenic potential between the product formu-

    lations.14,15 DBM is produced alone (Accell 100, PurosDBM)or in combination with osteocompatible carriers, such as glycerol

    (Optium DBM, Grafton), gelatin (Opteform, Optefil

    OsteofilDBM, BioSet), collagen (Progenix DBM Putty),lecithin (InterGro) and hyaluronic acid (DBX), which are all

    intended to increase the handling properties of DBM by turningThis journal is The Royal Society of Chemistry 2010

  • structures to allow bone ingrowth as well as biodegradability for

    a rapid turn-over to newly-formed osseous tissue.21 The stiffness

    and strength should be appropriate to offer an environment

    suitable for cell growth and, in the same time, allow the material

    itself to withstand the tractional forces and adapt to changes of

    surrounded tissue.18 Fig. 1 shows the possible strategies that can

    be adopted to achieve these instructive goals for the design of

    ideal 3rd generation bone substitute materials suitable for

    medical applications.

    2.2.1 Ceramic-based materials. Ceramic materials began to

    gain extreme popularity for biomedical applications in the late

    1960s. The huge advance in knowledge and technology during

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article OnlineThe next step for the development of 3rd generation bone

    substitute materials will consist of the integration of more far-

    reaching and powerful requirements into higher-level systems

    that instruct the biological surroundings into a desired response

    upon implantation. Indeed, future bone substitute materials will

    intervene actively to instruct the physiological environment

    toward desired biological responses (e.g. bone formation and

    blood vessels formation), for a real integration of the materials

    within the native bone.18 Hence, up-to-date approaches to meet

    these requirements include drug delivery systems as well as

    bioactive molecule carriers capable of triggering new bone tissue

    formation.19,11 Challenges in the field of bone substitute materials

    are to create gene-based or, even more ambitious, cell-based

    scaffolds to act as an additional source in vivo and to facilitate the

    remodeling process. However, the scarce availability of autolo-

    gous cells, risks of cell transplantation and several impediments

    of 3D scaffolds, such as biophysical complexity and cell-matrix

    interactions, make this strategy still too difficult to define in

    practice as well as time-, money- and labor-consuming.20

    Fig. 1 Current strategies to achieve 3rd generation bone substitute

    material requirements.Besides biochemical composition, there is growing apprecia-

    tion of the importance of other properties, including mechanical

    performance, mechanical durability and physical properties in

    guiding biological responses and providing the best chance for

    success. A bone substitute material should supply both sufficient

    mechanical stability to maintain structural integrity throughout

    the period of bone remodeling and interconnected porous

    Table 5 Chronological order of important milestones in the development of

    Date Event

    1960s Characterization of carbonate substituted HA (CHA) for bioapplication

    1967 Bioglass

    Early 1970s First bone bonding in bioactive glass ceramics1982 Apatite-wollastonite (A-W) glass-ceramic1980s Plasma-sprayed hydroxyapatite implants1980s Calcium phosphate bone cements1980s1990s Use of hydroxyapatite (HA) for implantation1980s1990s Use of calcium phosphate as a filler in polymer-matrix compo

    This journal is The Royal Society of Chemistry 2010the course of the last 40 years brought ceramics to be universally

    appreciated in terms of biocompatibility, osteoconductivity and

    bioactivity due to the close chemical and structural resemblance

    to the mineral phase of bone tissue.22 The historical landmarks of

    the development of ceramics for biomedical applications over the

    past four decades are summarized in Table 5.

    Currently, the most common types of calcium phosphate

    ceramics used as synthetic materials are porous hydroxyapatite

    (HA), b-tricalcium phosphate (b-TCP), their derivatives and

    their combinations, such as biphasic calcium phosphate ceramics

    (BCP).11,19 Calcium phosphate ceramics have consistently

    demonstrated an excellent tissue response in vivo, but some

    drawbacks of crystalline calcium phosphates, such as inherent

    brittleness and relatively slow degradation, limit their clinical

    application as synthetic bone substitute materials.23

    Current strategies for the fabrication of 3rd generation

    ceramic-based bone substitute materials will be discussed below

    according to modern requirements (as listed in Fig. 1) that are

    indispensable for a successful regeneration of bone tissue.

    Osteoinduction. Although the exact mechanism of osteoin-

    duction by calcium phosphate bioceramics is largely unknown,24

    the induction of bone formation at non-osseous sites in animals

    without the addition of any osteoinductive biomolecules is

    demonstrated to be feasible with calcium phosphate

    ceramics.25,26 These calcium phosphate materials have included

    synthetic porous hydroxyapatite, coral derived hydroxyapatite,

    a-tricalcium phosphate, b-tricalcium phosphate, biphasic

    calcium phosphate, a-pyrophosphate and b-pyrophosphate

    ceramics, calcium phosphate cements and calcium phosphate-

    containing biomaterials coatings.24 Their intrinsic osteoinductive

    capacity was proposed to be related to several design parameters

    (e.g. topography, geometry, composition, macroporosity and

    ceramics for biomedical applications during the past 40 years

    Pioneers

    medical LeGeros R.Z.134

    Hench L.L.135

    Hench L.L.136,137

    Kokubo T.138

    De Groot K.139

    Ferdandez E.,140,141 Chow L.C.142

    De Groot K., Jarcho M., Driessens F., Bonfield W.143

    sites Bonfield W.144J. Mater. Chem., 2010, 20, 87478759 | 8751

  • Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlinenano/microporosity) that allow entrapment and concentration of

    bone inducing substances and/or immature bone-forming cells

    from the biological surroundings onto the ceramic surface.2729

    Besides intrinsic osteoinductive activity of bioceramic mate-

    rials, engineered osteoinductivity can be introduced by grafting

    bioactive proteins, osteoinductive growth factors30 and/or bone-

    forming cells31,32 into the traditional bioactive materials. When

    cell binding-proteins, which allow cell attachment, are absorbed

    (e.g. fibronectin,28 vitronectin,33 fibrinogen and trombin34) or

    covalently or physicochemically incorporated (e.g. type I

    collagen35,36) onto the surface of bioceramics, these have been

    shown to improve the biological properties of these hybrid/

    composite materials.

    The most important growth factors in bone formation and

    healing are bone morphogenetic proteins (BMPs), belonging to

    the transforming growth factor (TGF)-b-superfamily. The

    primary function of BMPs is to induce the maturation of bone

    forming-cells through the binding to specific cell receptors. As

    a result, intracellular signaling will direct a gene-specific response

    that will transform the immature cell into a bone-forming cell.

    Several studies have demonstrated the powerful capacity of

    BMPs incorporated into bioceramic materials to provoke

    modification in the behavior of immature bone forming-cells,

    which are recruited to the site of injury and enhance new bone

    formation.37,38

    The delivery or presentation of growth factors to the biological

    surrounding is a critical step for engineered osteoinductive

    ceramics. Enrichment with growth factors can be based on

    simple adsorption,39,40 but more sophisticated entrapment

    approaches have been proposed, predominantly with the aim to

    prolong and intensify the exposure of the biological surround-

    ings to these factors.41,42

    The relatively high-strength and osteoconductive calcium

    phosphate cements (CPC), either in prefabricated blocks or

    injectable formulations, have been shown to be an effective

    delivery vehicle for growth factors to promote bone regeneration.

    Prefabricated block ceramics are commonly loaded with growth

    factors by adsorption,39,43 while growth factor loaded-polymeric

    microspheres (e.g. PLGA44 or gelatin38) can be incorporated

    inside injectable CPC as delivery vehicles. The clinical relevance

    of such microsphere structures is that these matrices can serve as

    appropriate carriers to control the release and maintain thera-

    peutic levels of diffusible growth factors at the repair site.

    Angiogenesis. Scaffolds not only provide a substrate onto

    which host cells can grow, but they can also be used to guide

    tissue formation, such as bone (see previous section) and

    vascular structures that provide nutrient and oxygen supply.45

    The exact requirements for a scaffold to induce or promote

    vascular tissue formation are still in part unsolved. Challenges

    in bone substitute materials research address to promote

    angiogenesis either by matching angiogenic growth factors,

    endothelial cells (i.e. cells that line the inner side of the blood

    vessels), or other biological components to a tissue engineered

    construct, or by defining an appropriate porosity within a scaf-

    fold. Recently, the constant release at low concentrations of

    vascular endothelial growth factor (VEGF) from biphasic

    calcium phosphate (BCP) ceramics has been shown to be

    beneficial for blood vessel in-growth and thus for bone8752 | J. Mater. Chem., 2010, 20, 87478759formation.46 Alternatively, inorganic copper ions incorporated

    within calcium phosphate scaffolds showed angiogenetic

    potential and represent an attractive alternative to the use of

    costly and complex proteinaceous factors.47

    Besides having an important effect on osteoinductive proper-

    ties, interconnecting macroporosity and microporosity of the

    scaffold are essential to allow vascularization. Pore size must be

    sufficient to provide efficient in vivo blood vessel formation

    within bioceramic scaffolds.19

    Mechanical properties. The development of ceramic bone

    substitute materials that show both good biological and

    mechanical performance is desired. However, the major short-

    coming of bioceramics relates to their inherent poor mechanical

    properties (i.e. brittleness) and non-matching to the elastic

    behavior of bone tissue. Different strategies, including sintering

    methods and biocomposite materials with polymers, can be

    adopted to confer favorable mechanical performance of

    ceramic scaffolds without compromising the biological prop-

    erties.

    Sinteringprocedures, inwhichagranularmaterial is subjectedto

    a thermal treatment with the purpose of increasing its strength,

    appear to be of major importance to produce bioceramics with

    appropriate mechanical properties.19 Sintering temperature is

    correlatedtodifferentscaffoldparameters,suchasdensity,porosity,

    grain size, chemical composition and strength. In a recent study,

    hydroxyapatite disks with four different porosities sintered at

    different temperatures (900,1100,1200or1250 C)showeddistinctmechanical properties, including elastic modulus and hardness.

    Indeed, with increasing sintering temperature, the bulk density,

    grain sizes and elastic modulus increased whilst the total porosity

    decreased.48

    Alternatively, in order to improve the mechanical properties of

    calcium phosphate bioceramics, ceramic-polymer composites

    have been synthesized.49 Composites can combine the advan-

    tages of both materials, namely (i) strength via a ceramic phase,

    and (ii) toughness and plasticity via a polymer phase. In analogy

    with living bone, which is a composite matrix of mainly collagen

    and biomineral (bone apatite crystals),27 the incorporation of

    polymer components within ceramic materials can provide

    improved elastic behavior of the bone substitute material.

    Substantial research efforts have already been focusing on the

    development of ceramic-polymer composites for bone graft

    applications, in which the major challenge relates to obtaining

    a good chemical and/or physical bond between the polymer and

    the ceramic phase. The most commonly used resorbable

    synthetic polymers in combination with ceramics materials are

    saturated poly-a-hydroxy esters, including (poly(lactic acid)

    (PLA) and poly(glycolic acid) (PGA), their copolymer (poly-

    (lactic-co-glycolide) (PLGA) and poly-e-caprolactone (PCL).

    These polymers can be mixed with ceramic scaffolds using

    different methods, such as porogen leaching, gas expansion,

    emulsion drying and, more recently, freeze-drying via thermally

    induced phase separation (TIPS).50

    Biodegradability. Development of bone substitute materials

    for the regeneration of living tissues leads to the requirement for

    higher resorbability, especially when combined with the release

    of pharmaceuticals to enhance tissue regeneration. AlthoughThis journal is The Royal Society of Chemistry 2010

  • bioresorbable ceramics are gradually degraded in bony defects,

    there is still need for ceramic scaffolds with higher resorbability.

    From a material point of view, the degradation speed of calcium

    phosphate ceramics depends on composition,51 crystallinity,

    porosity and preparation conditions. Modulation of these

    parameters improves the biodegradability of ceramic materials

    and facilitates new bone to invade the defect site.

    Modern strategies, including nanotechnology, have demon-

    strated the ability to reproduce superior structural properties

    compared to currently used implants, by creating materials that

    mimic the natural nanostructure of bone tissue.52 For instance,

    nanocrystalline calcium phosphates have demonstrated faster

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlinedegradation and enhanced bone cell functions compared to

    micron grain size calcium phosphate.53

    Porosity and interconnected pores within the scaffolds are also

    essential components that are introduced within bioceramic

    materials in order to enhance resorbability.54 Especially, gener-

    ating pores in injectable matrices is a current topic of research, in

    which investigators are examining fast-degrading particles that

    can dissolve within a short time after injection and hardening,

    leaving a porous but rigid structure behind.55 It has been

    reported that the optimal pore size for successful cell infiltration

    and bone tissue ingrowth is 100350mm.56 A number of fabri-

    cation techniques have been developed over the years for

    manufacturing porous bioceramics, according to the various

    physical forms (e.g. block, granular form, putty or paste).19 The

    most common techniques are incorporation of pore-creating

    additives (porogens),57,54,56 such as naphthalene, H2O2, poly-

    meric microspheres, or foaming methods.58 Habraken et al.

    conducted research on incorporation of different types of poly-

    meric microspheres (e.g. gelatin59,60 and PLGA61,62) within

    calcium phosphate cement to induce porosity, through the

    degradation of the polymeric microspheres, and to improve the

    degradation rate of the materials. The main advantage of these

    materials is that they degrade hydrolytically, resulting in

    a breakdown of the polymer chain, hence leading toward

    a controllable (bio)degradation. However, control of degrada-

    tion rate is a contributing factor to the success of bone substitute

    materials. High porosity and a too fast degradation rate may

    result in the loss of the surface that is needed for further facili-

    tation of bone formation. Therefore, an optimal balance between

    high surface reactivity and a low dissolution rate is required to

    regulate the in vivo bioresorbability of calcium phosphate

    ceramics.

    Handling properties. The clinical application of bone substi-

    tute materials requires optimal handling properties for reliable

    Fig. 2 Examples of injectable bone substitute materials: (A) CPC

    injection for maxillary sinus floor elevation in a goat model; (B) hydrogel

    applied in the medullar cavity of tibia in a guinea pig model.This journal is The Royal Society of Chemistry 2010and safe clinical application. Preparation of the bone substitute

    materials and implantation at the defect site should be simple,

    fast, and standardized under all conditions without surgeon-

    dependent variations.

    The current ceramic bone substitute materials are mainly

    available in dense or porous blocks or granules. However,

    ceramics in the hardened form are not easy to handle and to

    maintain in the surgical sites thus leaving empty spaces between

    bone tissue and filling material. Moreover, surgeons need to fit

    the surgical site around the implant or to carve the graft to the

    desired shape, increasing the probability of bone loss, trauma to

    the surrounding tissue and surgical time.

    Moldability of ceramic-based bone substitute has been intro-

    duced with the development of putty-like materials, that can be

    quickly sculptured during surgery in situ to provide intimate

    adaptation to the contours of defect surfaces.63 Additives, such as

    accelerants, cohesion promoters, and fluidificants have been used

    to improve workability, and setting and hardening properties.64

    For instance, the combination of fibrin glue with ceramic gran-

    ules has been used as a strategy to stabilize the material at the

    implantation site and produce a composite that can be molded

    into the defect without leaving empty spaces.65,66

    Even more sophisticated is the introduction of injectability

    with the development of calcium phosphate cement (CPC) paste

    that can be easily applied into the bone defects by using a syringe

    (Fig. 2A). The injectability of CPC is also of critical importance

    for use in minimally invasive techniques. The advantages of an

    injectable CPC include: (i) shortening the surgical operation

    time; (ii) minimizing the damaging effects of large muscle

    retraction; (iii) achieving optimal defect filling; (iv) reducing

    postoperative pain and scar size; (v) achieving rapid recovery;

    (vi) reducing cost.63 CPC consists of a powder containing one or

    more solid compounds of calcium and/or phosphate salts and

    a cement liquid that can be water or an aqueous solution.67 The

    powder and the liquid are mixed in an appropriate ratio prior to

    implantation to form a paste that subsequently hardens at body

    temperature within several minutes.55 Once injected, CPC form

    a low-crystallinity hydroxyapatite that can be adsorbed over

    time.

    2.2.2 Polymer based-materials. Polymers are increasingly

    gaining interest for the development of scaffolds for tissue

    engineering and regenerative medicine applications compared to

    other bone substitute materials, such as ceramics, because of

    their versatility in terms of chemical and physical properties.52

    Polymer based-materials mainly boast design flexibility associ-

    ated with a wide range of mechanical properties that make these

    materials to be easily manipulated into desired shapes and

    structures and to be degraded at a controlled rate in concert with

    tissue regeneration.68 In addition, polymers can be easily steril-

    ized without causing any chemical change and have the capacity

    to incorporate biological matrix components.

    The polymeric materials used in the biomedical field and, more

    specifically, in tissue engineering are of natural or synthetic

    origin. Natural polymers are categorized into proteinaceous

    polymers (e.g. collagen, gelatin, silk) and polysaccharidic poly-

    mers (e.g. chitosan, alginate, starch, and hyaluronic acid deriv-

    atives).69 Due to their extracellular matrix (ECM)-like properties

    suitable for cell survival and function, and low toxicity, naturalJ. Mater. Chem., 2010, 20, 87478759 | 8753

  • polymers are attractive materials for designing biocompatible,

    biodegradable and bioactive scaffolds. Nevertheless, natural

    polymers suffer from disadvantageous properties, including low

    mechanical strength and high degradation rates that limit their

    use for bone tissue replacement.

    On the other hand, synthetic polymers are appealing, and have

    a number of advantages over both natural polymers and other

    bone substitute materials. The chemical versatility of synthetic

    polymers permits the fabrication of materials and scaffolds in

    different forms as well as with variation in porosity and pore

    sizes, degradation kinetics and mechanical properties. Although

    synthetic polymers do not possess functional groups to stimulate

    cell adhesion, such groups can be easily introduced by the

    incorporation of bioactive molecules or protein sequences able to

    induce specific cellular responses. Moreover, synthetic polymer

    According to the mechanism of the crosslinking reaction,

    hydrogels can be classified into two main categories: (i) chemi-

    cally crosslinked hydrogels and (ii) physically crosslinked

    hydrogels.77 The so-called chemical gels are crosslinked by

    chemical interactions, including covalent (irreversible) or ionic

    (reversible) bonds,78 through the action of crosslinking agents

    under thermal,79 redox80 or photoinitiating81 (i.e. visible or UV

    light) conditions or enzymatic reactions.82,83 Chemical hydrogels

    have relatively strong and stable mechanical properties.

    However, high concentrations of initiators and crosslinking

    agents may compromise cell viability and be detrimental for

    surrounding tissues as a result of side-effect reactions. Unlike

    chemically crosslinked hydrogels, physical hydrogels lack

    crosslinking agents and thus polymerize spontaneously through

    a number of non-covalent interactions under physical stimuli,

    itro

    itropeppep

    ene

    ng d

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlineproduction can also be more easily scaled-up than natural

    polymers.70 The most frequently used biodegradable synthetic

    polymers for scaffolds in bone tissue engineering approved by the

    FDA are saturated poly-a-hydroxy esters, including (poly(lactic

    acid) (PLA) and poly(glycolic acid) (PGA), as well as (poly-

    (lactic-co-glycolide) (PLGA) copolymers.71

    Hydrogels. In addition to solid polymeric bone substitute

    materials, hydrogel-forming polymers are an emerging and

    physiologically relevant class of biomaterials. Hydrogels are

    a network of crosslinked polymer chains, which are able to swell,

    but not to dissolve, in aqueous solution.72 These properties

    confer native tissue-like viscoelasticity and provide a suitable

    physiologic micro-environment to house cells until the onset of

    ECM production by these cells.73 As early as 1960, hydrogels

    started to be used as biomaterials due to their major advantages,

    such as biocompatibility, permeability and ductile physical

    properties.74 However, only in the last 510 years hydrogel

    research has undergone sharp development, in which research

    interest has shifted from conventional prefabricated hydrogels,

    whose implantation requires large incisions (cuts), to injectable

    formulations that rapidly allow gelation under physiological

    conditions once injected into the body.75 Besides a minimally-

    invasive implantation procedure, these types of materials allow

    the incorporation of pharmaceutical agents by simply mixing

    them before injection.76 Following gelation, these gel-like

    matrices release their content at the target site triggering tissue

    regeneration.

    Table 6 Hydrogel functionalizations with different adhesive peptides

    Hydrogel Synthetic sequence Origin

    PEG RGD Fibronectin, v

    PEODA RGD Fibronectin, vOPF GRGD RGD derivedpHEMA RGDSK RGD derivedOPF DVDVPDGRGDSLAYG Osteopontin

    PLEOF KIPKA SSVPT ELSAI STLYL Bone morphog

    P(NIPAAm-co-AAc) FHRRIKA Heparin bindi

    PEGDM RGDS and Heparin8754 | J. Mater. Chem., 2010, 20, 87478759such as changes in temperature, pH, electric field, salt or ionic

    environment.77 For this reason, physical hydrogels can also be

    called self-assembling hydrogels.84

    Similar to ceramic based-materials, advanced strategies are

    being explored for the development of 3rd generation polymer-

    based materials. An overview of these strategies is provided

    below, listed by the criteria indicated in Fig. 1.

    Osteoinduction. New generation polymeric-based materials

    are engineered to direct the host cells to adhere to and degrade

    the synthetic matrix, and ultimately to regenerate new bone at

    the defect site. Release of biological signals from polymeric

    scaffolds or gene therapy, based on the localized delivery of

    naked DNA plasmid encoding osteoinductive proteins, are the

    current strategies to achieve these goals.

    Foremost, cell adhesion to the ECM or to a scaffold, through

    binding between specific cell membrane receptors and corre-

    sponding cell-adhesion-molecules, is an imperative requirement

    for survival and cell-cycle progression of certain types of cells, i.e.

    anchorage-dependent cells, which include bone-forming cells.8587

    Cell adhesion is a 4-step process that includes (1) initial cell

    attachment, (2) cell spreading, (3) cytoskeletal organization and

    (4) confocal adhesion formation. Cell attachment and cell

    adhesion are often used indistinctly, however, from the defini-

    tion it is evident that the first is just one part of the entire

    process.88 Although some hydrogels show appropriate structural

    characteristics suitable for a scaffold, they do not contain cell-

    adhesion-molecules and, hence do not support cell survival. To

    Functions Ref.

    nectin, collagen Osteoblast adhesion, proliferation andmineralization

    145

    nectin, collagen Osteoblast differentiation and mineralization 146tide Osteoblast adhesion 94tide Osteoblast adhesion and proliferation 147

    Osteoblast adhesion, proliferation, migrationand differentiation

    91,93

    tic protein-2 Osteoblast adhesion, proliferation anddifferentiation

    96

    omain Osteoblast adhesion, spreading andmineralization

    148

    Osteoblast viability and differentiation 97This journal is The Royal Society of Chemistry 2010

  • Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlineovercome this drawback, several modifications of design

    parameters have been made by chemical or physical entrapment

    of biological cues, ranging from inorganic minerals, such as

    calcium phosphate nanocrystals,89 to adhesive long or short

    ECM peptide sequences, derived from natural proteins. The

    resulting materials, containing biomimetic components, ensure

    cell-matrix interaction in order to control cell behavior and,

    subsequently, promote bone tissue growth. One successful

    strategy to improve the viability and activity of bone-forming

    cells in hydrogel scaffolds is the use of small peptides that acti-

    vate receptor-mediated cellular responses. The most commonly

    used peptide is the amino acid triplet RGD (Arg-Gly-Asp),

    which is the predominant ligand for cell-surface receptors (i.e.

    integrins) to achieve cell attachment. The RGD-sequence is

    abundantly present in many proteins that can be found in the

    extracellular matrix (e.g. fibronectin, laminin, fibrinogen,

    collagen). The RGD-sequence and other amino acid sequences,

    such as IKVAV and YIGSR from laminin, REDRV and LDV

    from fibronectin and DGEA from collagen I, have been cova-

    lently coupled to different types of hydrogel polymers that lack

    cellular adhesion72 (Table 6). Among synthetic hydrogels,

    poly(ethylene glycol) (PEG)- based polymer gels have been

    used abundantly because of their proven safety and FDA

    approval.90 However, due to inherent cell repellent properties (as

    a result of poor protein adsorption) PEG-based hydrogels have

    been functionalized through the incorporation of cell-binding

    peptides to modulate cell adhesion, morphology and migra-

    tion.9194

    The adhesion to synthetic peptide sequences is not always

    associated with changes in cytoskeletal organization and cellular

    behavior and thus in cell-cycle progression. More essential,

    models that ensure not only cell attachment but also succession

    of all the downstream biological pathways involved in cell

    function activation are required.85,86 According to the above

    consideration, synergism between adhesive proteins and selec-

    tion of bioactive molecules may be necessary to stimulate desir-

    able biological events.95 In a recent study, an RGD-sequence and

    a peptide derived from recombinant human bone morphogenetic

    protein (rhBMP-2) have been grafted in Poly(lactide-co-ethylene

    oxide- co-fumarate) (PLEOF) hydrogel. While RGD-sequences

    mediated cellular adhesion to the hydrogels, BMP-peptide

    promoted maturation of bone-forming cells and mineraliza-

    tion.96 Next to RGD-sequences, another extensive class of cell

    binding domains, as used to enhance cell adhesion into polymer

    based-materials, is represented by heparin, due to its intrinsic

    ability to enhance the osteogenic activity of some growth factors

    involved in bone formation.97

    The delivery of osteoinductive agents, such as the above-

    mentioned BMPs98,99 or other novel osteogenic proteins (e.g.

    Nell-1),100 has significantly enhanced the bone regenerative

    capacity of materials in animal models. However, the main

    concern of researchers is how to deliver these molecules at the

    appropriate site and how to maximize the therapeutic potential

    and efficacy. An effective strategy involves the encapsulation of

    osteoinductive factors within micro- or nanoparticles, subse-

    quently incorporated in the scaffold to be released to the local

    microenvironment in a controlled fashion by diffusion and/or

    scaffold erosion or degradation mechanisms.101 Although growth

    factor delivery has demonstrated to be a promising strategy, thisThis journal is The Royal Society of Chemistry 2010application can be limited by protein purification, short in vivo

    stability, low activity and prohibitive costs. Another attractive

    route to control the release of molecules stimulating bone growth

    at the site of interest is gene therapy via a DNA plasmid vector

    that integrates into the cells residing in the tissue. Thereby, the

    delivery of plasmid DNA promotes local protein production by

    the transfected cells.102 The success of therapeutic gene delivery,

    however, requires a system that both guarantees high level of

    transfection efficiency and long-term gene expression to produce

    physiological responses that lead to tissue formation.103 This has

    been shown in the context of a cranial critical-sized defect model

    in which PEI-condensed plasmid DNA encoding for BMP-4

    from macroporous PLGA scaffolds enhanced bone regeneration.

    This system provided a platform that potentially allows imma-

    ture bone-forming cells or other cell types (e.g. fibroblasts)

    surrounding the defect area to migrate into the defect site and to

    be transfected by the plasmid DNA associated with the scaf-

    fold.104 In another study, naked DNA plasmid encoding for

    vascular endothelial growth factor (pVEGF165) has been incor-

    porated into collagen/calcium phosphate scaffolds and has

    shown a significant increase in bone formation upon implanta-

    tion in an intra-femoral mouse model.105 Hence, DNA-present-

    ing polymeric surface gains long-term osteoinductivity by

    affecting the behavior of the cells that come in to contact with the

    material.

    Angiogenesis. As mentioned above, angiogenesis plays

    a pivotal role in skeletal development and bone fracture

    repair.45 Growth factor therapy as well as gene delivery from

    polymeric scaffolds are under evaluation and development to

    promote angiogenesis and thus to actively stimulate bone

    regeneration.

    Approaches involving single growth factor release by poly-

    meric vehicles (e.g. collagen106 or gelatin microparticles107) that

    allow for a localized and controlled delivery in the bone wound

    healing environment, have shown promising results on the

    formation of blood vessels. Although vascular endothelial

    growth factor (VEGF) is an important initiator of angiogenesis,

    the delivery of VEGF alone may lead to immature and leaky

    vasculature with poor function.108 Therefore, dual release

    approaches provide a more powerful tool to study and manip-

    ulate a wide array of developmental and regenerative processes.

    The addition of both fibroblast growth factor-2 (FGF-2) and

    VEGF to collagenheparin scaffolds led to an increased angio-

    genesis and blood vessel maturation at an earlier time point than

    with either FGF-2 and VEGF alone.109 Similarly, the interplay of

    VEGF and BMP-2 released from gelatin microparticles confined

    within porous poly(propylene fumarate) (PPF) scaffold resulted

    in a synergistic response to produce increased bony bridging and

    bone formation within a rat critical size defect model compared

    to delivery of BMP-2 alone.110 In another experiment, PLGA

    scaffolds containing combinations of condensed plasmid DNA

    encoding for BMP-4, VEGF, and human bone marrow stromal

    cells (hBMSCs) significantly enhanced bone formation compared

    to any single factor or combination of two factors.111

    More interestingly, therapeutic angiogenesis might be further

    improved by the sequential release of growth factors delivered

    by a biodegradable polymer, which achieves distinct release

    kinetics for each factor. A composite consisting of BMP-2J. Mater. Chem., 2010, 20, 87478759 | 8755

  • Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlineloaded-PLGA microspheres embedded in a PPF scaffold sur-

    rounded by a VEGF loaded-gelatin hydrogel (microspheres/

    PPF/gelatin composites) was implanted in a subcutaneous and

    femoral defect of rat model and used for the sequential release

    of the growth factors. The study demonstrated a significant

    contribution of VEGF to subcutaneous bone formation induced

    by BMP-2, whereas no effects of VEGF were observed in the

    bony defect.112

    Mechanical properties. The use of synthetic or natural polymer

    matrices with low mechanical properties and fast degradation

    kinetics results in bone substitute materials with high biological

    activity but poor structural properties, in particular low strength

    and stiffness.18 Polymers with better mechanical properties can

    be fabricated by altering design parameters or by incorporating

    organic or inorganic reinforcements within the polymeric matrix

    that increase strength and toughness.

    Anseth indicated several parameters that influence the

    mechanical properties of hydrogels: (i) the polymer composi-

    tion, (ii) the cross-linking density and (iii) the degree of

    swelling.113 However, it must be considered that changes of

    these parameters in the polymer will affect not only the

    mechanical properties but also other general material behavior.

    Temenoff and co-workers showed the possibility of altering

    crosslinking density and therefore mesh size (i.e. the free space

    between cross-linked macromolecules) of oligo (poly (ethylene

    glycol) fumarate) (OPF) hydrogels by changing the molecular

    weight of the polymeric chains.114 As a consequence, even

    physical properties such as water-adsorbable performance,

    mechanical strength, degradability and diffusivity were affected.

    Nevertheless, matching all the qualities suitable for both a given

    application and environmental conditions requires many

    attempts and efforts as some properties may affect others.115

    For example, hydrogels with high mechanical strength provide

    more stability, however swelling and diffusion are limited. On

    the other hand, lower mechanical strength guarantees higher

    swelling and thus a better molecular trafficking, oxygen supply

    and nutrient/waste transport. Therefore, it is imperative to find

    a compromise between mechanical properties, swelling and

    mass transport properties.116

    Besides altering crosslinking density, a variety of processing

    technologies have been developed to fabricate porous 3D poly-

    meric scaffolds for bone regeneration. These techniques mainly

    include solvent casting and particulate leaching, gas foaming,

    emulsion freeze-drying, electrospinning, rapid prototyping, and

    thermally induced phase separation (TIPS). Further details on

    the fabrication of polymeric scaffolds are available elsewhere.68

    Other strategies to increase the mechanical properties of poly-

    meric based-materials involve (i) blending polymer with other

    polymer(s) or (ii) reinforcement of the polymer matrix by

    minerals or, more recently, by carbon nanotubes117 or carbon

    nanoparticles.118

    Polymeric microspheres embedded within PLGA matrix have

    shown to tailor compressive strength as well as porosity and

    interconnectivity of a polymeric scaffolds.119 Likewise, polymer

    based-composites, such as nanocrystallites of inorganic biolog-

    ical compounds dispersed within polymer matrices or on the

    surface of the composites, can be designed to improve the

    mechanical performance and tissue integration and to provide8756 | J. Mater. Chem., 2010, 20, 87478759a suitable microenvironment that mimics the host tissues inor-

    ganic phase.89;120

    Liu et al. fabricated nanofibrous (NF)-gelatin/apatite

    composite scaffolds to mimic both physical architecture and

    chemical composition of natural bone ECM. Firstly, thermally

    induced phase separation (TIPS) was integrated with porogen

    leaching technique to fabricated (NF)-gelatin scaffolds with well

    defined micropores. Subsequently, bone-like apatite was incor-

    porated onto the surface of NF-gelatin scaffolds. The resulting

    biomimetic scaffolds showed high porosity, interconnectivity

    and good mechanical strength.121

    Biodegradability. Biodegradable polymers as bone substitute

    materials need to possess appropriate degradation rates to match

    new tissue formation under physiological conditions and non-

    toxic and neutral degradation products. The degradation of

    a polymer occurs (i) by absorption of water followed by hydro-

    lysis of the unstable bonds (hydrolytic degradation), (ii) by

    metabolic (i.e. cellular or enzymatic) pathways or (iii) dissolu-

    tion. The resulting non-toxic degradation products are metabo-

    lized and more easily excreted from the body compared to

    ceramics.71

    As illustrated for mechanical properties, biodegradability is

    influenced by design factors of polymers, including polymeric

    composition, molecular weight, the ratio of contents, pH, crys-

    tallinity and hydrophobicity.122 Therefore, control over these

    parameters determine the degradability and the tissue reaction

    that ultimately regulate bone formation. However, some poly-

    mers degrade too fast compared to the formation of new tissue,

    hindering the bone healing process. Degradation can be slowed

    down by chemical modifications. However, such modifications

    can lead to tissue toxicity and decrease biocompatibility.

    It has been only five years since stimuli-responsive nano-

    structured polymer materials have been developed. These

    sophisticated materials, also called smart materials, display

    a dramatic physicochemical change in response to small changes

    in their environment such as temperature, pH, solvent, magnetic

    field, ions, pressure, light, and magnetic fields.123 For example,

    degradable hydrogels have been designed by the addition of

    photolabile groups (e.g. nitrobenzyl ether-derived moiety124 or

    photoreactive acrylate group125) attached to the backbone of

    hydrophilic chains. Chemical and physical properties of these

    materials are tunable spatially and temporally with UV light even

    in the presence of entrapped cells. Gel degradation is externally

    triggered and directed upon light exposure by photolytic

    cleavage and removal of photosensitive macromolecules that

    compose the gel.

    Moreover, the development of smart biodegradable polymer-

    based injectable drug delivery systems has gained wide attention

    over the past few years. Drug-loaded biodegradable polymers are

    two-component systems (drug and polymer), which exhibit two

    functionalities: the capability of drug molecules to gradually

    escape from the matrix for a controlled release, and the ability of

    the matrix to subsequently degrade for complete secretion from

    the body. The application of these modern delivery systems

    allows a decreased body drug dosage with concurrent reduction

    in possible undesirable effects common to most forms of systemic

    delivery, maintain drug level in the therapeutic window and

    improve patient compliance and comfort.126This journal is The Royal Society of Chemistry 2010

  • logical systems.

    Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Onlinethe determination of mechanisms causing biomaterial failure,

    and the progress in the development of biomaterials that

    interact with the biological surroundings led to 2nd generation

    biomaterials. Because these biomaterials allow cell-surface

    interactions, they are responsive to external stimuli that promote

    direct bone-bonding and osteoconduction. The novel concepts

    for the generation of 3rd generation materials with instructive

    capacities are based on the combination of scientific knowledge

    and clinical needs. Scientifically, huge progress has been made

    toward the development of intelligent materials with unique

    properties regarding biological performances (e.g. osteoinduc-

    tion and angiogenesis), mechanical properties, degradation

    capacity and porosity characteristics. Especially, the almost

    unlimited possibilities of polymeric materials will offer valuable

    advantages. Furthermore, novel strategies for the delivery of

    growth factors will aid to orchestrate biological responses

    through the controlled temporal and spatial release of these

    factors. Although associated with major safety and regulatory

    issues, even concepts based on combinations of cells or genes

    with materials will evolve toward clinical applicability.Handling properties. The key advantages of polymers which

    make them preferable to other bone substitute materials are

    tunable physical properties that give excellent clinical charac-

    teristics. Indeed, polymer-based materials can be easily manip-

    ulated into various forms such as beads, micro/nanoparticles,

    and gels under mild conditions. In particular, injectable formu-

    lations, required to increase the scope of minimally invasive

    procedures, provide the ability to take shape of the cavity in

    which they are placed and can thus fill irregular defects75

    (Fig. 2B). Another category of stimuli-responsive materials in the

    forefront of polymer science for medical application is repre-

    sented by shape-memory materials (SMPs).127 These class of

    actively moving polymers change shape on demand in response

    to an external stimulus. The shape-memory effect (SME) is based

    on a suitable polymer network architecture in combination with

    a programming technology.128 In the future, these materials will

    be introduced into bone defects by minimally invasive proce-

    dures through a small incision leading to benefits for a more

    gentle treatment and reduction of costs in health care.

    3 Conclusion and final remarks

    The research on synthetic bone substitute materials has under-

    gone explosive growth over the past 40 years due to an increasing

    collaboration between experts from different scientific disci-

    plines. Despite this explosive growth, the translation of scientific

    knowledge on bone substitute materials has resulted in only a few

    commercially-available bone substitute products that are

    predominantly based on bioceramics and demineralized bone

    matrix (DBM). However, the development of synthetic bone

    substitute materials for commercial exploitation is in the initial

    phase of a revolutionary leap, in which novel concepts account

    for the generation of materials with instructive capacities.

    The 1st generation biomaterials were generally based on

    tolerant and inert materials that were only capable to withstand

    anticipated physiological stress while being passively accepted by

    the body. Although these materials provided an effective

    immediate solution for many patients, their long-term perfor-

    mance was often insufficient. The increasing research efforts onThis journal is The Royal Society of Chemistry 2010References

    1 K. A. Hing, Philos. Trans. R. Soc. London, Ser. A, 2004, 362, 28212850.

    2 D. F. Williams, Biomaterials, 2009, 30, 58975909.3 P. Chaudhari, C. Flemings, et al., US National Academy of Sciences,

    Washington, D C, 1990.4 D. F. Williams, Biomaterials, 2008, 29, 29412953.5 L. L. Hench, Science, 1980, 208, 826831.6 L. L. Hench and J. M. Polak, Science, 2002, 295, 10147.7 P. Ducheyne, A. Elghannam and I. Shapiro, J. Cell. Biochem., 1994,

    56, 162167.8 T. A. Gerds and M. Vogeler, Statistical Methods in MedicalResearch, 2005, 14, 579590.

    9 J. Wilson-Hench, Elsevier, Amsterdam, 1987, vol. 4, p 29.10 T. Albrektsson and C. Johansson, Eur. Spine J., 2001, 10, S96S101.11 M. Navarro, A. Michiardi, O. Castano and J. A. Planell, J. R. Soc.

    Interface, 2008, 5, 11371158.12 A. S. Greenwald, S. D. Boden, V. M. Goldberg, Y. Khan,

    C. T. Laurencin and R. N. Rosier, J. Bone Joint Surg. Am., 2001,83A, 98103.

    13 K. J. Lee, J. G. Roper and J. C. Wang, Spine J., 2005, 5, S217223S.14 J. C. Wang, A. Alanay, D. Mark, L. E. A. Kanim, P. A. Campbell,

    E. G. Dawson and J. R. Lieberman, Eur. Spine J., 2007, 16, 12331240.

    15 H. Bae, L. Zhao, D. Zhu, L. E. Kanim, J. C. Wang andR. B. Delamarter, J. Bone Jt. Surg., 2010, 92, 42735.

    16 S. M. Best, A. E. Porter, E. S. Thian and J. Huang, J. Eur. Ceram.Soc., 2008, 28, 13191327.

    17 M. N. Rahaman, A. H. Yao, B. S. Bal, J. P. Garino and M. D. Ries,J. Am. Ceram. Soc., 2007, 90, 19651988.

    18 D. W. Hutmacher, J. T. Schantz, C. X. F. Lam, K. C. Tan andT. C. Lim, J. Tissue Eng. Regener. Med., 2007, 1, 245260.

    19 S. V. Dorozhkin, Biomaterials, 2010, 31, 146585.20 M. P. Lutolf, P. M. Gilbert and H. M. Blau, Nature, 2009, 462, 433

    441.The widening of the classical view of bone substitute mate-

    rials will certainly have a positive impact on medicine, industry

    and more importantly on their clinical application. However,

    at present there is a large gap between the state-of-the-art in

    the field of materials science and the use of materials in clinical

    practice. The progression from laboratory concept to

    commercial application in patients faces several challenges

    including long and often undefined regulatory approval path-

    ways, high translational costs, assessing appropriate risk/

    benefit and cost/benefit ratios, and appropriate matching of

    technology and application. Moreover, it should be empha-

    sized that bone substitute materials ultimately are employed to

    address needs that the patient perceives in terms of rehabili-

    tation, comfort and convenience.129 Biomedical innovation is

    heading now into minimally invasive and quicker procedures,

    fewer hospital visits including shorter stays and reduced

    complications.

    Despite the recent advances, future progress in engineering

    disciplines (for example computer-aided design) together with

    an increased understanding of the processes through which

    inorganic and organic macromolecules self-assemble into

    organized architectures and complex forms will allow for the

    design of new materials with tailored structures at both the

    micro- and the nano-domain level. Such sophisticated mate-

    rials, so called bio-inspired, will be built to mimic nature not

    through presenting active fragments reproduced exactly from

    biological molecules, but rather through presenting sensor

    activities to monitor their function and interact with the bio-130J. Mater. Chem., 2010, 20, 87478759 | 8757

  • Publ

    ished

    on

    23 A

    ugus

    t 201

    0. D

    ownl

    oade

    d by

    UN

    IVER

    SID

    AD

    SA

    O P

    AU

    LO o

    n 09

    /05/

    2015

    16:

    57:2

    3.

    View Article Online21 G. C. Babis and P. N. Soucacos, Injury, 2005, 36, S38S44.22 A. El Ghannam, Expert Rev. Med. Devices, 2005, 2, 87101.23 J. O. Eniwumide, H. Yuan, S. H. Cartmell, G. J. Meijer and J. D. de

    Bruijn, Eur. Cell Mater., 2007, 14, 3038.24 P. Habibovic and K. de Groot, J. Tissue Eng. Regener.Med., 2007, 1,

    2532.25 M. Heliotis, U. Ripamonti, C. Ferretti, C. Kerawala, A. Mantalaris

    and E. Tsiridis, Br. J. Oral Maxillofacial Surg., 2009, 47, 5114.26 L. Chen, F. Ye, R. Yang, X. Lu, Y. Shi, L. Li, H. Fan and H. Bu,

    Acta Biomater., 2010, 6, 15691574.27 R. Z. LeGeros, Chem. Rev., 2008, 108, 47424753.28 G. Daculsi, D. J. Hartmann, M. Heughebaert, L. Hamel and

    J. C. Lenihouannen, J. Submicrosc. Cytol. Pathol., 1988, 20, 379384.

    29 P. Habibovic, H. P. Yuan, C. M. van der Valk, G. Meijer, C. A. vanBlitterswijk and K. de Groot, Biomaterials, 2005, 26, 35653575.

    30 M. Colilla, M. Manzano and M. Vallet-Regi, Int. J. Nanomedicine,2008, 3, 403414.

    31 M. B. Nair, H. K. Varma, K. V. Menon, S. J. Shenoy and A. John,Acta Biomater., 2009, 5, 17421755.

    32 J. C. Roldan, R. Detsch, S. Schaefer, E. Chang, M. Kelantan,W. Waiss, T. E. Reichert, G. C. Gurtner and U. Deisinger, J.Craniomaxillofac. Surg., 2010, 38, 423430.

    33 T. J. Webster, C. Ergun, R. H. Doremus and R. Bizios, J. Biomed.Mater. Res., 2002, 59, 312317.

    34 G. Bluteau, P. Pilet, X. Bourges, M. Bilban, R. Spaethe, G. Daculsiand J. R. Guicheux, Biomaterials, 2006, 27, 29342943.

    35 J. C. Brodie, J. Merry and M. H. Grant, J. Mater. Sci.: Mater. Med.,2006, 17, 4348.

    36 J. C. Brodie, E. Goldie, G. Connel, J. Merry and M. H. Grant, J.Biomed. Mater. Res., Part A, 2005, 73a, 409421.

    37 P. C. Bessa, M. Casal and R. L. Reis, J. Tissue Eng. Regener. Med.,2008, 2, 113.

    38 P. C. Bessa, M. Casal and R. L. Reis, J. Tissue Eng. Regener. Med.,2008, 2, 8196.

    39 J. Ziegler, D. Anger, F. Krummenauer, D. Breitig, S. Fickert andK. P. Guenther, J. Biomed. Mater. Res., Part A, 2008, 86a, 8997.

    40 H. Autefage, F. Briand-Mesange, S. Cazalbou, C. Drouet,D. Fourmy, S. Goncalves, J. P. Salles, C. Combes, P. Swider andC. Rey, J. Biomed. Mater. Res., Part B, 2009, 91b, 706715.

    41 D. P. Link, J. van den Dolder, J. J. van den Beucken, J. G. Wolke,A. G. Mikos and J. A. Jansen, Biomaterials, 2008, 29, 675682.

    42 E. Wernike, W. Hofstetter, Y. Liu, G. Wu, H. J. Sebald,D. Wismeijer, E. B. Hunziker, K. A. Siebenrock andF. M. Klenke, J. Biomed. Mater. Res. A, 2010, 92A, 463474.

    43 J. Tazaki, M. Murata, T. Akazawa, M. Yamamoto, K. Ito,M. Arisue, T. Shibata and Y. Tabata, Biomed. Mater. Eng., 2009,19, 141146.

    44 P. Q. Ruhe, E. L. Hedberg, N. T. Padron, P. H. M. Spauwen,J. A. Jansen and A. G. Mikos,J.Bone Joint Surg.Am., 2003, 85A, 7581.

    45 J. M. Kanczler and R. O. C. Oreffo, Eur. Cell Mater., 2008, 15, 100114.

    46 E. Wernike, M. O. Montjovent, Y. Liu, D. Wismeijer,E. B. Hunziker, K. A. Siebenrock, W. Hofstetter andF. M. Klenke, Eur. Cell Mater., 2010, 19, 3040.

    47 J. Barralet, U. Gbureck, P. Habibovic, E. Vorndran, C. Gerard andC. J. Doillon, Tissue Eng. A, 2009, 15, 16019.

    48 L. H. He, O. C. Standard, T. T. Y. Huanga, B. A. Latella andM. V. Swain, Acta Biomater., 2008, 4, 577586.

    49 S. V. Dorozhkin, J. Mater. Sci., 2009, 44, 23432387.50 Y. X. Huang, J. Ren, C. Chen, T. B. Ren and X. Y. Zhou, J.

    Biomater. Appl., 2007, 22, 409432.51 S. Y. Ni and J. Chang, J. Biomater. Appl., 2009, 24, 139158.52 N. Tran and T. J. Webster, Wiley Interdiscip. Rev. Nanomed.

    Nanobiotechnol., 2009, 1, 33651.53 T. J. Webster, C. Ergun, R. H. Doremus, R. W. Siegel and R. Bizios,

    Biomaterials, 2000, 21, 18031810.54 V. Karageorgiou and D. Kaplan, Biomaterials, 2005, 26, 54745491.55 Y. Khan, M. J. Yaszemski, A. G. Mikos and C. T. Laurencin, J.

    Bone Jt. Surg., 2008, 90, 3642.56 V. S. Komlev and S. M. Barinov, J. Mater. Sci.: Mater. Med., 2002,

    13, 295299.57 P. Habibovic, M. C. Kruyt, M. V. Juhl, S. Clyens, R. Martinetti,

    L. Dolcini, N. Theilgaard and C. A. van Blitterswijk, J. Orthop.Res., 2008, 26, 13631370.8758 | J. Mater. Chem., 2010, 20, 8747875958 S. Padilla, J. Roman and M. Vallet-Regi, J. Mater. Sci.: Mater.Med., 2002, 13, 11931197.

    59 W. J. E. M. Habraken, L. T. de Jonge, J. G. C. Wolke, L. Yubao,A. G. Mikos and J. A. Jansen, J. Biomed. Mater. Res., Part A,2008, 87a, 643655.

    60 W. J. E. M. Habraken, J. G. C. Wolke, A. G. Mikos andJ. A. Jansen, J. Biomed. Mater. Res., Part B, 2009, 91b, 555561.

    61 W. J. E. M. Habraken, J. G. C. Wolke, A. G. Mikos andJ. A. Jansen, J. Biomater. Sci., Polym. Ed., 2006, 17, 10571074.

    62 W. J. E. M. Habraken, H. B. Liao, Z. Zhang, J. G. Wolke,D. W. Grijpma, A. G. Mikos, J. Feijen and J. A. Jansen, ActaBiomater., 2010, 6, 220011.

    63 H. H. K. Xu, M. D. Weir and C. G. Simon, Dent. Mater., 2008, 24,12121222.

    64 G. Hannink, J. G. C. Wolke, B. W. Schreurs and P. Buma, J.Biomed. Mater. Res., Part B, 2008, 85b, 478488.

    65 F. Jegoux, E. Goyenvalle, M. B. DArc, E. Aguado and G. Daculsi,Arch. Orthop. Trauma Surg., 2005, 125, 153159.

    66 L. Le Guehennec, P. Layrolle and G. Daculsi, Eur. Cell Mater.,2004, 8, 110.

    67 E. M. Ooms, J. G. C. Wolke, J. P. C. M. van der Waerden andJ. A. Jansen, J. Biomed. Mater. Res., 2002, 61, 918.

    68 X. Liu and P. X. Ma, Ann. Biomed. Eng., 2004, 32, 477486.69 P. B. Malafaya, G. A. Silva and R. L. Reis, Adv. Drug Delivery Rev.,

    2007, 59, 207233.70 O. Z. Fisher, A. Khademhosseini, R. Langer and N. A. Peppas, Acc.

    Chem. Res., 2010, 43, 41928.71 P. A. Gunatillake and R. Adhikari, Eur. Cell Mater., 2003, 20, 116.72 N. A. Peppas, J. Z. Hilt, A. Khademhosseini and R. Langer, Adv.

    Mater., 2006, 18, 13451360.73 T. R. Hoare and D. S. Kohane, Polymer, 2008, 49, 19932007.74 O. Wichterle and D. Lim, Nature, 1960, 185, 117118.75 J. D. Kretlow, L. Klouda and A. G. Mikos, Adv. Drug Delivery Rev.,

    2007, 59, 263273.76 C. Migliaresi, A. Motta, A. Di Benedetto, Engineering of functional

    skeletal tissues, 2007, Berlin, pp. 95109.77 W. E. Hennink and C. F. van Nostrum, Adv. Drug Delivery Rev.,

    2002, 54, 1336.78 J. Berger, M. Reist, J. M. Mayer, O. Felt, N. A. Peppas and

    R. Gurny, Eur. J. Pharm. Biopharm., 2004, 57, 1934.79 J. S. Temenoff, H. Park, E. Jabbari, D. E. Conway, T. L. Sheffield,

    C. G. Ambrose and A. G. Mikos, Biomacromolecules, 2004, 5, 510.80 J. S. Temenoff, H. Park, E. Jabbari, T. L. Sheffield, R. G. Lebaron,

    C. G. Ambrose and A. G. Mikos, J. Biomed. Mater. Res., 2004, 70a,235244.

    81 K. T. Nguyen and J. L. West, Biomaterials, 2002, 23, 43074314.82 R. Jin, C. Hiemstra, Z. Y. Zhong and J. Feijen, Biomaterials, 2007,

    28, 27912800.83 N. Q. Tran, Y. K. Joung, E. Lih, K. M. Park and K. D. Park,

    Biomacromolecules, 2010, 11, 61725.84 C. Y. Xu and J. Kopecek, Polym. Bull., 2007, 58, 5363.85 S. Huang and D. E. Ingber, Nat. Cell Biol., 1999, 1, E131E138.86 E. Ruoslahti and J. C. Reed, Cell, 1994, 77, 477478.87 K. Anselme, Biomaterials, 2000, 21, 667681.88 R. G. Lebaron and K. A. Athanasiou, Tissue Eng., 2000, 6, 85103.89 S. C. G. Leeuwenburgh, J. A. Jansen and A. G. Mikos, J. Biomater.

    Sci. Polym. Ed., 2007, 18, 15471564.90 F. E. Bailey, J. V. Koleske, Academic Press, New York, 1976.91 H. Shin, K. Zygourakis, M. C. Farach-Carson, M. J. Yaszemski and

    A. G. Mikos, Biomaterials, 2004, 25, 895906.92 J. S. Temenoff and A. G. Mikos, Biomaterials, 2000, 21, 24052412.93 H. Shin, K. Zygourakis, M. C. Farach-Carson, M. J. Yaszemski and

    A. G. Mikos, J. Biomed. Mater. Res., 2004, 69a, 535543.94 H. Shin, S. Jo and A. G. Mikos, J. Biomed. Mater. Res., 2002, 61,

    169179.95 K. C. Dee and R. Bizios, Biotechnol. Bioeng., 2000, 50, 438442.96 X. Z. He, J. Y. Ma and E. Jabbari,Langmuir, 2008, 24, 1250812516.97 D. S. W. Benoit, A. R. Durney and K. S. Anseth, Biomaterials, 2007,

    28, 6677.98 P. C. Bessa, R. Machado, S. Nurnberger, D. Dopler, A. Banerjee,

    A. M. Cunha, J. C. Rodrguez-Cabello, H. Redl, M. vanGriensven, R. L. Reis and M. Casal, J. Controlled Release, 2010,142, 3128


Metabolismo do Glicogênio, Gliconeogênese, Cetogênese ... · carboidratos piruvato lipídeos acil-CoA mobiliz. ... Primeiro compartimento do estômago dos albergando um complexo

FUNDAÇÃO DO ABC - CENTRAL DE CONVÊNIOS COMPLEXO …

PLANTA SINTESE[1] · Title: PLANTA SINTESE[1].pdf Author: Jan Divine Home Created Date: 20190312115402Z

as variáveis intervenientes na produção do onset complexo

A SINTESE DAS TRÊS MONTANHAS - pdfMachine from Broadgun

Biografia e Sintese

sintese acetanilida

2006 yan revisao sintese ijms 07-00155

UMA ANÁLISE DO COMPLEXO INDUSTRIAL-MILITAR INDIANO …

Sintese Economica v - Abril

Tipia e Cor - Sintese

100 Anos de Sintese Da Amonia

Complexo oral canibal - UFPE

File1 3 sintese ac-cp

PP PGeneral - Sintese copy - Sesimbra · Title: PP_PGeneral - Sintese copy Created Date: 6/24/2008 10:12:47 AM

Reviw Sobre a Sintese de Fischer Tropsch

Sintese Ppc Bach Ciencias Biologic As UFABC

Sintese Da Carta a Diogneto

Biomecânica do Complexo Articular do Joelho

Tombamento dos Remanescentes do Complexo do Hospital

Edgar Morin a Sintese Possivel

Sintese Sonora

Brt Sintese Agropecuaria 2013 2014 Incaper

estudo do processo adsortivo do complexo Fe-eDTA na superfície do ...mtc-m21b.sid.inpe.br/col/sid.inpe.br/mtc-m21b/2016/06.03.12.40/doc/... · adsorção do analito na superfície

Sintese NP-Ag Com PVA - Incrivel - NanoArts

Cartografia Geológica do Complexo básico – ultrabásico de ...1051 Cartografia Geológica do Complexo básico – ultrabásico de Cabeço de Vide - Ensaio sobre a geometria e potencial

Pacification, Tourism, and Transformation in Complexo do Alem£o, Rio de Janeiro

Avaliação da sustentabilidade ambiental do entorno do Complexo ... · dos impactos ambientais do Complexo Portu-ário Industrial do Açu (São João da Barra - RJ), com ênfase

INSTITUTO POLITÉCNICO DO PORTO ESCOLA SUPERIOR DE ...recipp.ipp.pt/bitstream/10400.22/7886/1/DM_IsabelMilheiro_2015.pdf · Instabilidade Multidirecional do Complexo Articular do

INSTITUTO POLITÉCNICO DO PORTO ESCOLA SUPERIOR DE ... · Instabilidade Multidirecional do Complexo Articular do Ombro: Estudo de Caso Isabel Milheiro - 4 - ABSTRACT Backgroud: The

Sintese Dos Nucleotidos de Pirimidina

Aula3 sintese prtn

Sintese do-metodo-educacional-psicodramatico-maria-alicia-romaña

Portfolio - Complexo Educacional QI

Mediações no YouTube e o caso 'Ocupação do Complexo do