Drug Delivery Reviews

Controlled drug delivery in tissue engineering
Marco Biondi, Francesca Ungaro, Fabiana Quaglia, Paolo Antonio Netti
Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, Naples, Italy
Received 1 August 2007; accepted 9 August 2007
Available online 11 October 2007
Abstract
The concept of tissue and cell guidance is rapidly evolving as more information regarding the effect of the microenvironment on cellular
function and tissue morphogenesis become available. These disclosures have lead to a tremendous advancement in the design of a new generation
of multifunctional biomaterials able to mimic the molecular regulatory characteristics and the three-dimensional architecture of the native
extracellular matrix. Micro- and nano-structured scaffolds able to sequester and deliver in a highly specific manner biomolecular moieties have
already been proved to be effective in bone repairing, in guiding functional angiogenesis and in controlling stem cell differentiation. Although
these platforms represent a first attempt to mimic the complex temporal and spatial microenvironment presented in vivo, an increased symbiosis of
material engineering, drug delivery technology and cell and molecular biology may ultimately lead to biomaterials that encode the necessary
signals to guide and control developmental process in tissue- and organ-specific differentiation and morphogenesis.
2007 Elsevier B.V. All rights reserved.
Keywords: Tissue engineering; Drug delivery; Biomaterials; Growth factors; Scaffold
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
2. Extracellular matrix mimicry as guideline for scaffolds design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
3. Tissue engineering scaffolds as controlled release matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
3.1. Interspersed signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
3.2. Immobilized signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
3.3. Signal delivery from cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
4. Delivery systems for proteins of potential interest in tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.1. Continuous delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.1.1. Non biodegradable systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.1.2. Biodegradable systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.2. Onoff delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
4.2.1. Programmed delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
4.2.2. Triggered delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
5. The issue of delivery system integration in three-dimensional scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Available online at www.sciencedirect.com
Advanced Drug Delivery Reviews 60 (2008) 229242
www.elsevier.com/locate/addr
Abbreviations: bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BSA, bovine serum albumin; CASD, computer-aided scaffold design;
DS, delivery systems; ECM, extracellular matrix; EGF, epidermal growth factor; EVAc, ethylene-vinyl acetate copolymers; GF, growth factor; HBDS, heparin-based
delivery systems; NT-3, neurotrophin-3; PA, peptide amphiphile; PCL, poly(-caprolactone); PDGF, platelet derived growth factor; PEG, poly(ethylene glycol); PEO, poly
(ethylene oxide); PLA, poly(lactide); PLGA, poly(lactide-co-glycolide); POE, poly(ortho esters); PTH, parathyroid hormone; SFF, solid free-form fabrication; TE, tissue
engineering; TGF-1, transforming growth factor-beta1; VEGF, vascular endothelial growth factor.
This review is part of the Advanced Drug Delivery Reviews theme issue on Emerging Trends in Cell-Based Therapeutics.
* Corresponding author. Tel.: +39 817682408; fax: +39 817682404.
E-mail address: nettipa@unina.it (P.A. Netti).
0169-409X/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2007.08.038
1. Introduction
Tissue engineering (TE) aims at the repairing and restor-
ing damaged tissue function employing three fundamental
tools, namely cells, scaffolds and growth factors (GFs)
which, however, are not always simultaneously used [1,2].
On the other hand, summoning recent experimental and cli-
nical evidences indicate that the success of any TE approach
mainly relies on the delicate and dynamic interplay among
these three components and that functional tissue integration
and regeneration depend upon their sapient integration [3,4].
Future generation of scaffolds will have to provide not only
the adequate mechanical and structural support but also have
to actively guide and control cell attachment, migration, pro-
liferation and differentiation. This may be achieved if the
functions of scaffold are extended to supply biological signals
able to guide and direct cell function through a combination
of matricellular cue exposition and GF sequestration and
delivery [2,5]. Therefore an ideal scaffold should possess a
three-dimensional and well defined microstructure with an
interconnected pore network, mechanical properties similar to
those of natural tissues, be biocompatible and bio-resorbable
at a controllable degradation and resorption rate as well as
provide the control over the sequestration and delivery of
specific bioactive factors to enhance and guide the regener-
ation process [6,7].
Recent advances in micro- and nano-fabrication technologies
offer the possibility to engineer scaffolds with a well defined
stereoregulated architecture providing a control of cellular spa-
tial organization, mimicking the microarctitectural organization
of cells in native tissues [6,813]. Furthermore, by combining
material chemistry and processing technology, scaffold degra-
dation rate can be tuned to match the rate of tissue growth in such
a way that the regenerated tissue may progressively replace the
scaffold [1416]. Enhancing further the functionality of these
already complex matrices by encoding in them the capability
to expose an array of biological signals with an adequate dose
and for a desired time frame, represents the major scientific and
technological challenge in tissue engineering today. Bolus ad-
ministration of GFs would not be effective in these cases since
they rapidly diffuse from the target site and are readily enzy-
matically digested or deactivated. Moreover, local delivery and
prolonged exposition of the bioactive molecules is necessary to
minimize the release of the agent to non-target sites, and support
tissue regeneration which normally occurs in long time frames
[17]. Thus, it has been soon realized that by integrating con-
trolled release strategies within scaffolding materials may lead to
novel multifunctional platforms able to control and guide the
tissue regeneration process [1822]. Through the recapitulation
of the spatial and temporal microenvironments presented by
natural extracellular matrix (ECM), it is hoped to successfully
guide the evolution of the construct towards neotissue formation,
inducing on-demand different pathways to cell response. In this
perspective, TE can be viewed as a special case of controlled
drug delivery in which the presentation of bioactive molecules is
finely tuned to dynamically match the needs of the ingrowing
tissue.
The control over the regenerative potential of TE scaffolds
has dramatically improved in recent years, mainly by using drug
releasing scaffolds or by incorporating drug delivery devices
into TE scaffolds [17,19,23]. For example, on-demand respon-
sive matrices based on enzymatically-triggered release of GFs
have been realized by introducing enzyme-cleavable linkers for
covalent interaction between the released molecule and a bio-
active protein [8]. Furthermore, potent morphogenetic factors
have been loaded in polymeric depots and included into various
biomaterials to enable a sustained and controlled point source
release while preserving bioactivity as reviewed extensively in
the literature [1922]. Despite the impressive enhancement in
tissue guidance and regeneration offered by GF releasing scaf-
folds, several challenges have yet to be broadly resolved. These
include the tight control over time and space of tiny quantities
of multiple biomacromolecular factors and of their gradients
within the interstitial space of the scaffold as well as at the
scaffold-tissue interface. Moreover, there is a paucity of studies
regarding the effective dose in the local microenvironment, the
magnitude of the spatial and temporal gradients and the de-
velopment of technological strategies to integrate and position
drug delivery devices with a submicrometric spatial resolution
within the scaffolds.
In this review we will first summarize the complex processes
of cell guidance occurring within native ECM along with the
most updated strategies to design biomimetic scaffolds able to
recapitulate in part these processes. A synthetic overview of the
most promising approaches in controlling the release of the
relevant factors in TE will follow. Finally, the main challenges
to design novel scaffolds with time and space orchestrated
exposure of biomacromolecular moieties will be presented and
critically discussed.
2. Extracellular matrix mimicry as guideline for scaffolds
design
ECM, the natural medium in which cells proliferate, differ-
entiate and migrate, is the gold standard for tissue regeneration
[24]. Cell-ECM interaction is specific and biunivocal. Cells
synthesize, assembly and degrade ECM components respond-
ing to specific signals and, on the other hand, ECM controls
and guides specific cell functions. The continuous cross-talk
between cells and ECM is essential for tissue and organ
development and repair, providing both a structural guidance
(i.e. directional cell migration) and cell guidance at a molecular
level (i.e. signaling molecule delivery).
ECM is a highly organized dynamic biomolecular environ-
ment in which many proliferationadhesiondifferentiation
motifs, governing cell behaviours, are continuously generated,
sequestered and released, inducing matrix synthesis and degra-
dation (Table 1). These motifs are locally released according to
cellular stimuli, generally occurring upon degradation of the
adhesion sites binding them to the ECM [25]. Cells are
attached to ECM through molecules belonging to the integrin
family [26] and recognize specific amminoacid sequences
through cell surface receptors. Integrin receptors are recruited
in microdomains of cell membrane, and in these areas integrins
230 M. Biondi et al. / Advanced Drug Delivery Reviews 60 (2008) 229242
communicate with structural and signaling molecules influ-
encing transport, degradation and secretion of ECM mole-
cules, endocytosis and cellular fate [27,28]. Moreover, solid-
state, structural ECM molecules act as reservoirs for secreted
signaling molecules for their on-demand release [29,30].
Besides insoluble factors and proteins presented on the surface
of adjacent cells, the molecular cues that mainly define the
microenvironment consist of soluble macromolecules, such as
GFs [31].
GFs are protein molecules specific for intercellular and
cell-ECM signaling, which are involved in ECM dynamic
properties through specific surface receptors, driving GFs
regulatory activity [31,32]. GFs are locally secreted by ECM,
in which they are stored in insoluble/latent forms through
specific binding with glycosaminoglycans (e.g. heparins), and
can elicit their biological activity once released. During tissue
morphogenesis the presence of soluble GFs guides cellular
behaviours, thus governing neo-tissue formation and organi-
zation. The sequestration of GFs within ECM in inert form is
necessary for rapid signal transduction, allowing extracellular
signal processing to take place in time frames similar to those
inside cells. Moreover, concentration gradients of GFs play a
major role in ECM maintenance and equilibrium because
the gradients direct cell adhesion, migration and differentia-
tion deriving from given progenitor cells and organize pat-
terns of cells into complex structures such as vascular
networks and nervous system [3335]. Thus, spatial patterns
in tissues are dictated by both the architectural features of
ECM and concentration profiles/gradients of diffusible bioac-
tive factors [36].
Recent research in biomaterial science has been driven by
biomimicry-inspired design of materials to recreate the natural
three-dimensional architecture. Several micro- and nano-
fabrication strategies have been applied in an attempt to mimic
the spatial distribution of the fibrillar structure of ECM, which
provides essential guidance for cell organization, survival
and function [913]. These technologies include gas foaming,
solid free-form fabrication (SFF) (3D printing, 3D plotting),
molecular and nanoparticulate self assembly, electrospinning,
molecular and nano-templating [6,9,12,13,37]. Albeit the
influence of scaffold microarchitecture and stereomorphology
on cell function and guidance has been proved in several
systems and with different cell types, the underlying mechan-
isms by which cells recognize and decode topological infor-
mation are still unclear. A wide variety of biodegradable and
biocompatible polymers have been processed to fabricate
stereoregulated scaffolds, including synthetic polymers, such
as poly(lactide) (PLA), poly(glycolide) and their copolymers
poly(lactide-co-glycolide) (PLGA), poly(-caprolactone)
(PCL), and natural polymers, such as collagen, protein, and
Table 1
Main molecular components of ECM and their role
Component Description Function Location Ref.
Collagens Fibrillar protein forming ECM backbone Resistance to tensile strength Ubiquitous [148,149]
Matrix scaffolding
Cellcell interaction
CellECM interaction
Modulation of ECM morphology
Modeling the framework of connective tissues
Proteoglycans Carbohydrate polymers composed of
a polypeptide backbone covalently attached to
glycosaminoglycan chains
Filler substance between cells Ubiquitous [150]
Binding to cations/water
Transport of small molecules in ECM
Resistance to conprhessive stresses
Cell adhesion, migration and proliferation
Ligand/receptors of signaling molecules
Hyaluronan Negatively charged glycosaminoglycan polymer Cell proliferation Ubiquitous [151,152]
Cell differentiation
Cell migration
Transport of small molecules in ECM
Laminins Glycoproteins of basal lamina Development and maintenance of basement membranes Basement
membranes
[153]
Receptor-mediated cell attachment
Cell signaling/migration
Fibronectin High molecular weight glycoprotein binding to
ECM components
Cell adhesion Cell surfaces [154]
Cell migration
Cell proliferation
Matrix adhesion
Fibroblast activation
Elastin Hydrophobic, cross-linked insoluble protein Tissue resilience and elasticity Blood vessels [155]
Lungs
Ligaments
Skin
Bladder
Growth factors Proteins associated to ECM or heparin sulphate able to
induce cell migration, proliferation, differentiation,
in soluble form, upon activation of latent forms
Information processing Ubiquitous [33,156]
Cell signaling
ECM synthesis/remodeling
fibrinogen [1416] (Table 2). Most of the technologies used to
date suffer from the limitation that the scaffolds are preformed
and cell have to be loaded within the interstitial spaces which
are often smaller than the cell size.
3. Tissue engineering scaffolds as controlled release
matrices
Future generations of TE scaffolds with extended function-
ality and bioactivity require an increased integration with cell
and molecular biology, to identify novel design parameters and
bio-inspired design approaches (Fig. 1). Synthetic bio-inspired
ECM should broadcast specific cellular events, such as the
recruitment and the enhancement of migration of peripheral host
cells into the scaffold, or should guide morphogenetic processes
taking place within its interstices through the fine tuning of
spatial and temporal gradients of growth and morphogenetic
factors [3842].
The local concentration and the spatio-temporal gradients of
a molecule depend upon a delicate balance between the
transport properties of the scaffold, the binding and degradation
rate of the molecule and its generation rate. Once transport
mechanisms and biological decay time constant of the bioactive
molecule are known, it is virtually possible to engineer any
complex static or dynamic gradient distribution within the
scaffold by including artificial reservoirs able to deliver the
relevant biomacromolecules at a predefined rate. However, the
magnitude of the gradients and the optimal time frame to elicit
the desired cell response are yet unknown. Significant
advancement in scaffold design can be achieved through a
deeper understanding of the quantitative aspects of the influence
of amount of morphogens and their gradient on cell fate.
One of the possible attempts to control molecular microen-
vironment is to use a TE scaffold as a controlled release
platform. This can be achieved by the incorporation of signaling
molecules in three-dimensional scaffolds through their simple
dispersion in the matrix, or their immobilization by electrostatic
interactions with and covalent bonding to the scaffold. A gene-
mediated approach is also feasible by introducing into target
cells nucleic acid encoding for a specific protein signal inducing
tissue regeneration. In this way, each cell can act as a single
source point for release of the signaling proteins.
3.1. Interspersed signals
Signaling molecules can be integrated within scaffolds by
simply interspersing them in the matrix. Although this method
presents several shortcomings, it has been widely used in the
literature. Most of these approaches are carried out by hydrogel-
based scaffolds in such a way that the hydrogel acts
simultaneously as a scaffold and a controlled delivery platform.
Both naturally-derived (collagen, fibrin, chitosan) [14,15] or
Table 2
Materials for tissue engineering scaffolds
Material Relevant features and application Ref.
Naturally-derived Collagen-based scaffolds Soft tissue repair [16,157,158160]
Cell differentiation
Capillary engineering
Dermis engineering
Vascularized adipose tissue
Hyaluronic acid (HA) and derivatives Regeneration of skin, cartilage [161]
Patterning of cell growth
Collagen-HA gels Control of vascular sprouting [162]
Chitosan Chitosan microsphere-integrated scaffold [163,164]
Cartilage engineering
Fibrin Vessel engineering [165,166]
Release of fibroblasts
Gelatin Trachea engineering [167,168]
Bone engineering
Alginate Vascular engineering [100]
Synthetic Poly(glycolic acid) (PGA) Musculoskeletal tissue [8,169]
Poly(lactic acid) (PLA) engineering
Polylactide-co-glycolide (PLGA) Cartilage regeneration
Fibrovascular engineering
Poly(-caprolactone) (PCL) Skin engineering [170]
Polyethylene glycol (PEG) Bone formation [171]
Oligo(poly(ethylene glycol) fumarate) (OPF) Cartilage engineering [172]
GF release from gelatin microspheres
Inorganic Tricalciumphosphate (TCP) Bone substitute [173,174]
Hydroxyapatite (HA)
Semi-synthetic Cross-linked thiolated HA Neurite growth and support [175,176]
Vocal fold repair
Esterified hyaluronan (HYAFF derivatives) Cartilage engineering [160]
synthetic hydrogels (poly(ethylene glycol) (PEG)) have been
used [14,16]. Natural materials possess innate capacity of
cellular interaction and undergo a mainly cell-related degrada-
tion profile, while synthetic materials lack the cellular/
biomolecular recognition but can be more readily manipulated
in terms of macro- and microscopic properties [8].
Under particular conditions, cells and other bioactive entities
can be safely encapsulated in hydrogels before gelation, the
structural parameter that mainly controls transport properties
being the cross-linking density and stability [43,44]. The
dispersed factors move through the mesh network of the
crosslinked gel by a combination of diffusion and degradation
mechanisms. Particularly, when the hydrodynamic diameter of
the diffusing species is smaller than the average hydrogel mesh
size, diffusion mechanisms prevail. This leads to a fast, not
sustained release (hours to days) of the dispersed molecules,
which is not very useful for a TE approach. On the other side,
when the molecular size approaches hydrogel mesh size, the
release is mainly controlled by the degradation of the polymer
backbone, which can occur either by hydrolytic or enzymatic
(i.e. on cell demand) mechanisms [45].
A variety of synthetic and natural polymers have been
utilized for the design of hydrolytically degradable hydrogels
in which chemical or physical cross-linking offers the possi-
bility to control the diffusion of solubilized hydrophilic
macromolecules [4648]. An important formulation challenge
when fabricating hydrogels for protein delivery is the choice of
the cross-linking method, which must not involve steps with
potentially adverse effects on protein stability. Naturally-derived
hydrogels such as collagens, hyaluronic acid and derivatives are
frequently used due to their well-known biocompatibility. For
example, collagen-based scaffolds can induce transgene expres-
sion and physiological improvements for bone regeneration [49]
and wound healing [50]. Furthermore, collagen modified with
poly(L-lysine) has been shown to promote aspecific interactions
between plasmid molecules and collagen, resulting in plasmid
binding and release [51].
Among synthetic hydrogels, PEG-based materials are widely
applied in TE. Release mechanism and degradation rate can be
tailored through chemical modifications by inserting units
affecting PEG functionality, such as fumarate, lactic acid,
caprolactone, hyaluronic acid units [5254]. Pseudo-zero-order
release kinetics can be attained by modulating the degradation
rate of cross-links and cross-link density by inserting additional
double bonds [55], or through the optimization of cross-linking
agent amount [56,57]. Proteolytically-sensitive PEG-based net-
works have been engineered by the group of Hubbell by
encoding signals able to finely control the release of bioactive
agents based on a cell-demanded logic [58]. These synthetic
analogs of ECM can thus represent a first step towards the
reproduction of the dual-reciprocity mechanism (i.e. cell-material
cross-talk) occurring in a native ECM.
Synthetic solid biodegradable materials have been tested for
drug delivery in TE, especially for hard-tissue repair. However,
also the fabrication of protein-loaded solid scaffolds poses
serious issues regarding protein leaching and stability [19],
such as: i) the use of organic solvents during the manufac-
turing processes; ii) protein elution occurring during scaffold
processing (e.g. hydrosoluble porogen leaching processes);
iii) exposure of the protein to high temperatures (e.g. melt
processing); iv) generation of organic-aqueous interfaces during
scaffold processing (e.g. scaffolds made of lipophilic synthetic
biodegradable polymers). Thus, direct encapsulation of proteins
in solid scaffolds should be preferentially carried out under mild
techniques, such as gas foaming and electrospinning, eventually
combined with particulate leaching. Despite these limitations,
Fig. 1. The concept of multifunctional TE scaffolds based on controlled delivery technology. A hydrogel or solid state three-dimensional scaffold (A) releases GFs,
either encapsulated in controlled delivery systems, or dispersed/tethered within the scaffold, according to predetermined spatial gradients and with controlled kinetics,
occasionally with retarded delivery onset (B). The broadcasting of the appropriate molecular signals towards the surrounding defective tissue induces the desired
cellular responses (C), which are triggered by the spatio-temporally controlled presentation of the proper tissue-inductive signals.
scaffolds made of PLGA have been successfully used to
engineer tissues. Release through PLGA occurs by a combined
diffusion-degradation mechanism which leads to the progres-
sive generation of acids, particularly in the bulk regions of the
scaffold [59], and have been produced for the prolonged release
of vascular endothelial growth factor (VEGF) for bone TE [60]
and vascular bed generation [61], or a plasmid encoding for
platelet-derived growth factor (PDGF) in vascular induction
[62].
The few examples described above show that scaffolds in
which the bioactive agent is simply dispersed may not offer the
necessary control over release kinetics and extent. Therefore,
new designing approaches have been exploited to provide a
control over spatial and temporal release pattern.
3.2. Immobilized signals
Polymer scaffolds can be modified to interact with signaling
molecules, thereby hindering their diffusion out of the polymer
platform, thus prolonging their release. Signal immobilization
can occur through reversible association with the scaffold (i.e.
binding/de-binding kinetics), irreversible binding to the poly-
mer. Alternatively, signals can be released upon degradation of a
linking tether or the matrix itself, which immobilize the
molecule within the scaffold. The number of binding sites, the
affinity of the signal for these sites, and the degradation rate of
the scaffold are key determinants of the amount of bound signal,
as well as the release profile [40,63].
In the case of GFs, the most common approach to improve
release kinetics of the immobilized molecule relies on the use of
heparin-immobilized scaffolds. Actually, heparin grafted on the
surface or chemically bound to the polymer can interact with
heparin-binding GFs [6470]. Heparin-based delivery systems
(HBDS) have been largely employed to control GF concentration
with fibrin scaffolds, in which a synthetic linker peptide, capable
of binding heparin, is covalently attached to fibrin [64,66,67].
Conjugating capacity and release rate were found to be dependent
upon the number of binding sites, the affinity of factors towards
binding sites and the degradation rate of the scaffold. HBDS for
the controlled release of neurotrophin-3 (NT-3) were fabricated
using a linker peptide containing a Factor XIIIa substrate to
sequester heparin within fibrin gels [67]. The authors showed that
heparin not only binds NT-3, hindering its diffusion, but also
allows an active release mechanism, which is triggered by cell-
associated enzymatic activity. In so doing, release of NT-3 was
extended for 9 days, and the neural fiber density was increased in
spinal cord lesions. Also heparinized cross-linked collagens for in
vivo endothelial cell seeding have been studied with respect to GF
binding and release [65,70]. Collagen matrices were modified
with heparin for binding and release of basic fibroblast growth
factor (bFGF), by a conjugation reaction between carboxyl
groups of heparin and amino groups of collagen [65]. In a
systematic study, Nillesen et al. [70] prepared and characterized
five porous scaffolds consisting of collagen, collagen with
heparin, and collagen with heparin plus one or two GFs (bFGF
and VEGF). The scaffolds obtained by collagenheparin
conjugation and GF incorporation displayed the highest density
of blood vessels and most mature blood vessels after subcutane-
ous implantation in rats. Also synthetic scaffolds based on
heparinconjugated PLGA fabricated by a gas-foaming/salt-
leaching method showed the ability to sustain bFGF release over
20 days and promote blood vessel formation in vivo [68]. More
recently, a heparin-conjugated PLGA scaffold for the sustained
delivery of bone morphogenetic protein (BMP)-2 was used to
enhance ectopic bone formation [69]. The amount of heparin
conjugated to the PLGA scaffolds could be increased up to 3.2-
fold by using scaffolds made from star-shaped PLGA, as
compared to scaffolds made from linear PLGA.
Immobilization of GFs within the scaffold can be also
achieved by their covalent conjugation to the polymer [7173].
Covalent conjugation of GFs was intuitively thought not feasible
since chemical bond could negatively affect their biological
activity. However, if appropriately designed, covalent conjuga-
tion, or tethering, of GFs, have been proven a valuable strategy to
retain GFs for longer time periods at the delivery site, offering an
important control over the amount and spatial distribution of these
molecules in solid matrices. Whether covalently conjugated GFs
act directly in the immobilized form, mimicking the heparin-
bound complex, or act upon release occurring via hydrolytic
cleavage of the tether is still to be ascertained. Early studies
demonstrated that epidermal growth factor (EGF) covalently
tethered to aminosilane-modified glass via star poly(ethylene
oxide) (PEO) could elicit DNA synthesis and cell responses of
primary rat hepatocytes, whereas the simple physical absorption
of EGF on modified glass was not effective [71]. Similarly,
tethering transforming growth factor-beta1 (TGF-1) to adhesive
ligand-modified glass surfaces resulted in a significant increase in
ECM production over the same amount of soluble TGF-1 [73].
Using carbodiimide chemistry, BMP-2 was directly immobilized
on silk fibroin films [72]. Human bone marrow stromal cells
cultured on unmodified silk fibroin films in the presence of
osteogenic stimulants exhibited little if any osteogenesis, whereas
the same cells cultured on BMP-2 decorated films in the presence
of osteogenic stimulants differentiated into an osteoblastic
lineage. In another study, covalent attachment of VEGF and
RGD-containing synthetic oligopeptides to PEG hydrogels could
generate complete vascularization of the construct by a cell-
demanded release of the angiogenic factor [74].
These studies demonstrate that signaling by immobilized
GFs may be more potent than signaling by soluble GFs directly
interdispersed within the scaffold. In particular, presentation of
GFs by covalent immobilization on scaffold surface may permit
a greater control of their temporal and spatial availability in the
extracellular environment [75]. Nevertheless, the immobiliza-
tion strategy must consider protein structure and active region
topology when designing suitable delivery system (DS) in order
to maximize GFs bioactivity. Furthermore, some signals takes
advantage of sustained release, while others benefit from direct
attachment to the biomaterial substrate [76].
3.3. Signal delivery from cells
An alternative and more sophisticated approach to overcome
issues related to tissue regeneration (i.e. local and controlled
delivery of GFs) and to elicit the desired biological responses
within the scaffold relies on the use of nucleic acid-releasing
systems. In principle, nucleic acids containing a sequence
encoding for specific proteins can be introduced into target
cells, which are thus prompted to produce the desired proteins.
Alternatively, oligonucleotides can be used to return abnormal
gene expression to a certain state in antisense and interference
therapies based on silencing RNA [77]. In this way, cells
genetically induced to secrete proteins may act as point-source
DS, allowing a prolonged and more specific effect.
For a successful gene-mediated TE approach, synthetic
oligopeptides containing the adhesion site of fibronectin (the
RGD sequence) have been used [36,78]. The nanoscale
distribution of the oligopeptides chemically coupled to adhesion
substrates was found to mediate the efficiency of gene delivery
[36]. Gene expression levels increase with increasing oligopep-
tide density (i.e. stiffness of adhesion), which enhances the
ability of cells to internalize plasmid DNA. The use of
biomaterial-based devices modified with specific cell-adhesion
molecules can maximize the population of stimulated cells [78].
In perspective, coupling oligopeptides containing the RGD
sequence to protein-loaded DS may improve the cellular
response to GFs by exposing cells to genes according to a
predetermined scheme, thus activating the tissue-inductive cell
machinery.
Synthetic gene-activated matrices, loaded with plasmid, may
also play a prominent role as cell-activating scaffolds for TE.
Time-controlled release of the plasmid encoding for tissue-
inductive PDGF from porous PLGA matrices lead to matrix
deposition and vascular bed formation [62]. Similarly, the
dispersion of plasmid DNA encoding for both angiogenic and
osteogenic factors within PLGA scaffolds resulted in a gene-
activated cell recruitment from peripheral tissue promoting
osteogenesis [79]. Bioengineered tissues secrete recombinant
proteins and act as long-term DS when implanted in vivo.
Myoblasts retrovirally transduced to locally secrete recombi-
nant VEGF induced the regrowth of a functional capillary bed
in the bulk of a bioengineered tissue substrate and in the
adjacent muscle ischemic tissue [80]. More recently, genetically
activated rabbit bone marrow stromal cells engineered to
express BMP-4 in a porous PCL scaffold effectively promoted
new bone tissue formation [81].
The concept of gene-mediated protein expression has been
put forward by immobilizing DNA at the pericellular level, thus
triggering molecular signal broadcasting from cells, probably
due to high local concentration of DNA in the cellular niche
[82]. Combining GF delivery with covalent attachment of DNA
able to dictate protein release from the cells may improve tissue
response. Actually, dual delivery of BMP-2 and a plasmid
encoding the same protein may induce a feedback mechanism
by which the transcription efficacy of the plasmid was further
increased, thus opening the way to possible pathways eluding
viral gene transfer [83].
The results of combined GF and gene delivery suggest the
opportunity of a sequential release of multiple signaling
molecules, allowing the recapitulation of tissue formation
steps at predetermined time intervals and/or after induction
times. However, the synchronization between the intracellular
transport of gene material and the activation of protein
expression by cells is still far from being optimized. Current
understanding suggests that the properties of the pericellular
space, the delivery method and the biomolecule structure must
Fig. 2. Protein release from non-biodegradable (A), soluble/biodegradable (B) and pulsatile (C) delivery systems. Continuous, delayed and pulse-like delivery may be
achieved with non-biodegradable and soluble/biodegradable delivery systems (D). Onoff delivery (single or multi-pulses) may be achieved with pulsatile (pre-
programmed or triggered) delivery systems (E).
be carefully controlled to achieve a synergistic effect able to
lead to an overall improvement of the therapeutic approach.
Probably, cellular signaling and genetic manipulation should
work in concert to promote a full mimicry of the natural
sequences governing tissue regeneration.
4. Delivery systems for proteins of potential interest in
tissue engineering
Drug delivery technologies can be of help in designing
bioactivated scaffolds in which low or high molecular weight
molecules should be released in a specific area at pre-
programmed rates [17,18,84]. First of all, a DS can offer to its
protein cargo adequate protection from inactivation occurring
in biological environments and guarantee the preservation of
bioactivity during the whole release duration [85]. On the other
hand a fine tuning of release rate can be realized by regulating
platform composition, shape and architecture. DS offering a
time-control of the delivered dose can be useful to trigger off the
release of a bioactive molecule and maintain a specific
concentration for extended duration. Furthermore, this strategy
gives the opportunity to deliver more than one protein at
different pre-programmed rates according to the needings of a
specific application.
DS can be designed in different shapes (particles, implants),
architectures (reservoirs, matrices) and made with different
biodegradable and non-biodegradable materials offering a tunable
control over release rate. Two main possible rates are feasible that
is continuous delivery and pulsatile delivery (Fig. 2). In the
following we describe a number of DS for proteins of potential
interest in TE highlighting the type of control over release rate
offered.
4.1. Continuous delivery
4.1.1. Non biodegradable systems
Pure diffusion-controlled systems based on non-biodegrad-
able polymers, such as ethylene-vinyl acetate copolymers
(EVAc) and silicones, have been firstly tested/used for the
controlled release of drugs. At present, the pharmaceutical use
of EVAc for the controlled release of proteins is relatively rare,
even if its potential has been long investigated and a particular
attention has been devoted to GF release [8688]. In such
systems, protein transport out of the device is driven by a
concentration gradient and limited by the presence of an
insoluble polymeric matrix which regulates drug diffusion.
Mass transport occurring through polymer chains or pores
is the only rate-limiting step of the process. Reservoir or
matrix systems can be designed to respectively achieve zero-
or first-order release kinetics, with different biological
implications. In this perspective, EVAc-based systems may
be of help in applications where the effect of GF release
kinetics within the scaffold on tissue regeneration process
needs to be highlighted.
Recent advances in the field of micro- and nanotechnology
has given a new strength also to the application of silicon tubing
in protein delivery. Actually, novel delivery and sensing silicon-
based platforms for long-term integration of cells may be
achieved, forming the so-called nanoporous micromachined
biocapsules' [89]. These systems are specifically intended for
encapsulation of pancreatic islet cells, able to release insulin, or
other cells of interest. In perspective, applications other than
peptide and protein delivery may involve the restoring of organ
functions.
4.1.2. Biodegradable systems
Even if EVAc and other non-biodegradable polymers are still
investigated as protein DS, current studies have been directed
towards the development of soluble/biodegradable systems
requiring no follow-up surgical removal once the drug supply is
depleted. Amongst synthetic biodegradable polymers, thermo-
plastic aliphatic polyesters like PLA and PLGA have generated
tremendous interest due to their excellent biocompatibility as
well as the possibility to tailor their in vivo life-time from weeks
to years by varying composition (lactide/glycolide ratio),
molecular weight and chemical structure (i.e. capped and
uncapped end-groups) [90]. Protein encapsulation within PLGA
micro- and nano-carriers is regarded as a powerful tool to
protect the biological activity of generally labile macromolec-
ular therapeutics and sustain their release over long time frames
[91]. Different PLGA formulations for protein release are
already on the market (Lupron Depot, Sandostatin LAR
Depot, Nutropine Depot and Zoladex) and several examples
of successful protein and GF delivery through PLGA micro-
spheres are reported in the literature [92102]. PLGA-based
particles can be engineered in terms of composition, size (i.e.,
microparticles or nanoparticles), size distribution and morphol-
ogy to tailor the release rate on the specific application, and their
surface functionalized to enhance their interaction with cell and
tissues [103105].
Drug-containing solution of PLGA copolymers in biocom-
patible organic solvent have been proposed as in situ forming
DS [106107]. This technology (Atrigel) is used in Eligard
(QLT Inc.), a leuprolide acetate-containing formulation for the
treatment of prostate cancer able to sustain protein release from
one to six months. Analogously, thermally induced gelling
systems based either on PLGA-PEG-PLGA or PEG-PLGA-
PEG triblock copolymers have also been used as in situ forming
protein delivery platforms [106] and recently tested for the
sustained release of interleukin-2, human growth hormone and
insulin [108110]. Along with in situ forming biodegradable
polymers matrices, another class of biodegradable polymers is
attracting research attention as protein DS due to their peculiar
chemicalphysical properties. The new generation of poly
(ortho esters) (POE) have evolved through a larger number of
families spanning from injectable viscous biomaterials, where
the protein can be directly incorporated by simple mixing,
without the use of heat or solvents, to a low melting temperature
polymer (POE IV) that can be extruded at temperatures
compatible with protein biological activity [111,112]. Both
semi-solid and solid extruded POE have been proved as highly
flexible and programmable matrices for controlled protein
delivery [113115]. These systems are particularly relevant in
TE since the polymer formulation can be easily adopted with the
most promising micro- and nanotechnologies adopted to fabri-
cate scaffolds such as 3D printing and SFF.
A quite recent class of biodegradable polymers currently
investigated for protein delivery are polyanhydrides, character-
ized by an hydrophobic backbone carrying hydrolytically labile
anhydride linkages [116]. Differently from PLGA copolymers,
polyanhydrides are believed to undergo predominantly surface
erosion providing a better and easier control over the protein
release kinetics through the material formulation [117].
Polyanhydrides can be processed in several usable form, such
as particulate (i.e. microspheres, nanoparticles and beads) and
matrix systems (i.e. implant, films, surgical paste and sheets)
[116,117] and have been proven to preserve the biological
stability of protein therapeutics [118120].
4.2. Onoff delivery
Protein and peptide release can be engineered to occur in a
pulsatile mode, intended as the rapid and transient release of a
certain amount of drug molecules within a short time-period
immediately after a pre-determined off-release interval. One
way to classify pulsatile DS is based on the physicochemical
and biological principles that trigger the release [121]. These
devices are classified into programmed and triggered DS. In
programmed-DS, the release is completely governed by the
inner mechanism of the device (for example lag-time prior to
drug release in some DS). In triggered-DS, the release is
governed by changes in the physiologic environment of the
device (i.e. self-regulated DS or biologically-triggered DDS) or
by external stimuli (i.e. externally-triggered systems). In the
latter case, external stimuli, such as temperature changes,
electric or magnetic fields, ultrasounds and irradiation, activate
drug release [122,123].
4.2.1. Programmed delivery systems
In the case of programmed-DS, precisely timed drug delivery
can be accomplished by the spontaneous hydrolysis (i.e. bulk
and surface eroding systems) or enzymatic degradation of the
polymer comprising the device.
Bulk- and surface-eroding systems may be engineered to
achieve pulsed protein delivery slightly modifying the compo-
sition of the device, which can be based on PLGA [124126],
cross-linked hydrogels [127], polyanhydride [128,129], and all
those biodegradable polymers discussed above. In case of
PLGA-based microparticles, more than an effective pulsed
drug delivery, a booster release occurring over a period of
several weeks after a typical lag-phase has been realized
[124,125,130,131]. A real pulsed protein delivery from PLA/
PLGA-based devices has been achieved by Langer et al., who
developed a resorbable microchip based on PLA [132]. In
perspective, the implant can enable the patterned delivery of
multiple agents.
Also surface eroding polymers, such as poly(anhydrides),
can be of help when pointing to pulsed protein delivery [129].
Recently, a polyanhydride-based laminated device has been
applied to the pulsatile release of parathyroid hormone (PTH)
[129]. The implantable DS, consisting of drug layers, isolation
layers and sealant filling, allowed multi-pulse release profiles of
PTH and bovine serum albumin (BSA) in their bioactive forms.
This implant can be produced in various shapes and deliver
more than one drug. Furthermore, the load of therapeutics can
be easily tailored over a broad range in the drug layers.
4.2.2. Triggered delivery systems
Self-regulated DS (i.e. biologically-regulated DS) are
closed-loop controlled release devices in which the release
rates are adjusted by the system, in response to feedback
information, without any external intervention. This is the case
of pH-responsive systems, which have been mainly investigated
for oral protein delivery [133,134].
The most interesting class of self-regulated DS for TE
applications is probably represented by biomolecule-sensitive
hydrogels, a kind of biologically-inspired materials able to
response to specific physiological stimuli, such as increase of
glucose levels or the presence of special proteins and/or enzymes
[122,123]. These systems can be potentially manufactured in
form of fibers, gel, sheets or microparticles to fabricate scaffolds.
A great deal of interest has been focused to glucose-responsive
insulin delivery since the development of pH-responsive
polymeric hydrogels that swell in response to glucose [135].
The intelligent systemconsists of immobilized glucose oxidase
in a pH-responsive polymeric hydrogel, enclosing a saturated
insulin solution. As glucose diffuses into the hydrogel, glucose
oxidase catalyzes its conversion to gluconic acid, thereby
lowering the pH in the microenvironment of the membrane,
causing swelling and insulin release. Recent progresses have been
made in designing smart hydrogels able to specific recognize a
biomolecule through molecular imprinting techniques [136].
Contrariwise to self-regulated DS, externally-regulated DS
are open-loop controlled devices in which drug release can be
activated by an external stimuli, including temperature changes,
magnetism, ultrasound, electrical effect and irradiation
[122,137,138]. These systems make use of smart polymeric
materials, which respond with a considerable change in their
properties to small changes in their environment. The incorpo-
ration of these devices into polymer scaffold may potentially
offer the opportunity to control the time and space environmen-
tal conditions by mean of an externally-imposed field.
5. The issue of delivery systemintegrationinthree-dimensional
scaffolds
A clinically effective TE approach requires a chrono-
programmed scaffold, able to trigger the on-demand release of
molecular agents fulfilling the specific needs of the bio-
integrating tissue. This approach is part of the flourishing
concept of chrono-biotechnology which may be accomplished
through the tight control over dosing and localization of
signaling molecule exposure in a complex, mostly anisotropic,
dynamic three-dimensional environment [17,78].
To date, several attempts have been made to obtain systems
integrating delivery devices and TE templates able to mimic
ECM and directionally reorganize tissue [139]. In the case of
microsphere-integrated scaffolds, however, a very limited
number of the possible protein delivery strategies described
above have been exploited. Some relevant results have been
achieved by the group of Mooney [17], who developed PLGA
scaffolds for the sequential release of multiple GFs by mixing
free VEGF with empty and PDGF-loaded polymer particles and
subsequently assembling them into a porous scaffold [140].
More recently, they have also presented an anisotropic system
based on a porous bi-layered PLGA scaffold able to expose only
VEGF in one spatial region, and deliver VEGF and PDGF in an
adjacent region [141]. In a similar attempt, PLA microparticles
plasticized with PEG were sintered into scaffolds formed by
protein-free and protein-loaded layers, thus allowing a release
of different bioactive molecules restricted to specific regions
within the scaffold [142]. These scaffolds may find utility in
applications where GF gradients or a region-dependent tissue
growth are required.
Despite these encouraging results, important technological
limitations exist. The major issue relies on the use of GF-loaded
microspheres which are partly modified when formed into
scaffolds, thus altering their architecture and consequently
release features. Furthermore, to engineer dynamic gradients of
a signaling molecule, the detailed understanding of release
kinetics at the single microsphere level is necessary.
An alternative approach to create microsphere-integrated
scaffolds able to regulate both temporally and spatially GF
release kinetics may take advantage of micromanipulation-based
techniques. The simple dispersion of microspheres within gel-like
scaffolds is a well-established approach to achieve a temporal
control over GFs release [139]. It has been recently demonstrated
that through the fine tuning of microsphere formulation and
scaffold properties it is possible to realize platforms able to control
the microenvironmental conditions in terms of time evolution of
bioactive molecules delivery [143]. Possible developments of
these findings may benefit from advances in micro- and
nanotechnologies so as to engineer templates embedding micro-
spheres releasing GFs at known release rates in a predetermined
and optimized spatial distribution within the scaffold. Actually,
devices acting as single point source may be micropositioned by
3D printing and soft lithography to obtain highly regulated
structures able to trigger the extent, and possibly the architecture/
structure of tissue formation [10,144]. The combination of
micropositioning systems and mathematical modeling describing
the complex and multiple mechanisms governing the release
kinetics from single microspheres within the scaffold can be of
help to realize scaffolds with highly controlled architecture by
computer-aided scaffold design (CASD) [10,145].
A possible limitation of DS-integrated scaffolds derives from
their pre-defined nature. In fact, once pre-programmed in vitro,
they will not be able to interactively modify release kinetics
according to the needs of the surrounding tissue. As underlined
above, a more effective biomimicry could be obtained if a dual-
reciprocity scheme could be encoded in the matrix. In this way,
cells can trigger the on-demand development of ECM and, in
turn, the engineered scaffold could stimulate cell behaviors
through the controlled release of bioactive molecules. In
perspective, the use of bottom-up strategies based on molecular
self-assembly appears very promising. On this matter, the group
of Stupp has developed a class of peptide amphiphile (PA)
molecules that self-assemble into three-dimensional nanofiber
networks under physiological conditions in the presence of
polyvalent metal ions [37]. While PA self-assembly entraps
cells in the nanofibrillar matrix, the entrapped cells internalize
the nanofibers and possibly utilize PA molecules in their
metabolic pathways. The method is not limited to uniaxial
alignment but can be used to guide self-assembling nanofibers
around corners and in complex patterns. It is also versatile
enough to be used in the alignment of other self-assembling
supramolecular systems starting from solutions of small
molecules [146].
It should also be mentioned that a multifunctional scaffold
should not only provide a controlled administration of relevant
biomacromolecules and their gradients, but also present such
molecules in a suitable conformation state, mimicking ECM-
GFs binding. Indeed, it has been shown that molecularly
decorated materials enhance tissue formation through the
modulation of the interaction between protein signaling and
biomaterials appears to be fundamental to provide a better
integration of the scaffold with the neo-forming tissue [147].
These emerging approaches suggest that the next-future
scaffolds will not be realized by simply integrating DS within
the scaffolds. Indeed, taking advantage of the current
knowledge of drug delivery and biomaterial science, multi-
functional scaffolds, where the polymer three-dimensional
template itself acts as a biomimetic, programmable and multi-
drug delivery device, should be designed.
6. Conclusions
Extraordinary progresses have been made in the last decade
towards the design of scaffolds with a suitable multiscale
hierarchical structure and the design of DS able to release active
proteins according to virtually any complex delivery pattern.
The integration of cutting-edge scaffold production technolo-
gies and DS may lead to significant advances in both
therapeutic applications of TE and basic knowledge on cell
guidance and tissue morphogenesis. However, technological
and scientific challenges have still to be overcome to realize a
faithful mimic of the complex orchestration of structural and
molecular signals presented by the natural ECM. For instance,
micro- and nano-technologies should be further exploited in
order to organize a DS-integrated biomaterial scaffold with the
required spatial resolution. On the other hand, design of future
biomaterial platforms should benefit from a quantitative
understanding of the influence of the amounts of morphogens
and their gradients on cell fate, as well as the influence of
different microenvironments on their action.
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Controlled drug delivery in tissue engineering

Marco Biondi, Francesca Ungaro, Fabiana Quaglia, Paolo Antonio Netti

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