Journal of Controlled Release 161 (2012) 680–692

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review

Hydrogels for protein delivery in tissue engineering

Roberta Censi a,⁎, Piera Di Martino a, Tina Vermonden b, Wim E. Hennink b
a
School of Pharmacy, University of Camerino, via S. Agostino 1, 62032, Camerino (MC), Italy
b
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences,Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Tissue defects caused by diseases or trauma present enormous challenges in regenerative medicine. Recently, a
Received 13 January 2012 better understanding of the biological processes underlying tissue repair led to the establishment of new ap-
Accepted 2 March 2012 proaches in tissue engineering which comprise the combination of biodegradable scaffolds and appropriate
Available online 8 March 2012
cells together with specific environmental cues, such as growth or adhesive factors. These factors (in fact pro-
teins) have to be loaded and sustainably released from the scaffolds in time. This review provides an overview
Keywords:
Hydrogels
of the various hydrogel technologies that have been proposed to control the release of bioactive molecules of in-
Growth factors terest for tissue engineering applications.
Protein delivery In particular, after a brief introduction on bioactive protein drugs that have remarkable relevance for tissue
Tissue engineering engineering, this review will discuss their release mechanisms from hydrogels, their encapsulation and immobiliza-
Controlled release tion methods and will overview the main classes of hydrogel forming biomaterials used in vitro and in vivo to release
them.
Finally, an outlook on future directions and a glimpse into the current clinical developments are provided.
© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680
2. Proteins in the healing process: an introduction to their delivery approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
3. Protein release strategies from hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
3.1. Physical entrapment of growth factors into hydrogels: general principles and release mechanisms . . . . . . . . . . . . . . . . . . 681
3.1.1. Synthetic polymers for the physical encapsulation of growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
3.1.2. Natural polymers for the physical encapsulation of growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
3.2. Covalent binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
3.3. Dual/multiple delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
4. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690

1. Introduction
Controlled drug delivery has seen rapid advances in the last few de-
cades with the introduction of novel biomaterials and technologies that
found application in all fields of pharmaceutical and biomedical sciences.
Particularly, with the advent of protein therapeutics, the need for con-
trolled delivery systems able to enhance protein's pharmacokinetic and
pharmacodynamic properties became more urgent. Nowadays, proteins
are used in the treatment of many diseases but also in the area of tissue
engineering, where supply of biomolecular cues that mimic the environ-
ment of natural tissues and promote the communication between cells
⁎ Corresponding author. Tel.: + 39 0737 402215; fax: + 39 0737 402457.
E-mail address: roberta.censi@unicam.it (R. Censi).
proved crucial for achieving effective tissue repair or replacement. There-
fore, modern tissue engineering aims at assisting the re-growth of func-
tional tissues by combining cells and engineering materials with
signaling biomolecules [1,2]. Biomolecular signals that are mainly
growth factors or other cytokines, chemoattractants, adhesion proteins
and many others, must be locally delivered in their active form and
with a sustained release profile. Among the existing technologies, hydro-
gels – water-swollen, cross-linked polymer networks – have emerged as
particularly promising materials for tissue engineering, as they can act
both as scaffolding materials and/or releasing matrices for biologically
active and cell modulating substances. Their water content, soft nature
and porous structure mimic biological tissues and make them suitable
to accommodate cells and to encapsulate and release water-soluble com-
pounds like proteins in a controlled fashion.

0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2012.03.002
 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
After highlighting the rationale for using of delivery technologies to
improve the effectiveness of biomolecular cues, this mini-review briefly
describes the main hydrogel systems that have been studied recently as
releasing matrices for biomolecules involved in healing processes. Strat-
egies for encapsulation, (transient) immobilization and controlled re-
lease are also discussed. Finally, hydrogel-based releasing technologies
with potential clinical impact in tissue engineering are outlined and
an outlook on future directions is provided.
2. Proteins in the healing process: an introduction to their
delivery approaches
Cell fate and behavior are highly influenced by a number of factors
and interactions with the surrounding microenvironment. Signaling
molecules create an effective communication between cells and extra-
cellular matrix (ECM) and orchestrate the complex cascade of events
that leads to successful regeneration of damaged tissues [3]. Of all mol-
ecules currently identified as key components of the wound healing
process, growth factors play a pivotal role in the information transfer
mechanism between cell and ECM. Although the natural dynamics of
the cellular microenvironment are difficult to emulate in an artificial
setting, it is currently widely accepted that the self-healing capacity of
an organ or tissue can be augmented by the integration of growth fac-
tors. In fact, they accelerate the proliferation and differentiation of
recruited or implanted cells and promote the re-growth of tissues and
organs not capable of self-healing otherwise [4–6].
Growth factors are large polypeptides that modulate cellular prolif-
eration, differentiation, migration, adhesion and gene expression upon
binding to specific receptors on the surface of target cells. Examples of
growth factors and their applications in tissue engineering are listed
in Table 1. Unlike other proteins (i.e. hormones), growth factors act lo-
cally and possess short diffusion distances through the ECMs, owing to
their short half-lives. Typically, growth factors are enzymatically or
chemically degraded or deactivated in physiological conditions in a
very limited time frame. To mention, basic fibroblast growth factor
(bFGF) has a half-life of only 3 min after intravenous administration;
[7] similarly, less than 30 min are needed to halve the plasma concen-
tration of vascular endothelial growth factor (VEGF) [8] and platelet-
derived growth factor (PDGF), isolated from platelets, has a half life of
less than 2 min when injected intravenously [9].
Therefore, the exogenous administration of growth factors for tissue
engineering faces important limitations associated to their rapid inacti-
vation and/or elimination after intravenous delivery, their poor trans-
dermal adsorption due to the large size and the hydrolytic and
proteolytic degradation upon oral administration. Hydrogels are fre-
quently used to release growth factors in a controlled and effective
manner and to direct the protein specifically to the wound site
[10,11]. Spatiotemporal control over the delivery of these key signaling
Table 1
Typical examples of growth factors and their applications in the field of tissue engineering [11,14].
Growth factor Abbreviation
Vascular endothelial growth factor VEGF
Insulin-like growth factor IGF-1
Hepatocyte growth factor HGF
Epithelial growth factor EGF
Nerve growth factor NGF
Bone morphogenetic protein BMP-2/3/7
Platelet derived growth factor (2 polypeptide PDGF-AA/BB/AB
chains A and B forming homodimers
AA and BB and heterodimer AB)
Transforming growth factor TGF-α/β
Angiopoietin fibroblast growth factor Ang-1/Ang-2/bFGF
681
molecules not only enhances tissue regeneration, but also prevents
unwanted and potentially harmful side-effects at other locations than
the target. Various strategies have been investigated to achieve con-
trolled protein delivery from hydrogels. They comprise ‘direct’ and ‘in-
direct’ delivery approaches. Direct release occurs through physical
encapsulation, non-covalent binding, covalent immobilization to the
delivery system via hydrolytically or enzymatically degradable linkers
and the use of double carriers, where protein loaded micro/nano-
spheres are embedded in hydrogels and the release of the active is
based on a combination of mechanisms (diffusion and or degradation).
‘Indirect’ approaches rely on gene therapy and cell transplantation.
Gene therapy is realized through the expression of genetic material
encoding for the desired protein that is delivered in the target tissue,
while in cell transplantation, specific proteins are secreted by cells
that are encapsulated in a hydrogel [12,13].
In the following section the ‘direct’ approaches for protein deliv-
ery in tissue engineering will be discussed.
3. Protein release strategies from hydrogels
Proteins can be loaded into hydrogels through a manifold of mecha-
nisms and strategies. They can be physically entrapped in hydrogels or
adsorbed to the matrix by specific interactions, non-covalent or covalent
binding via degradable linkers and subsequently released via diffusion,
swelling, erosion, degradation or a specific trigger such as pH, tempera-
ture, etc. Also, a combination of multiple delivery systems can be used to
achieve modular release of multiple proteins. Depending on the release
mechanism, different release profiles can be obtained. Table 2 overviews
the technologies further described in the following sections.
3.1. Physical entrapment of growth factors into hydrogels: general
principles and release mechanisms
Physical encapsulation of growth factors in order to obtain a con-
trolled release of the active is a frequently applied technique for local
growth factor delivery in tissue engineering. Its relatively simplicity
represents a clear advantage over more sophisticated encapsulation/
release methods and the majority of hydrogel systems relies on this
loading process. Loading is done by incubation of the preformed gel
with the protein of interest or by adding the protein to the hydrogel
forming monomers/prepolymers. In recent years, attention is shifted
to in situ gelling systems which preferably are liquid before adminis-
tration and gellify at the site of injection [42]. For these gels, loading
with proteins can easily be established by dissolution of the biother-
apeutic in the gel forming polymer solution before administration.
Typically, the mechanisms that govern the drug release from these
hydrogels are controlled by diffusion, swelling, erosion, external
stimuli or combinations thereof [43].
Action Tissue engineering applications
Migration, proliferation and survival of endothelial cells Blood and lymphatic vessels
Proliferation, apoptosis inhibition Cartilage, skin, nerve, kidney,
bone, muscle
Proliferation, migration, differentiation of mesenchymal stem cells Liver, muscle, bone
Proliferation and differentiation of fibroblast, epithelial, Skin, nerve
mesenchymal and glial cells
Proliferation and survival of neural cells Nerve, brain, spine
Differentiation and migration of osteoblasts, renal development Bone, cartilage, kidney
Embryonic development, proliferation and migration of Bone, skin, muscle, blood vessels
endhotelial and smooth muscle cells
Proliferation and differentiation of basal, neural, bone-forming Brain, skin, cartilage, bone
cells, keratinocytes, anti-proliferation epithelial cells
Blood vessels maturation Hearth, muscle, blood vessels,
Proliferation endothelial cells bone, skin, nerve
682 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692

Table 2
Overview of the main release mechanisms and strategies to load and modulate the kinetics of proteins released from hydrogels.

Release mechanism from hydrogels Characteristics Some examples

✓ Fickian diffusion PEG/p(HPMAmlac) [15–17]

✓ Mt/M∞ = k √t PLA-PEG-PLA [18]
✓ Concentration gradient RADA 16 networks [19]
✓ Generally limited to relatively short time spans alginate [20,21]
✓ Protein size b mesh size dex-VS/PEG-4-SH [22]
✓ Geometry determines release rate
✓ Swelling degree controls release rate dextran/P(D,L)LA [23]
✓ Water uptake allows protein to diffuse out oppositely charged dextran microspheres [24]
✓ Swelling can be mediated by degradation

✓ Controlled by physical or chemical erosion PLGA-PEG-PLGA [25,26] PEG/chol-PEG/β-CD [27].

✓ Surface erosion mostly for physical gels PEG-P(D,L)LA [28]
✓ Constant release rate
✓ Hydrolytical or enzymatic degradation
✓ Protein size > mesh size

✓ Stimuli lead to gel changes and drug release nitrilotriacetic acid/GyrB [29]
✓ Stimuli: temperature, pH, biomolecules, drugs, magnetic field, etc… PEG-VS-MMP [30]
PEG acrylate-MMP [31]

✓ Adsorption of proteins through weak interaction (i.e. electrostatic, hydro- Fibrin-heparin [32,33]
phobic, H-bonding) PEG-IDAA/Ni+ 2Cu+ 2 [34]
✓ Affinity with binding sites: heparin, antibodies, chelating ions, peptides, thiol acrylate PEG [35]
molecular imprinting

✓ Attachment of drugs to matrix via covalent bonds polyacrylamide [36,37]

✓ Cleavable linker PEG-RGD-MMP [38]
✓ Hydrolytical or enzymatic cleavage

✓ Combination of multiple carriers: i.e. protein-loaded micro/nanoparticles PEG-GMA/gelatin [39]

embedded in hydrogels Fibrin-PLGA [40]
✓ Release of multiple proteins through combination of mechanisms and rates HAMC/PLGA [41]

The release mechanism depends on both the characteristics of the
polymeric network and the protein. When the hydrogel pores are big-
ger than the hydrodynamic radius of the protein, diffusion is the driv-
ing force for release, with a diffusion rate depending on the protein
size and the water-content of the gel (‘free-volume’) [44]. On the
other hand, when the hydrogels pores are smaller than the protein di-
ameter, swelling or erosion/degradation (bulk or surface) is needed
for release. Generally speaking, the majority of the gel matrices
reported to date exhibit diffusion controlled release, following Higu-
chi's kinetics, implying that the release is proportional to the square
root of time [45]. This release profile was demonstrated particularly
beneficial for the delivery of several growth factors for tissue engi-
neering applications. For example, Brown et al. and Boerckel et al.
demonstrated that bone regeneration was enhanced when rhBMP-2
was released down a concentration gradient [46,47]. The diffusional
spatiotemporal growth factor presentation proved effective not only
for bone regeneration, but also for other engineered tissues like
blood vessels [20,21].
The Ritger–Peppas equation is often used to fit release data and de-
termine the underlying release mechanism: M t/M∞ = ktn with M t/M∞
the fractional drug release at time, t and k a constant incorporating
structural and geometric characteristics of the device. If n = 0.5, the re-
lease is governed by Fickian diffusion. If n = 1, molecules are released by
surface erosion, while both mechanisms play a role if n has a value be-
tween 0.5 and 1 [48,49].
Swelling-controlled systems depend on water uptake and changes in
drug diffusivity within the matrix. Swelling increases polymer flexibility
and makes pores bigger, resulting in higher drug mobility with some-
times n values that exceed 1. As a consequence, drug release depends
on Fickian diffusion, polymer disentanglement and dissolution in water
[50].
Erosion-controlled systems have increased in number since the
development of synthetic biodegradable polymers. In these systems,
the mobility of the drug in the homogeneous non-degraded polymer
matrix is limited and drug release is then governed by the degrada-
tion rate of the polymer, porosity increase, and drug diffusion mainly
in the surface of the polymer matrix.
The possibility to tailor the release kinetics of proteins from hydrogels
can be achieved through the use of excipients or by changing the cross-
linking density of the polymer network. In this particular instance, the
use of synthetic polymers is extremely advantageous because they offer
the opportunity to fine tune their chemical structure to achieve modular
release. However, also natural polymer networks can be tailored to some
extent by changing polymer concentration and crosslink density. Also ex-
tensive work on the development of hybrid networks in which natural
polymers are synthetically modified or combined with synthetic poly-
mers, has been proposed to combine beneficial properties of both kinds
of polymers with respect to mechanical properties, tailorability in terms
of biodegradability as well as biocompatibility and cytocompatibility.
The geometry of the hydrogel-based depot also affects the release
rate and the duration. Generally, the bigger is the device, the longer the
diffusion distances become and the longer the release consequently
lasts. In recent years, patterning techniques utilized in tissue engineering
(i.e. stereolithography, bioprinting) have been used to adjust surface area
and consequently modulate release profiles of the biomolecules [51–53].
Deviation from the described release behaviors is observed when a
specific stimulus – typically temperature, pH, presence of certain mole-
cules – leads to a physical or chemical change in the network structure,
 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
for instance, hydrogels swell or shrink in response to a certain trigger
thereby modulating the release of encapsulated drugs/proteins.
3.1.1. Synthetic polymers for the physical encapsulation of growth factors
Among the various synthetic polymers studied for the delivery of
growth factors in tissue repair, PEG-based networks play a prominent
role.
Burdick et al. investigated the feasibility of a PEG-based hydrogel
platform for the replacement and delivery of neurotrophins to the cen-
tral nervous system. For this purpose, triblock copolymers based on
PEG and acrylated poly(lactic acid) (PLA-b-PEG-b-PLA) were synthesized
and hydrogels formed by photopolymerization. Three neurotrophins, cil-
iary neurotrophic factor (CNTF), brain-derived neurotrophic factor
(BDNF) and neurotrophin-3 (NT-3) were encapsulated and released in
vitro mainly by diffusion in 20 to 80 days depending on polymer concen-
tration (10 to 30%) and neurotrophin size. In vitro, the released proteins
stimulated the proliferation of a TF-1 cells transfected with the α-subunit
of the CNTF receptor and stimulated outgrowth of a greater number of
neuritis from retinal explants than in control culture conditions [18].
These types of hydrogels, initially described by Hubbell and coworkers
[54], have been extensively investigated for growth factor delivery
[55,56] because they offer several benefits, including the possibility to
easily modify the network properties by changing the macromer chem-
istry and solution concentration. For instance, the degradation and swell-
ing of the hydrogels are dictated by the number of lactic acid repeat units,
the acrylation efficiency, the molecular weight of the PEG core and the
concentration of macromer in the prepolymer solution [57]. Also, the
type of degradable unit (e.g., lactic vs caproic acid) alters network degra-
dation rates [58].
Similar release and network concepts apply to other PEG/polyesters
hydrogels, like those based on PEG-poly(lactic-co-glycolic acid) (PLGA)-
PEG triblock copolymers [59]. These materials display lower critical solu-
tion temperature behavior and were processable avoiding the use of high
temperatures to dissolve the polymer. It was shown that upon subcuta-
neous injection (rat model) the hydrogels were stable for one month
[60]. TGF-β1 was loaded into these hydrogels and used as a slow releas-
ing drug reservoir aimed for wound healing purposes. Significant levels
of re-epithelialization, cell proliferation and collagen organization were
observed [61]. Porcine growth hormone (pGH) and Zn-pGH were sus-
tainably released from PLGA-PEG-PLGA hydrogels for 10–14 days in
vitro with no initial burst and a good correlation with in vivo data, that
confirmed a weight gain in hypophysectomized rats equivalent to con-
ventional daily injections of 5 mg pGH for 14 consecutive days. Approxi-
mately 85% of the glycosylated granulocyte colony stimulating factor
loaded in PLGA-PEG-PLGA hydrogels was released over a 12-day period
and similarly to pGH, showed similar efficacy in rats as compared to
daily i.v. injections [62]. Censi et al. studied the protein release from
photopolymerized thermosensitive PEG-based networks formed upon
gelation of methacrylated poly(hydroxypropyl methacrylamide lac-
tate)-PEG- poly(hydroxypropyl methacrylamide lactate) (p(HPMAm-
lac)-PEG-p(HPMAm-lac)) triblock copolymers [63]. Proteins are released
from this network by Fickian diffusion and, similarly to previously de-
scribed systems, network properties and release behavior can be tailored
by tuning polymer chemistry and concentration [16,17,64]. The gels
showed a good biocompatibility in rats [65] and their potential for carti-
lage tissue engineering application has been demonstrated [51].
Peptide based hydrogels were also used for protein delivery and re-
lease. A class of ionic self-complementary oligopeptides named RAD 16
and commercialized as PuraMatrix was introduced by Zhang et al. These
peptides consist of alternating hydrophilic and hydrophobic amino
acids that form β-sheet structures having one polar surface with com-
plementary charged ionic side chains and a non-polar surface with ala-
nines. These peptides are able to spontaneously self-assemble into
stable macroscopic matrices or nanofibers. The monovalent cations
that are needed for the peptide assembly are sequestered from the
physiological environment [19]. The described family of peptides has
683
been used to encapsulate and deliver several proteins for intramyocar-
dial delivery. This peptide-based hydrogel has also been used to deliver
platelet-derived growth factor BB (PDGF-BB) [66], stromal cell-derived
factor-1 (SDF-1) [67] and insulin-like growth factor I (IGF-I) [68] to de-
crease myocardial infarct. The observed slow and controlled release of
the active proteins has been ascribed to diffusion and to some extent
to interaction between protein and polymer. The amphiphilic nature
of the self-assembling polypeptide leads to interactions with the loaded
protein, resulting in reduced diffusivity and slower release kinetics.
Hydrogels that showed degradation mediated release of model
proteins and that potentially can be used to physically encapsulate
and release growth factors with a zero order release kinetics are for
example those formed by stereocomplexation between star PEG copo-
lymerized with the stereoforms D or L of PLA [28] or those based on
PLGA-PEG-PLGA (ReGel) systems [25,26] and inclusion complexes of
PEG/cholesterol-PEG/β-cyclodextrin [27].
Stimuli sensing hydrogels have also been investigated for growth fac-
tor release in tissue engineering. Recently, Weber et al. proposed a drug-
sensing hydrogel based on gyrase sub-unit B (GyrB), reversibly cross-
linked by coumermycin and able to release VEGF upon addition of an ami-
nocoumarin antibiotic, novobiocin. The polymer forming the hydrogel is
based on polyacrylamide functionalized with nitrilotriacetic acid chelat-
ing a Ni2+ ion to which GyrB can bind through a hexahistidine sequence.
The addition of novobicin causes the cross-links to be partly broken,
resulting in opening of the network structure and release of VEGF [29].
Hubbell et al. synthesized hydrogels comprising multiarm vinyl
sulfone-terminated PEG, a monocysteine containing adhesion pro-
tein, and a bis(cysteine) metalloprotein substrate protein (MMP).
These hydrogels were studied for tissue engineering purposes and
both cells and vascular endothelial growth factor (VEGF105) were en-
capsulated in the hydrogel. The release of the encapsulated factor is
triggered by matrix metalloproteinases (MMPs) [30]. MMPs are are
zinc-dependent endopeptidases capable of degrading extracellular
matrix proteins and they influence cell behavior such as cell prolifer-
ation, migration (adhesion/dispersion), differentiation, angiogenesis,
apoptosis, and host defense. MMPs sensing hydrogels were devel-
oped with the aim to mimic the natural processes involved in tissue
repair. Other examples of semi-synthetic hydrogels were described
by Phelps et al. whose design provides incorporated VEGF, enzyme-
degradable sites and arginine-glycine-aspartic acid (RGD) cell adhe-
sive ligands. Similarly to the system proposed by Hubbell, these ma-
trices use PEG as the main building block because it has been shown
to act as a protein-resistant and cell non-adherent background. The
network is based on PEG diacrylate which is photocrosslinked in the
presence of MMP-degradable polypeptide functionalized with two
PEG-acrylate groups, a mono-PEG-acrylate RGD and a mono-PEG-
acrylate VEGF. Recently, Phelps et al. have demonstrated the benefits
of this approach in a mouse hind limb femoral artery ligation model.
After 7 days, mice treated with implanted hydrogels showed a 50% in-
crease in perfusion to the limbs and 100% increase in perfusion to the
feet as compared to untreated mice. The matrix alone (not containing
VEGF) performed as well as soluble VEGF injections, indicating that
the engineered degradable and adhesive hydrogel has itself a benefi-
cial healing or supportive effect. Engineered matrix containing VEGF
performed better than injections of soluble VEGF, acting synergisti-
cally as a directive scaffold and a growth factor delivery vehicle [31].
3.1.2. Natural polymers for the physical encapsulation of growth factors
Hydrogels designed with natural polymers as building blocks display
multiple advantages over synthetic polymer networks with respect to
their biocompatibility, biodegradability and good cell adhesion proper-
ties. Therefore, extensive work is available on biopolymer-based hydro-
gels for cell and growth factor encapsulation in regenerative medicine
[69–72].
The main classes of natural polymers studied in hydrogel formula-
tions are polysaccharides and proteins/polypeptides.
684 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692

its C-5 epimer α-L-guluronic acid (G), respectively, covalently linked

Fig. 1. Most commonly used polysaccharides for hydrogel preparation for biomedical
applications (M = mannuronic acid, G = guluronic acid).
3.1.2.1. Polysaccharides. Polysaccharides are in general hydrophilic
polymers and are therefore very suitable for the design of hydrogels.
The most commonly used polysaccharides in recent hydrogel research
aimed for protein delivery are alginate, hyaluronic acid, chitosan and
dextran (Fig. 1) [73,74]. Representatives of the hydrogel technologies
based on these polysaccharides are briefly discussed below with respect
to their use for growth factor delivery.
3.1.2.1.1. Alginate. Alginate is a linear polysaccharide composed of
homopolymeric blocks of 1-4′-linked β-D-mannuronic acid (M) and
in different sequences or blocks. This polymer block consists of consec-
utive G-residues, consecutive M-residues or alternating M and G resi-
dues. Depending on the block composition, alginate has different
conformational preferences and behavior. G-rich blocks of the polymer
are able to bind divalent cations of which Ca2+ is frequently used for
crosslinking alginate to obtain hydrogels. Ca2+ ions bind to the G units
of different polymers chains cooperatively in a so-called egg-box ar-
rangement. The partial oxidation of alginate using sodium periodate
makes the polysaccharide biodegradable [75,76], and these oxidized al-
ginates are therefore particularly appealing for biomedical applications.
An alginate hydrogel patch to deliver stromal cell-derived factor-1
(SDF-1), a naturally occurring chemokine that is rapidly overexpressed
in response to tissue injury, was described by Rabbany et al. Alginate
patches were loaded with either purified recombinant human SDF-1
protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release
were measured both in vitro and in vivo in mice. They demonstrated
that SDF-1 plasmid- and protein-loaded patches were able to release
therapeutic product over hours to days, with faster release for SDF-1
protein (in vivo K(d) 0.55 days) than SDF-1 plasmid (in vivo K(d)
3.67 days). Prolonged (induction of) SDF-1 release of SDF-1 resulted
in accelerated healing (9 days) and reduced scarring of acute surgical
wounds in Yorkshire pigs [77].
Although the biological properties of alginate are well recognized,
some limitations in tailoring the mechanical properties, the pore size
and the release kinetics of proteins from these gels still remain. Many re-
searchers investigated several modified alginates to overcome these lim-
itations [78–83]. For example, Ruvinov et al. examined the release of
hepatocyte growth factor (HGF) from modified alginate hydrogels and

Fig. 2. Biophysical properties and vascular endothelial growth factor (VEGF) release from alginate gels. Degradation of gels formed from high molecular weight non-oxidized algi-
nate (triangles), binary molecular weight non-oxidized alginate (white squares) and binary molecular weight partially oxidized alginate (black squares) in phosphate-buffered sa-
line (A). The viscosities of solutions of high (white bar), low (black bar) and binary (grey bar) molecular weight partially oxidized alginate were assessed (B). Release kinetics of
VEGF165 from gels formed from binary molecular weight alginate, either partially oxidized (black squares) or non-oxidized (white squares) (C). Greater endothelial cell prolifer-
ation was observed in cells cultured with VEGF released from binary molecular weight partially oxidized alginate gels (grey), as compared to medium without VEGF (black) and
control VEGF added to medium (white), as determined from the cell number counts from day 0 to day 4 (D). Reproduced from ref. [85].
 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
studied its potential to induce angiogenesis in vivo. They developed a
cross-linkable affinity-binding alginate by introducing sulfoester groups
into the uronic acids of alginate. It was shown that alginate-sulfate was
capable to bind heparin-binding proteins, like growth factors, with equi-
librium binding constants similar to that of heparin. The affinity binding
to alginate-sulfate retarded the release of HGFThe bioconjugate HGF-
alginate-sulfate was combined with alginate in an aqueous solution
and a hydrogel was formed ex vivo by partial cross-linking in presence
of CaCl2. Upon injection into ischemic myocardial tissue the injectable
hydrogel completed its cross-linking by sequestration of endogeneous
Ca2+. A cumulative protein release of 75% was observed after 6 h and
in the following 5 days a cumulative release of approximately 85% was
achieved [84]. This profile is typical of diffusion-based delivery systems
that release the drug down a concentration gradient, as described for an-
other alginate hydrogel reported in Fig. 2 [85]. A recent method
employed to make alginate networks more stable and tailorable is
through the preparation of interpenetrating polymer networks (IPN's)
[83]. An IPN is defined as a network comprising two or more polymers
that are partially interlaced on a molecular scale but not chemically
bounded to each other and that can be separated only after chemical
bond breakage [86]. IPNs are designed to combine the advantageous
properties of different polymers. This combination often translates in
good modulation of the mechanical and biological characteristics of the
final hydrogel, derived from the additive or synergistic effect of the poly-
mers' properties. Pescosolido et al. developed IPN hydrogels based on
Ca2+-alginate and photocross-linked dextran-methacrylate derivatives,
of which the mechanical properties were notably improved by the syner-
gistic effect of the polymers composing the IPN system. These hydrogels
proved effective in the controlled release of proteins [83]. In another
study, it was demonstrated that complete delivery and a better control
over VEGF release kinetics were achieved by cross-linking alginate mi-
croparticles with Zn2+ instead of Ca2+ [87,88].
Silva et al. described alginate hydrogels that were covalently modi-
fied with RGD sequences and loaded with growth factor VEGF, and
that were lyophilized to form microporous scaffolds to induce
Fig. 3. Analysis of angiogenesis in ischemic hindlimbs after OEC transplantation. (A) Implantation of blank scaffolds, bolus injection of OECs and VEGF (same quantities as placed in
scaffolds), transplantation of OECs on scaffolds lacking VEGF (alginate scaffold OEC), and transplantation of OECs on scaffolds presenting VEGF121 [alginate scaffold (VEGF) OEC].
Photomicrographs of tissue sections from ischemic hindlimbs of SCID mice at postoperative day 15, immunostained for the mouse endothelial cell marker CD-31 (B), and
human CD-31 (C). Reproduced from ref. [89].
685
neovascularization. The delivery system, modified with adhesive se-
quences and loaded with growth factor and cells, was used to induce
the formation of a depot of vascular progenitor cells (outgrowth endo-
thelial cells (OECs)) in vivo. It was observed that OECs were viable dur-
ing the studied time frame and that the encapsulated VEGF induced the
cells to migrate out of the scaffold. Local and controlled delivery of the
morphogen was effective in stimulating the cells to repopulate the dam-
aged tissue and participate in regeneration of a vascular network, while
bolus injections was ineffective in preventing necrotic toe (Fig. 3) [89].
3.1.2.1.2. Dextran. Dextran consists of α-1,6-linked D-
glucopyranoses with some degree of 1,3-branching. Various methods
to cross-link dextran hydrogels have been developed and its high num-
ber of available hydroxy groups presents many options for derivatization
and subsequent physical or chemical crosslinking [90]. For example,
Hennink et al. developed a hydrogel system potentially suitable for the
delivery of proteins relevant for tissue engineering, where a dextran
backbone is derivatized with hydroxyethyl methacrylate (HEMA) moie-
ties. This polymer in aqueous medium was chemically cross-linked by
radical polymerization of the dextran-bound methacrylate groups to
yield hydrogel based microspheres [91,92]. These microspheres released
model proteins as well as therapeutic relevant proteins (IL2 [93]; hgH
[94]) in a controlled manner for days to weeks. The same group also de-
veloped a physically crosslinked hydrogel system by grafting the dextran
backbone with either D- or L-oligo-lactate chains containing 5 to 15 lactic
acid units. The hydrogel formation was driven by stereocomplexation
and preclinical studies demonstrated the suitability of this hydrogel for
the controlled delivery of IL-2. The density of oligo-lactates and the
grafting density allowed tailoring of the release and degradation rate.
The release of the encapsulated protein was dependent on diffusion
[23]. Further efforts were addressed by Hennink et al. towards the devel-
opment of dextran based delivery platforms for proteins, potentially suit-
able for growth factor and cytokine controlled release. Self-assembled
macroscopic hydrogels based on oppositely charged dextran micro-
spheres were prepared and their release behavior investigated. It was
found that native proteins were released by diffusion/swelling [24].
686 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
Fig. 4. (A) Reaction of dextran with p-maleimidophenyl isocynate (PMPI) and (B) preparation of dextran-peptide hydrogel through the Michael-type reaction of peptide cross-
linker to Dex-PMPI. Reproduced from ref [95].
More recently, dextran-peptide bioconjugates were synthesized by
Shoichet et al. In their approach, the polysaccharide was modified
with p-maleimidophenyl isocyanate (PMPI) thereby introducing malei-
mide functionalities in the backbone (Dex-PMPI). A peptide cross-
linker, derived from collagen and susceptible to gelatinase A digestion,
was synthesized with bifunctional cysteine termini and used to cross-
link Dex-PMPI (Fig. 4). It was demonstrated that this type of hydrogel
was cytocompatible and mimicked the degradation and remodeling of
the ECM through the activity of cell-secreted enzymes [95].
Michael addition cross-linking reaction was used to induce network
formation between dextran vinyl sulfone conjugates (dex-VS) and tet-
rafunctional mercapto poly(ethylene glycol) (PEG-4-SH). The release
of several proteins including bFGF from hydrogels of different polymer
concentrations was studied. Also these networks showed diffusional
release and a certain extent of tailorability of the bFGF [22].
In a recent study, Sun and coworkers demonstrated that the ma-
nipulation of the cross-link density of dextran based hydrogels
loaded with multiple growth factors affects the neovascularization
of ischemic and wounded tissues. Dextran-allyl isocyanate-
ethylamine (Dex-AE) derivatives with different degrees of substitu-
tion were synthesized and mixed with PEG diacrylate at different ra-
tios and subsequently, hydrogel formation was induced by
photopolymerization. It was found that tissue ingrowth was favored
by reducing the degree of substitution of cross-linking groups, which
resulted in reduced rigidity, increased swelling, increased VEGF re-
lease rate, and rapid hydrogel disintegration. Furthermore, the re-
lease of multiple angiogenic GFs increased the size and number of
newly formed functional vessels [96]. In another study, hybrid
hydrogels based on combinations of glycidyl methacrylated dextran
and gelatin, processed into different physical structures (micro-
spheres, cylinders) have been synthesized and used to deliver
growth factors, including BMP-2and IGF-1 [97].
3.1.2.1.3. Hyaluronan. Hyaluronan or hyaluronic Acid (HA) is a
linear glycosaminoglycan composed of repeating disaccharide units
 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
of D-glucuronic acid and N-acetylglucosamine [98,99]. This naturally
occurring polysaccharide is negatively charged under physiological
conditions has molecular weights up to 10 7 Da. It is found mainly in
the extracellular matrix (ECM) and in the synovial fluids of joints
where it acts as a lubricant by which it reduces the friction of bones
due to its unique viscoelastic properties [100].
Because of its biocompatible, attractive physical properties and
possibilities for further chemical modifications, HA is an extensively
studied polysaccharide for the development of delivery systems for
tissue engineering applications [101–107]. However, unmodified
hyaluronic acid in aqueous solution displays poor mechanical proper-
ties. Therefore, to be suitable for protein delivery, HA-gels need phys-
ical and/or chemical stabilization. Usually, the carboxylic groups of
hyaluronic acid are derivatized to introduce functionalities that
make the polymer suitable for cross-linking [108–113].
In situ photopolymerization was applied by Park et al. to crosslink
thermosensitive hyaluronic acid/Pluronic composite hydrogels that re-
leased human growth hormone with kinetics correlated with the mass
erosion [105]. Subsequently, Patterson et al. synthesized glycidyl methac-
rylate modified hyaluronan hydrogels with different crosslink densities
and thus of different degradation rates and used them to prepare photo-
polymerized hydrogels as BMP-2 and/or VEGF releasing matrices for bone
regeneration. They demonstrated that all matrices displayed diffusion-
like release behavior of entrapped proteins and their degradation and re-
lease rates modulated the formation of mature bone, specifically affecting
the organization of the collagen matrix. Additionally, the co-delivery of an
angiogenic molecule (VEGF) in conjunction with an osteoinductive mol-
ecule (BMP-2) increased the extent of formed mineralized tissue [102].
Crosslinked, PEG diacrylate/thiolated hyaluronan hydrogels were
studied for the delivery of multiple growth factors (VEGF and/or
Ang-1) in both presence or absence of heparin, a molecule that forms
complexes, stabilizes the structure and modulates the release of
growth factors, according to the dissociation constant of the complex
growth factor/heparin. Greater neovascularization was observed
when the hydrogels were loaded with both growth factors [106,113].
Similar studies were conducted by the same group both in vitro and
in vivo using in situ gelled thiolated gelatin/thiolated hyaluronic acid/
PEG diacrylate based networks with or without disulfide bridged hep-
arin as part of the network and releasing combinations of growth fac-
tors like VEGF, Ang-1, PDGF and KGF. It was found that release
followed first order kinetics, with gelatin slightly increasing the
growth factor release rate, and heparin, slowing down the kinetics, be-
cause of its complexation with the growth factors. The extent of revas-
cularization and blood vessel maturation was found greater in gels
containing both heparin and gelatin than in those containing only gel-
atin or only heparin [104,114].
Other cross-linked hyaluronic acid networks developed for tissue en-
gineering purposes are for example those studied by Varghese et al. based
on aldehyde-HA/hydrazide-HA for the burst-free release of rhBMP-2
[107] or those developed by Leach et al. composed of photocross-linked
glycidyl HA or photocross-linked glycidyl HA/acrylate 4-arms PEG [108].
Other HA cross-linkable hydrogels for protein delivery that have been
proposed recently are based on tyramine-HA [109], on disulfide bridged
thiolated-HA [111], divinyl sulfone-HA/divinyl sulfone PEG [110] and adi-
pic acid dihydrazide-HA/methacrylate-HA [112].
Since HA is negatively charged at physiological pH, the protein re-
lease rate will be affected by the charge of the protein. Generally
speaking, cationic proteins are slower released than anionic or neu-
tral proteins. The release of proteins is not only influenced by charge
interactions with the polymer matrix, but also by the enzymatic deg-
radation of HA-based gels in the presence of hyaluronidase, which is
present in biological tissues [109,112].
3.1.2.1.4. Chitosan. Chitosan is a copolymer of glucosamine and N-
acetylglucosamine and is derived from the natural polymer chitin
(=poly N-acetylglucosamine) by (partial) deacetylation. The polymer
is positively charged at low pH's and uncharged and insoluble at neutral
687
and high pH's. Chitosan has mucoadhesive properties originating from
its cationic nature [92]. To make this polymer suitable for hydrogel for-
mation, water-soluble and cross-linkable derivatives were synthesized.
One of these approaches was realized by grafting 4-azidobenzoic acid to
the available free amine groups of lactose-modified chitosan. In the
presence of UV light the azido groups are converted into nitrenes that
are highly reactive species with the free amine groups on chitosan
yielding networks [115]. VEGF and FGF-2 were loaded into photo
cross-linkable modified chitosan hydrogels and their release and effica-
cy in inducing neovascularization in ischemia models were studied
[116–118]. The interactions between the nitrene groups and the
amine groups of the proteins were identified as the reason for the in-
complete release from these types of hydrogel [119,120]. Yeo et al.
reported that 80% of initially loaded VEGF was retained in the network
after cross-linking [118]. In situ gelling double networks of oxidized
dextrans and thiolated chitosan (semi-interpenetrating networks)
were recently proposed for healing purposes by Chen et al. These net-
works showed faster gelation kinetics as compared to auto-gelling thio-
lated chitosan hydrogels [121]. Recently, comprehensive and complete
reviews on chitosan hydrogels were published concerning their drug/
protein delivery aspects [122–125].
3.1.2.2. Proteins. Collagen, fibrin and gelatin are naturally occurring, en-
zymatically degradable proteins that have been extensively exploited as
main component in matrices for drug delivery and tissue engineering
purposes. Many of the hydrogel systems based on gelatin and collagen
are cross-linked using harsh conditions that exploit gluteraldehyde or
water-soluble carbodiimides and that can be detrimental for the encap-
sulated proteins [126]. However, also milder preparation conditions
were investigated. For instance, non-covalently crosslinked fibrillar col-
lagen can be used to create hydrogels by entanglements of collagen fi-
bers [127]. The mesh sizes of these entangled collagen fibers are quite
large and therefore diffusion of only very large proteins can be con-
trolled. Nevertheless, the protein release rate may still be lower than
expected based on diffusion coefficients due to weak interactions be-
tween collagen and loaded proteins [127].
Gelatin has a long history in clinical use with examples of gelatin
based systems with a variety of physical geometries. Gelatin derived
from collagen can be positively or negatively charged depending whether
collagen depolymerization is carried out at acidic or basic conditions. The
charge of gelatin network can be utilized to complex oppositely charged
proteins serving as additional feature to control release rate [128]. Gelatin
based hydrogels have been used for the delivery of FGF-2 [129], HGF and
VEGF [130]. Nowadays, gelatins can be produced in yeast cells recombi-
nantly leading to slightly different polymer properties. The advantage is
that properties such as molecular weight, amino acid sequences and iso-
electric points can be tailored precisely. Degradation times, swelling
properties and ultimately also drug release kinetics are affected by the de-
sign of recombinant gelatin. Only a few recombinant gelatin hydrogels
have been investigated for their protein delivery properties [131,132]. Fi-
brin is a fibrous, non-globular protein derived from fibrinogen, a protein
of the blood coagulation cascade. Fibrinogen, a water soluble protein, is
converted into fibrin that polymerizes to yield a gel due to the action of
thrombin. Fibrin networks are gradually resorbed in vivo by the secretion
of fibrinolytic enzymes and as such has been widely used for formulating
depot systems for proangiogenic growth factor delivery. Commercial fi-
brin glues are also used in the clinic for in situ application.
In an early clinical trial, VEGF was injected in an in situ forming fi-
brin matrix into the right popliteal region of a man suffering from
claudication. Although substantial growth of newly formed vessels
was observed, a high initial burst occurred within the first 24 h. To
overcome this issue, VEGF was later covalently bound to the matrix
and released by enzymatic cleavage [133,134]. Similarly, FGF-2 was
electrostatically immobilized in fibrin hydrogels using heparin
[135]. These strategies showed in animal model enhanced neovascu-
larization and reduced fibrosis and inflammation.
688 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
Fig. 5. Gelation mechanism and protein encapsulation methods for PEGylated fibrin
gels. Reproduced from ref. [136].
Recently, Drinnan et al. reported on an elegant approach on the
multimodal release of different proteins from the same hydrogel.
They developed an injectable PEGylated fibrin gel designed for the re-
lease of PDGF-BB and TGF-β1 with distinct kinetics (Fig. 5). Growth
factors were loaded into PEGylated fibrin gels via 3 mechanisms: en-
trapment, conjugation through a homobifunctional amine reactive
PEG linker, and physical adsorption on the fibrin matrix. PDGF-BB
was entrapped during thrombin-mediated crosslinking leading to its
diffusion-controlled release over 2 days. TGF-β1 was both conjugated
through the PEG linker and bound to the matrix via physical adsorp-
tion, delaying the release rate of TGF-β1 up to 10 days. Further, the
release rate was highly correlated to gel degradation rate, indicating
that TGF-β1 release was degradation-controlled [136].
Fibrin hydrogels were also used to fabricate circuits for the con-
trolled release of chondroitinase ABC (a bacterial enzyme capable of
digesting the glycosaminoglycan (GAG) side chains from chondroitin
sulphate proteoglycans) in spinal cord lesion treatment. Sustained de-
livery of the protein in vivo for about 3 weeks was demonstrated as
compared to bolus injection. Furthermore, after 3 weeks, six times
more bioactive chondroitinase ABC and 37% lower levels of inhibitory
glycosaminoglycans (GAG) were found in the spinal cord when hydro-
gels were used vs intraspinal injection of enzyme solution [137].
3.1.2.2.1. Affinity-based drug release. Affinity based drug delivery sys-
tems utilize physical interactions between the therapeutic drug and the
delivery system to manipulate drug loading and control release kinetics.
The easiest method to modulate release kinetics of loaded proteins is
physical adsorption, involving the formation of ionic complexes by elec-
trostatic interactions between charged polymers oppositely charged pro-
teins [138–140]. Many charged polymers like hyaluronan, chitosan,
alginate, acidic gelatin can be potentially used to retard drug release [89].
Heparin sulfate is a naturally occurring, highly sulfated anionic
glycosaminoglycan found in the ECM that is a natural matrix responsi-
ble for immobilizing and releasing various proteins that influence the
natural processes of adhesion, migration, proliferation, differentiation.
This glycosaminoglycan possesses a specific binding domain for many
growth factors that interact with heparin via non-covalent bonding
[32,141]. Besides enhancing matrix ability to retain the encapsulated
protein, it has been shown that affinity binding with heparin also stabi-
lizes the drug, preserving its structure and functionality during handling
of the material [142]. Heparin is generally incorporated within the de-
livery system through many strategies as addressed in recent state of
art reviews of affinity-based drug delivery [11,143,144]. Therefore,
this paper deals only with some general aspects of this topic.
One approach to incorporate heparin in a hydrogel structure is to co-
valently link the heparin moiety to the polymer. Many examples of
growth factor delivery from heparin modified polymer hydrogels have
been reported. Hubbell used a bi-domain peptide that was covalently
attached to fibrin on one site and to heparin on the other extremity.
Growth factors of different affinity for heparin (bFGF >bNGF, BDNF,
neutrophin-3) [32,33] were loaded and released with slower kinetics
as compared to fibrin hydrogels. In a similar approach adopted by Kiick
et al. four arms thiolated PEGs were reacted with monomaleimide-
functionalized low molecular weight heparin and cross-linked in aque-
ous medium after addition of heparin-binding peptide modified PEGs.
This delivery system as well other similar hydrogels have been exten-
sively used for the controlled delivery of many growth factors for differ-
ent applications [145–148].
Heparinization was applied also to collagen [149,150], alginate
[151], hyaluronan [152] and pluronic [153] using different chemis-
tries and cross-link methods.
Polymers were complexed with growth factors binding heparin also
by electrostatic interactions. Sulfonation of hyaluronan and uronic acid
monomers of alginate allowed non-covalent interaction with heparin-
binding proteins [80,84]. Although heparin bound hydrogels are defi-
nitely the most studied affinity systems to date, some challenges still re-
main such as unstable protein-heparin bonding in vivo, large amounts
of heparin necessary to achieve sufficient drug loading and heparin-
induced thrombocytopenia [154]. Affinity hydrogel systems based on
the mechanism of metal-ion-chelation and binding of histidine tagged
proteins have been proposed as an effective alternative to the
heparin-containing systems. To mention, Metters et al. developed
PEG-co-methacrylated iminoadiacetic acid displaying affinity binding
for histidine tagged proteins through divalent metal ions such as nickel
and copper. The authors also elaborated a mathematical model to pre-
dict the release rate of proteins from the hydrogels [34].
Phage display is a technique that has been exploited to identify pep-
tides with affinities for growth factors that could be coupled to the gel-
ling polymer [155].
As an example Anseth et al. developed an affinity peptide-
functionalized PEG hydrogel with the ability to sequester monocyte che-
motactic protein 1 (MCP-1), a chemokine that induces the chemotaxis of
monocytes, dendritic cells, and memory T-cells. Affinity peptides were
immobilized in PEG hydrogels via a thiol-acrylate photopolymerization.
The release of encapsulated recombinant MCP-1 from PEG hydrogels
was tailored by altering the spacer distance between the affinity peptide
and the crosslinking site [35]. Antibodies were also exploited for the spe-
cific binding of proteins onto scaffolds [156].
Molecular imprinting is a technique used to synthesize biomimetic
polymer networks with template-shaped cavities that have affinity for
specific molecules of interest (the templates). The nature of the interac-
tion can vary from non-covalent bonds to metal coordination and even
covalent bonds. This technique has been applied for drug and protein
delivery and recognition. However, although some success has been
achieved in the molecular imprinting of small molecule drugs, huge
challenges still exist with imprinting of proteins [42,77,125,157].
3.2. Covalent binding
Proteins, mostly exploiting their reactive amine and thiol groups,
can be covalently bound to polymer matrices via functional groups
like hydroxy, amine, carboxyl groups that, if not naturally present in
the structure of the polymer, have to be introduced by functionalization
reactions, blending or co-polymerization. The release of the protein is
mediated either via hydrolysis or reduction reactions or (cell-mediated)
enzymatic cleavage. This type of mechanism leads to on-demand re-
lease of loaded proteins, mimicking the enzymatic activity naturally
occurring in ‘healthy’ ECM. However, stability and maintenance of
biological activity of the protein may represent an issue.
Verheyen et al. recently reported on an efficient strategy to intro-
duce methacrylamide groups on the lysine residues of a model protein
(lysozyme) for immobilization and triggered release from a polyacryl-
amide or dextran hydrogel network. They designed a novel spacer
 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
unit containing a disulfide bond, such that the release of the protein can
be triggered by reduction [36,37].
PEG hydrogels containing pendant RGD peptides can be formed
using amine-functionalized MMP ligands as a cross-linker. Close prox-
imity of the gel to cells was mediated by RGD peptide and BMP-2
bound to the matrix was delivered to the site of the bone defect [38].
Other popular systems that rely on covalent cross-linking are those con-
taining peptide linkers susceptible to the enzymatic activity [158–160].
3.3. Dual/multiple delivery systems
Biomedical hydrogels that can deliver multiple growth factors in a
multimodal mode and provide desirable pore structure and porosity
to potentially encapsulate cells, have considerable potential as future
therapeutic tools in tissue engineering. The use of growth factors
loaded microspheres embedded in the hydrogel structure is a com-
mon approach to multimodal protein delivery.
Microparticles of acidic and basic gelatin were used as carriers for the
individual delivery of two different growth factors and embedded in a hy-
drogel matrix, composed of glycidyl methacrylated dextran (Dex-GMA)/
gelatin. This hybrid system allowed the independent release of BMP-2
or IGF-1(Fig. 6) that facilitated cell attachment, proliferation, metabolism,
and osteoblastic differentiation of cells in a synergistic manner [39,161].
Fibrin hydrogels were complexed with heparin-functionalized PLGA
nanoparticles for the release of VEGF for revascularization purposes [40].
A similar approach was adopted by Burdick et al., who formulated
PLGA microspheres into PLA-PEG-PLA hydrogels for the delivery of mul-
tiple neutrophins with individual release rate [18]. Similarly, Biondi
et al. used PLGA microspheres in collagen gels and collagen-HA semi-
interpenetrating networks. The protein release kinetics and delivery
onset strongly depended on the complex interplay between protein
transport through the PLGA matrix and in the collagen-based release
media, and water sequestration within the scaffold [162,163]. Physical
hydrogel blends composed of hyaluronan (HA) and methyl cellulose
Fig. 6. Cumulative protein(s) release of BMP-2 and/or IGF-1 from scaffolds containing
BMP-2/IGF-1 combination (BMP-2 and IGF-1) (C); or from scaffolds containing a mix-
ture of microparticles loaded with BMP-2 or IGF-1 (F). Reproduced from ref. [39].
689
(MC) were designed by Shoichet et al. for independent delivery of one
or more therapeutic proteins, from 1 to 28 days, for the ultimate appli-
cation of spinal cord injury repair. To achieve a diversity of release
profiles, they exploited the combination of fast diffusion-
controlled release of dissolved solutes from the HAMC itself and
slow drug release from PLGA particles dispersed within the gel
[41]. Methacrylated hyaluronan networks encapsulating alginate
microparticles enhanced mesenchymal stem cell chondrogenesis
following delivery of TGF-β3 in vitro and in vivo [164].
4. Conclusions and outlook
The importance of growth and adhesive factors and their synergistic
interplay during healthy tissue development is nowadays a well-
established concept. However, their administration in the form of bolus
injection to promote tissue regeneration and repair has shown to be
often ineffective and potentially harmful due to the short duration of ac-
tion of the encapsulated protein drug. Such a delivery approach of
growth factors has been applied in a number of clinical trials of unsatis-
factory outcome. While some phase I clinical trials (angiogenic gene
therapy and human growth factor FGF-I infusion for coronary artery dis-
ease) [165,166] have reported promising results, phase II clinical trials,
recruiting a larger number of patients, have not shown the expected
benefits to patients. To mention, VIVA (Vascular endothelial growth fac-
tor in Ischemia for Vascular Angiogenesis) trial conducted on 178 pa-
tients by intravenous and intracoronary infusion of VEGF [167] and
FIRST trial comprising intracoronary infusions of FGF-2 on 337 patients
[168], did not result in effective commercially available formulations
for the treatment of cardiovascular diseases. On the other hand, positive
outcomes resulted from clinical trials on growth factors administered
through the use of delivery systems that controlled to some extent the
protein release rate. In this way the problems associated with the appli-
cations of bolus injections are overcome. For example, after successful
clinical trials, two growth factors BMP-2 (clinical trial BESTT) [169,170]
and BMP-7 (clinical trial OP-1 Putty) [171,172] immobilized in collagen
sponge and matrix, respectively, are currently commercially available
for the treatment of bone fractures and defects. Also in the field of cardio-
vascular diseases, encapsulation of FGF-2 into alginate microcapsules led
to some positive results in clinical trial phases (clinical trial Polymer)
[173,174]. Finally, Regranex® Gel is the only growth factor delivery sys-
tem of PDGF that obtained full FDA approval for clinical use.
Generally speaking, formulation of proteins in controlled delivery
systems allow to sustain the release and lower the doses of potent
growth factors, that, if administered by infusion, need supraphysiolo-
gical concentrations, leading to severe side-effects and do not assure
therapeutic efficacy over a sufficient time-span, because of rapid deg-
radation/elimination. Moreover, the design of smart delivery systems
may offer the possibility to deliver multiple growth factors with inde-
pendent release rates and to load or attract cells for the regeneration
of damaged tissues. This review summarizes the efforts that have
been addressed on facilitating the delivery of single or multiple
growth factors with appropriate control over temporal and spatial
presentation of these biomolecular cues. Current available technolo-
gies that have demonstrated potential in the modulation of release
profiles, according to the specific therapeutic needs, including tradi-
tional diffusion/swelling/degradation mediated, on-demand, affinity
and covalent binding based delivery are presented.
However, some challenges still exist. For example, highly inconve-
nient encapsulation methods often involving post-loading techniques
and low encapsulation efficiency, the stability of the protein, the safety
and biocompatibility of the delivery devices and the difficulties to trans-
late in vitro results to in vivo situation. Further efforts in the design or
optimization of materials with minimal tissue response, sufficient stiff-
ness, appropriate degradation and release profiles are needed in order
to make the translational step between academia and clinics successful.
690 R. Censi et al. / Journal of Controlled Release 161 (2012) 680–692
Acknowledgement
This project was financially supported by L'Òreal – Unesco, “For
Women in Science 2011”.
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