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Review

Future of local bone regeneration e Protein versus gene therapy

  J. Fischera,1, A. Kolk b,*,1, St. Wolfart a, C. Pautke b, P.H. Warnke d,e, C. Plank c, R. Smeets f 

a Department of Prosthodontics, RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germanyb Department of Oral and Maxillofacial Surgery, Technische Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, D-81675 Munich, Germanyc Institute of Experimental Oncology and Therapy Research, Technische Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, D-81675 Munich, Germanyd Department of Oral and Maxillofacial Surgery, University of Kiel, Arnold-Heller-Strasse 16, 24105 Kiel, Germanye Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland, Australiaf Department of Oral and Maxillofacial Surgery, RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany

a r t i c l e i n f o

 Article history:

Paper received 30 July 2009

Accepted 11 March 2010

Keywords:

Bone morphogenic protein

Gene transfer

Bone regeneration

Drug delivery

Nanotechnology

a b s t r a c t

The most promising attempts to achieve bone regeneration artificially are based on the application of 

mediators such as bone morphogenetic proteins (BMPs) directly to the deficient tissue site. BMPs, as

promoters of the regenerative process, have the ability to induce de novo bone formation in various

tissues, and many animal models have demonstrated their high potential for ectopic and orthotopic bone

formation. However, the biological activity of the soluble factors that promote bone formation in vivo is

limited by diffusion and degradation, leading to a short half-life. Local delivery remains a problem in

clinical applications. Several materials, including hydroxyapatite, tricalcium phosphate, demineralised

bone matrices, poly-lactic acid homo- and heterodimers, and collagen have been tested as carriers and

delivery systems for these factors in a sustained and appropriate manner. Unfortunately these delivery

vehicles often have limitations in terms of biodegradability, inflammatory and immunological rejection,

disease transmission, and most importantly, an inability to provide a sustained, continuous release of 

these factors at the region of interest. In coping with these problems, new approaches have been

established: genes encoding these growth factor proteins can be delivered to the target cells. In this way

the transfected cells serve as local“

bioreactors”

, as they express the exogenous genes and secrete thesynthesised proteins into their vicinity. The purpose of this review is to present the different methods of 

gene versus growth factor delivery in tissue engineering. Our review focuses on these promising and

innovative methods that are defined as regional gene therapy and provide an alternative to the direct

application of growth factors. Various advantages and disadvantages of non-viral and viral vectors are

discussed. This review identifies potential candidate genes and target cells, and in vivo as well as ex vivo

approaches for cell transduction and transfection. In explaining the biological basis, this paper also refers

to current experimental and clinical applications.

Ó 2010 European Association for Cranio-Maxillo-Facial Surgery.

1. Introduction

Despite recent progress in minimally invasive surgical proce-

dures, problems remain in the treatment of injuries due to thelimited self-healing capacity of most tissues of the musculoskeletal

system. Oral bone loss and subsequent tooth loss are substantial

worldwide health problems and in the US alone the cost has been

estimated to be $5e6 billion/year for the surgical treatments

related to oral-bone loss (Eke and Genco, 2007). The market

potential of the 16 publicly traded tissue engineering startups’amounts approx. US $2.6 billion/year (Lysaght and Reyes, 2001) and

the overall annual costs of regenerative medicine account for over

US $80 billion (Niklason and Langer, 2001).In the treatment of tissue loss, three main approaches to tissue

regeneration and engineering are defined: conduction, induction,

and cell transplantation (Langer and Vacanti,1993). The conduction

and induction techniques use the regenerative capacity of the

existing tissue to restore small amounts of tissue, but their use is

dependent on the defect size, the condition of adjacent cells, cell

migration speed, and the availability of surrounding vasculature

(Alsberg et al., 2001).

The conductive approach acts passively, allowing cells to

migrate from host tissue into a scaffold, while preventing the

invasion of unwanted cell types (Kaigler and Mooney, 2001).

* Corresponding author. Department of Oral and Maxillofacial Surgery, Techni-

sche Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, D-81675

Munich, Germany.

E-mail address: kolk@mkg.med.tum.de (A. Kolk).1 These authors contributed equally to this work.

Contents lists available at ScienceDirect

 Journal of Cranio-Maxillo-Facial Surgery

j o u r n a l h o m e p a g e : w w w . j c m f s . c o m

1010-5182/$ e see front matter Ó 2010 European Association for Cranio-Maxillo-Facial Surgery.

doi:10.1016/j.jcms.2010.03.016

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The inductive techniques use bioactive factors to stimulate tissue

regeneration and control cellular behavior. One common technique

is the delivery of soluble mediators, such as cytokines or growth

factors (Alsberg et al., 2001). The inductive method arose from the

discovery of bone morphogenetic proteins (BMPs), a sub-family of 

the transforming growth factor-b (TGF-b) superfamily in 1965

(Urist, 1965). Following purification and subsequent molecular

cloning (Wozney et al., 1988; Celeste et al., 1990; Ozkaynak et al.,

1990), recombinant proteins with similar activity were identified

(Wang et al., 1990) and made available for preclinical use (Smeets

et al., 2009). These proteins are synthesised by various cells at

the defect site (e.g., fibroblasts, endothelial cells, or mesenchymal

stem cells (MSCs)) or by infiltrating repair and inflammatory cells

(e.g., platelets, macrophages, or monocytes).

Since then, many subtypes of the BMP-family have been iden-

tified, along with other growth and differentiation factors (GDFs)

that inducebone formation (Ripamonti and Reddi,1997). The major

problems of sustaining their expression and controlling their

delivery seem to have been overcome by the development and use

of carriers such as poly(D,L-lactide) (PDLLA) coating (Schmidmaier

et al., 2002, 2003; Deppe et al., 2003; Deppe and Stemberger, 2004)

or bone substitutes like hydroxyapatite, Bio-OssÓ (Terheyden et al.,

2001; Warnke et al., 2004) or calcium phosphate as a naturalrelease system (Smeets et al., 2009). However, the problems of 

unwanted ectopic bone formation as well as the high costs asso-

ciated with the application of recombinant growth factors have not

yet been solved. There is increasing interest in alternative means of 

exploiting the effects of growth factors. Among these potential

alternatives, one very promising procedure is gene delivery, also

called nucleic acid transfer. Instead of the respective proteins, the

DNA encoding these molecules is integrated into cells using either

in vivo or ex vivo approaches, enabling these cells to produce the

therapeutic proteins for specific periods (Bonadio et al., 1999).

The ex vivo approach represents tissue engineering in the

classical sense (Bianco and Robey, 2001). This approach, such as cell

transplantation, is required if the bone defect is extensive or lacks

active cells capable of stimulating regeneration, and is associatedwith improved results (Alsberg et al., 2001) (Fig. 1). In vivo

approaches include classic gene integration, the use of nucleicacids

to increase the expression of a target protein (De Laporte et al.,

2006).

A number of current tissue replacement strategies use

a combined approach. One example is the use of a synthetic bone

graft with a cancellous bone-like pore system, that combines

conductive properties (for strength) with an inductive potential as

a carrier for BMP (Tancred et al., 1998). Furthermore, some studies

have used transplanted cells not for ex vivo tissue formation, but

solely as carriers for inductive signals (Gysin et al., 2002).

This article reviews the use of different osteoinductive proce-

dures utilised in local bone repair and regeneration, including the

move from protein application to regional gene therapy, with an

emphasis on bone morphogenic proteins. The development of the

field of local gene transfer is described in detail. In particular, the

challenges, benefits, and risks associated with the various

gene therapy strategies and gene transfer alternatives will be

discussed.

2. Osteoinductive approaches

  2.1. Bioactive mediators

Many GDFs e such as BMPs, basic fibroblast growth factor (FGF)

(bFGF), insulin-like growth factors (IGFs), TGF-b, platelet-derived

growth factor (PDGF), and vascular endothelial growth factor

(VEGF) e have been found to induce new bone through their effects

on the recruitment, proliferation, and differentiation of bone

forming cells (Hollinger et al., 2000; Oringer, 2002; Seeherman

et al., 2002).

BMPs enable skeletal tissue formation during embryogenesis,growth, and healing, as well as throughout adulthood (Reddi, 1992;

Ripamonti and Reddi, 1994; Kirker-Head, 2000). More than 30

BMPs have been identified so far. Of the known BMPs, BMP-2, -4,

and -7 are each individually able to induce de novo bone formation

at ectopic sites in vivo (Kirker-Head, 2000; Warnke et al., 2004). As

members of the TGF-b super family, BMPs themselves can be

classified into subgroups according to either their functional or

structural similarities (Ducy and Karsenty, 2000). Further studies

have shown that BMPs exhibit functions in embryogenesis that are

not restricted to skeletal development. Thus, the name “GDFs”

would be more accurate in describing this class of proteins than the

historical term (Ducy and Karsenty, 2000).

Their function is regulated from the extra-cellular compartment

by BMP-binding proteins, such as noggin and chordin, whichprevent BMPs from binding to their surface receptors. Following

the activation of a BMP receptor, the intracellular effect is trans-

mitted by Smad proteins, which induce the transcription of specific

genes (Kawabata et al., 1998). An active, mature BMP is a disul-

phide-linkeddimer that consists of eithertwo different members of 

the BMP family (heterodimer), or two similar members (homo-

dimer). The dimer composition affects the function: e.g., hetero-

dimers consisting of BMP-2 (or BMP-4) and BMP-7 are more potent

morphogens than their corresponding homodimers (Kawabata

et al., 1998).

The clinical use of BMPs has been limited for years because of 

the expensive and dif ficult purification process. In addition, these

proteins, isolated from allogenic or xenogenic bone, carried

potential health risks as transmission of slow virus diseases or theinduction of malignancies (Wang et al., 1988; Kirker-Head, 2000).

The identification of the corresponding human RNA and comple-

mentary DNA (cDNA) coding for human BMPs (Wozney, 1992)

allowed the production of recombinant human BMP (rhBMP)

(Kirker-Head, 2000). Despite the unlimited availability of rhBMP,

only a few human clinical studies on its use have been published

(Giannoudis et al., 2007, Schmidmaier et al., 2007). The fact that

doses far above physiological levels have to be used for bone

formation may have reduced clinical enthusiasm (Hollinger et al.,

2000). A number of animal models have demonstrated the

general beneficial effect of BMPs on the healing of bone defects. The

therapeutic potential of rhBMPs (especially rhBMP-2, -4, and -7)

has been proven in selected fracture repair models, craniomax-

illofacial-, periodontal-, and dental diseases periodontal, and dentalFig. 1. Survey of the components involved in bone formation.

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diseases (Kirker-Head, 2000). Studies have revealed that different

effects of BMPs can be determined for specific cell types in a dose-

related manner (Sykaras and Opperman, 2003). For example,

increased concentrations of BMPs result in faster bone growth

(Wang, 1993), while ankylosis of teeth has been noted, dependent

on the BMP-2 dose, following application for periodontal regener-

ation (Sigurdsson et al., 1996). Studies have demonstrated the

important role of carriers in maintaining the BMP concentration at

the target site for a suf ficient period of time to promote chemotaxis

and the migration of bone-forming cells to the target site, as well as

the proliferation and differentiation of these cells (Seeherman et al.,

2002, Dai et al., 2005). Under clinical conditions, bone regeneration

requires three components: morphogenetic signals, responding

cells that are capable of assuming an osteogenic phenotype, as well

as a matrix to deliver the signal (Fig. 1). The matrix also acts, in

certain cases, as a scaffold to allow cell recruitment, attachment,

proliferation, and differentiation (Ripamonti and Duneas, 1998).

A completely new osteoinductive approach is given by the in

vivo tissue engineering techniques, such as endocultivation. They

may offer the potential to cultivate customised, vascularised bone

replacements for skeletal reconstruction. The key feature of endo-

cultivation is the use of the patient as his own bioreactor to prevent

problems with immune reactions and to allow vascularisation of the implanted matrices. Computer aided design (CAD) is used to

shape matrices for individual defects in endocultivation. The first

computer planned, complex and vascularised bone replacement e

a mandible e was grown in the latissmus dorsi muscle of man and

successfully transplanted to repair his mandibular defect (Warnke

et al., 2004). Even though this procedure was mainly successful,

drawbacks were seen and lead the focus onto developments of new

matrix designs for improved hosting of stem cells (Warnke et al.,

2006).

  2.2. Delivery strategies

After the discovery of the important role of cytokines in bone

formation, much research has been invested on the controlled andcontinuous delivery of these proteins. The biological activity of 

soluble factors in vivo is very short, due to proteolytic degradation

and rapid diffusion of the water-soluble factors (Winn et al., 1999;

Crombleholme, 2000). Kirker-Head (2000) defined three principle

BMP delivery strategies for clinical use: systemic administration of 

BMP, delivery by means of a carrier matrix, and transfer of BMP

encoding DNA (gene therapy). The observation of elevated serum

BMP levels in growing children and decreased levels in patients

suffering from osteoporosis (Einhorn, 1992) gave rise to the idea of 

the systemic application of BMPs to treat osteoporosis and poly-

traumatised patients. However, the systemic application of BMPs

still requires the development of an appropriate carrier molecule

that protects BMPs from proteolytic deactivation without impeding

their function (Kirker-Head, 2000). The major concern regardingsystemic delivery is that only a small amountof the injected protein

arrives at the diseased target tissue, while surrounding tissue

bearing BMP receptors might be adversely affected (Bonadio,

2000). In response to this, the strategy of local cytokine and

growth factor delivery directly to the target tissues was developed

(Langer, 1998). Currently, the local delivery of BMPs by carrier

matrix is at an advanced stage of development. Because of the short

lived biological activity of the factors, the ideal matrix should allow

for sustained BMP release through chemical interaction or physical

impediment, protection of the BMPs from proteolysis, rapid inva-

sion of vascular and mesenchymal cells, as well as subsequent

osteogenesis. Under clinical conditions, concentrations far above

a physiological level of osteogenic factors seem to be required to

increase the retention time and overcome the strict regulation by

inhibitors. The ef ficiency of a carrier system is often influenced by

site and species (Seeherman et al., 2002). The material used should

be non-immunogenic, non-toxic, bio-absorbable, malleable, steri-

lisable, and easily manufactured (Kirker-Head, 2000). Variousattempts have been made to develop a suitable substance that

combines all or many of these properties into one material. Organic

and inorganic materials have been used both separately and in

combination to create an appropriate matrix (Table 1).

Combinations of some of the listed materials have been used to

enhance the beneficial properties. Hydroxyapatite, as an example

of an inorganic matrix, has been used as a delivery vehicle alone

(Horisaka et al., 1991) and as a composite carrier with tricalcium

phosphate (Boden et al., 1999), collagen (Asahina et al., 1997), and

coral (Gao et al., 1996). The majoradvantages of an inorganic matrix

are its strength in supporting the surrounding tissue and

preserving bone function, and its ability to allow osteoconductive

bone formation. Additional benefits are immunological inertness

and the slow biodegradation of some of the materials. Tancredet al.(1998) demonstrated the further development of a synthetic

calcium phosphate-based material that has a cancellous bone-like

pore structure that combines optimal strength and suitability as

a carrier for BMP (Tancred et al., 1998). A novel approach was to

implant matrices that, rather than store and deliver rhBMP, actively

concentrate endogenous BMPs at the site of implantation at levels

that allow bone induction to take place. Native BMP complexes are

much more active than their constituent BMP molecules, which

means that the concentration of the native complex required for

bone induction is about 1000 times lower (De Groot, 1998;

Schliephake et al., 2005). A biodegradable, porous carrier system

is convenient for the clinician, as it limits and protects the release of 

proteins or genes in a predictable and time-controlled manner,

permits cell growth, and acts transiently as an extra-cellular matrixuntil suf ficient cells are present to build a new substratum

(Hollinger and Schmitz, 1997). Among the organic matrices,

collagen and synthetic polymer delivery vehicles currently show

the greatest potential for clinical use (Kirker-Head, 2000).

Collagen, the industry’s favoured material (Hollinger et al.,

2000), has been used to create various delivery vehicles: collagen

sponges, strips, gels, membranes, and others (Kirker-Head, 2000).

In 1997, the feasibility of an rhBMP-2 absorbent collagen sponge

was evaluated for the use in local alveolar ridge preservation/

augmentation (Howell et al., 1997) and in maxillary sinus

augmentation (Boyne et al., 1997). rhBMPs bind differently to

absorbable collagen sponges, depending on their isoelectric point

(pI). It has recently been reported that rhBMP-2 and rhBMP-6 are

retained better than rhBMP-4 (Uludag et al., 1999). Unfortunately,

 Table 1

Carrier matrices for delivery of BMPs (data adapted from Kirker-Head, 200 0).

Carrier matrices for delivery of BMPs

Organic Inorganic

Bone derived:

- Demineralised bone matrix

- Autolyzed antigen

extracted allogenic bone

Bone derived:

- Natural bone mineral

- Thermoashed bone mineral

Synthetic polymers:- Polylactic acid

- Polyglycolic acid homo-/

heterodimer

Metals:- Titanium

Natural polymers:

- Collagen (types I and IV)

- Non-collagenous proteins

- Fibrin

- Hydrogels

Other:

- Hydroxyapatite

- Tricalcium phosphate and other

bioceramics

- Bioactive glass

- Coral

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in such a delivery system, the highly potent osteoinductive heter-

odimers of the rhBMPs are less well retained than the homodimers.

A small percentage of patients develop antibodies to bovine

collagen (4e6%) or rhBMP-2 (Hollinger et al., 2000). The problems

of bovine material have been addressed with the development of 

synthetic polymers, which have been made biodegradable and

assessed in various preparations (Kirker-Head, 2000). Polylactide-

co-glycolide (PLGA) not only combines the absorptive ability of 

polylactide with the mechanical strength of polyglycolide (Winet

and Hollinger, 1993), making this material particularly interesting

for craniofacial surgery (Hollinger and Winn, 1999; Hollinger et al.,

2000), it can also be manufactured in various forms, depending on

the intended clinical use (Kirker-Head, 2000).

The pharmacokinetics of BMP release from a matrix is generally

characterised by two phases. After application, a short initial burst

of BMP is followed by a second phase of delayed release (Bonadio

et al., 1999; Winn et al., 1999; Kirker-Head, 2000; Sykaras and

Opperman, 2003). In the first phase, concentrations far above

a physiological level of BMP doses may diffuse, causing systemic

(Terrell et al.,1993) and local toxicity (Bonadio, 2000). Furthermore,

dose escalation seems to cause a decrease in the number of 

responding cells, resulting in a slower rate of bone formation

(Seeherman et al., 2002). During the second phase, an effectivefactor level must be maintained over time, supporting mitosis and

cell differentiation, and resulting in bone formation. The long-term

delivery of BMP-2 seems to enhance the in vivo osteogenic ef ficacy

of the protein compared to short-term delivery at an equivalent

dose (Ripamonti and Duneas, 1998; Jeon et al., 2008).

These problems demonstrate the narrow therapeutic window

for the in vivo use of many recombinant cytokines and growth

factors, and provide a possible explanation for some of the disap-

pointing results in human clinical trials (Bonadio, 2000). An alter-

native strategy, regional gene therapy, attempts to overcome these

problems by providing a time- and dose-controlled delivery of 

growth factors, cytokines, or morphogens for inducing bone

formation (Bonadio et al., 1999; Bonadio, 2000; Hollinger et al.,

2000; Oakes and Lieberman, 2000; Alsberg et al., 2001; Jenkinset al., 2003; Jin et al., 2003).

  2.3. Gene therapy

Originally, the therapeutic application of genes was proposed

for the correction of genetic defects, such as single mutations.

Recently, gene therapy has been used to induce the expression of 

molecules that are normally involved in the regenerative response

in the tissue of interest (Bonadio et al.,1999). This method provides

a potential alternative to protein therapy (Franceschi et al., 2004).

Gene therapy requires three steps to be successful: transduction

(transfection), transcription, and translation (Anderson, 1998).

The process by which the desired gene is introduced into the cellis called transduction or transfection. The introduced genes can

either integrate into the DNA of the host cell or remain in the

nucleus without integration (episomal position). In the first case,

the gene is replicated together with the host genes, and may

provide prolonged expression of the desired protein (Oakes and

Lieberman, 2000). Most research efforts have focused on trans-

duction and enhancement of its ef ficiency. Transcription, a process

while coding DNA servesas a template for mRNA synthesis in target

cells, has recently becomea focus of interest. Substantial synergistic

activity in gene therapy induced osteogenic activity is achieved by

the use of gene combination therapies to express complimentary

osteogenic signals e.g., specific combinations of BMPs, or BMPs and

their related transcription factors. The latter can regulate them-

selves and act as their own suppressor: if the transcription factor

protein binds the DNA of its own gene, it will down-regulate its

own production (Franceschi et al., 2004).

Gene therapy can be classified as in vivo or ex vivo (Fig. 4). The

desired gene can either be introduced directly into the target site

(in vivo technique), or target cells can be harvested, expanded in

culture, genetically manipulated, and (re)-implanted (ex vivo

technique) (Kirker-Head, 2000). In the ex vivo technique, cells can

be obtained from the recipient, or generated from a pre-existing

osteoprogenitor cell line (Hollinger et al., 2000).

The DNA transfer can be either native, or through a viral or non-

viral vector (Scaduto and Lieberman, 1999). The simplest trans-

duction method is the direct injection of DNA, or “naked DNA

insertion”, into the target tissue without using a vector ( Wiethoff 

and Middaugh, 2003). The electroporation technique uses pulsed

electrical fields to form holes in the plasma membrane in order to

increase DNA diffusion. However, these methods are limited by

a low rate of transduction due to DNA instability and inef ficient

delivery to the target cell nucleus (Wiethoff and Middaugh, 2003).

 2.4. Vector types

Most gene therapy models use vectors to enhance DNA entry

into target cell nuclei and expression of the desired genes (Scadutoand Lieberman, 1999). An ideal vector would possess the following

characteristics: avoidance of an immunological host response,

preferential binding to specific target cells, transduction of dividing

and non-dividing cells, integration of genes into cell DNA without

disruption of normal cell function, expression of genes at an

appropriate therapeutic level, ability to allow external control of 

protein expression, and ease of production at a reasonable cost

(Evans and Robbins, 1995; Anderson, 1998; Oakes and Lieberman,

2000). However, the perfect vector has yet to be developed. Many

of the currently used vectors partially fulfil the above criteria. The

choice of vector for gene therapy depends on the desired duration

of protein function, anatomical location, condition to be treated,

and whether an in vivo or ex vivo approach is favoured (Oakes and

Lieberman, 200 0). The vector systems can be classified into non-viral and viral vectors.

Different viruses have been introduced as gene delivery vectors,

with adenoviruses and retroviruses among the most common,

lentiviruses and adeno-associated viruses (AAVs) are among the

more promising vectors for future therapies (Kootstra and Verma,

2003; Roth and Sundaram, 2004; Pack et al., 2005; Zhang and

Godbey, 2006). The major advantage of viral vectors is their high

frequency of transduction due to the natural tropism of viruses for

living cells ( Jenkins et al., 2003). The main disadvantages of viral

vectors are their immunogenic potential (Mahr and Gooding,1999)

and, in the case of retroviruses and certain AAVs, the threat of 

disturbing normal gene function (Noguchi, 2003).

  2.5. Non-viral vectors

Non-viral vectors, such as DNA plasmids, lipoplexes, or poly-

plexes mimic functions of viral cell entry but avoid many problems

associated with viral vectors, though generally possess a lower rate

of transfection (Franceschi et al., 2000; Blum et al., 2003; Wiethoff 

and Middaugh, 2003). In addition, physical methods, such as

electroporation, sonoporation, magnetofection, hydrodynamic

methods, and ballistic methods (the so-called gene gun) have been

developed that support non-viral nucleic acid delivery to cells.

Naked plasmid and non-viral vectors can initiate inflammatory

responses that are different in nature and often milder than the

ones seen with viral vectors. These techniques have yet to achieve

the intrinsic ef ficiency of viral vectors. Although the induction of 

bone in a non-union fracture model has been reported using the

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direct transfer of plasmid DNA encoding BMP-4 and PTH (Fang

et al., 1996; Bonadio et al., 1999), the amount of plasmid DNA

necessary to induce bone in these studies was approx. 109 times

higher than the number of gene copies necessary in recent studies

using virus transduction (Franceschi et al., 2000). Nucleic acids are

complexed with cationic polymers or lipids (Spagnou et al., 2004;

Putnam and Doody, 2006), which protect them against degrada-

tion, create positively charged particles, and facilitate internal-

isation, intracellular traf ficking, and processing (De Laporte et al.,

2006; Storrie and Mooney, 2006).

Cationic lipid and adenoviral vectors have also been tested for

the delivery of BMP-2 to bone marrow stroma cells (BMSCs). Park

et al. (2003) demonstrated that both liposome- and adenoviral-

mediated BMP-2 gene transfers to primary BMSCs are suitable

methods for achieving considerable healing of critically-sized bone

defects in rats. Despite shorter healing times with adenoviral

vectors, the authors recommended liposomes as one of the most

useful vectors because of their simple preparation, the theoretically

unlimited size of the DNA insert, and fewer immunological and

safety problems (Blum et al., 2003; Park et al., 2003).

Magnetofection, another novel gene transfer method uses

magnetic force acting on gene vectors associated with magnetic

particles to enhance and target gene delivery. The major potentialof this gene transfer principle lies in the extra-ordinarily rapid and

ef ficient transfection at relatively low vector doses, and the possi-

bility of remotely controlled vector targeting in vivo (Plank et al.,

2003a,b,c). Magnetic force-derived transfection of non-viral gene

vectors is especially appropriate for cell lines that are dif ficult to

transfect, such as stem cells. By sedimenting the particles directly

onto the cells, transfection and its ef ficiency are enhanced up to

several thousand-fold compared with transfections carried out by

other gene vector procedures (Mykhaylyk et al., 2007).

Another promising non-viral gene transfer strategy uses

biodegradable structural matrix carriers to deliver plasmid DNA in

a combination referred to as a gene activated matrix (GAM) (Fang

et al., 1996). The structural matrix holds the DNA in situ until

endogenous cells, such as fibroblasts, colonise the matrix, aretransfected, and secrete the plasmid-encoded proteins that

enhance tissue regeneration (Bonadio, 2000). The carrier matrix

can gradually be replaced by the endogenous matrix deposited by

the colonising cells (Ueblacker et al., 2007). The feasibility of this

concept has been proven in animal studies. Fang et al. implanted

GAMs with genes encoding BMP-4 or human parathyroid hormone

(hPTH 1e34), resulting in bone deposition and enhanced fracture

repair, as compared to controls (Fang et al., 1996). Bonadio et al.

(1999) used a collagen sponge loaded with parathyroid hormone

cDNA in a canine tibia to improve bone formation in a dose- and

time-dependent manner. The authors argue that the retention and

expression of a plasmid gene at the site of GAM implantation is

prolonged compared to a directly delivered recombinant protein

(Bonadio et al., 1999). Despite the promising results in models(Huang et al., 2005), the delivery of DNA needs to be improved,

alternative scaffolds require evaluation, and the safety of GAM must

be further validated (Bonadio, 2002).

In general, significant technical hurdles to non-viral gene

transfer remain with regard to DNA instability, inef ficient delivery

to target cells, variable clearance by lysosomes, unpredictable

cytosolic transport, and inconsistent transcription of desired genes

(Wiethoff and Middaugh, 2003).

 2.6. Viral vectors

So far, viruses are considered to be the most ef ficient vectors for

gene transfer (Chen et al., 2003). The viral vectors currently in use

are predominantly retroviruses, adenoviruses, AAVs, herpes

viruses, and lentiviruses (Evans and Robbins,1995; Anderson, 1998;

Scaduto and Lieberman, 1999; Asahara et al., 2000; Frolova-Jones

et al., 2000; Franceschi et al., 2004) in which the genes required

for viral replication are deleted and the desired gene is inserted. A

great challenge is to avoid a strong host immune response to the

viral proteins, one approach is to create a “gutless” or “gutted” virus

from which most of the viral genes are removed (Hartigan-

O’Connor et al., 1999). In current osteoinductive approaches,

retroviruses, adenoviruses, or AAVs are most commonly used to

transfer genes of bone inducing and promoting factors (Oakes and

Lieberman, 2000; Franceschi et al., 2004; Zhao et al., 2005).

The selection of its carrier is important for the direct clinical

application of a viral vector which mayprotectthe vector andenhance

its delivery to the appropriate anatomic site (Oakes and Lieberman,

2000). Schek et al. (2004) demonstrated enhanced transduction

rates and bone formation using biocompatible hydro gels as a carrier

vehicle. Other investigators have also examined strategies to tempo-

rarily control gene expression. For example, Moutsatsos et al. (2001)

reported the ability of a tetracycline-regulated vector to control

BMP-2 expression in MSCs in vitro and in vivo by administration of 

doxycycline.

  2.7. Retroviruses

Retroviruses are an example of viruses contained in envelopes

consisting of a lipid bilayer that encloses the viral capsid containing

viral RNA and RNA transcriptase (Fig. 2).

The lipid membrane originates through budding from the

plasma membrane when the virus is released from the host cell.

The budding process is reversed when the virus attaches to target

cells through receptor-binding proteins that are anchored in its

envelope. After entering the host cell, the RNA is transcribed into

DNA by the viral reverse transcriptase, and a complementary strand

of DNA is subsequently synthesised, resulting in double-stranded

DNA that is integrated into the host cell chromosome by the viral

enzyme integrase. This allows the virus to use the replication and

translation mechanisms of the cell to assemble and release newviral particles.

Retrovirus vectors are constructed in such a way that they are

only ableto produce the desired mRNA, thus eliminating the hazard

of regaining infectiousness.

Retroviral vectors offer the advantage of being incorporated into

the host genome, providing the potential for prolonged transgene

expression (Marx et al., 1999). Furthermore, the viral proteins

produce only a weak immune response, as compared to those from

an adenoviral vector (McCormack et al., 1997). The most obvious

limitation in the application of retroviruses, e.g., the murine

leukaemia virus (MLV), is that they are only able to transfect

dividing cells (Miller et al., 1990). Rundle et al. took advantage of 

this and used an MLV-based vector for direct gene therapy in

fracture repair. The vector, containing a BMP-2/4 hybrid gene, wasselectively expressed in the proliferating cells that are character-

istic for the initial stages of wound healing at the fracture site.

Although the investigators could detect an increase in the amount

of bone formed, no acceleration of the fracture union by the bone

tissue was observed (Rundle et al., 2003). Ueblacker et al. (2007)

demonstrated a highly ef ficient transduction and persistent gene

transfer to chondrocytes in vitro and in vivo by using a retroviral

based vector. Apart from this, retroviral vectors have mostly been

applied to the ex vivo transduction of target cells that show a sus-

tained expression of the incorporated BMP gene (Lieberman et al.,

1998; Breitbart et al., 1999; Gysin et al., 2002; Lee et al., 2002).

These cells, when implanted at orthotopic sites, have successfully

promoted bone healing in clinical models. Despite promising clin-

ical results, retroviral insertion can entail unexpected risks:

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“insertional mutagenesis” can occur, when the retroviral vector

integrates into the genome at a random site ( Jenkins et al., 2003).

This happens particularly at transcriptionally active sites and

occasionally in the neighbourhood of an oncogene, driving tran-

scription of the oncogene. A tragic example of this was reported in

the human trial of a modified Moloney retroviral vector. After the

successful cure of 11 children suffering from severe combined

immunodeficiency X1 (Cavazzana-Calvo et al., 2000), several

patients developed a leukaemia-like condition caused by “inser-

tional mutagenesis”

(Hacein-Bey-Abina et al., 2003). A similarsevere adverse event occurred in an independent trial in 2006

(Shou et al., 2006).

  2.8. Adenoviruses

Currently, the adenovirus vector (AV) is the most commonly

used vehicle for locally administered BMP gene therapy because of 

its high ef ficiency in infecting both dividing and non-dividing cells

(Chen et al., 2003). Two major advantages of adenoviral vectors are

the high rate of transduction ( Jenkins et al., 2003) and the ability to

transfect a wide range of cell types (Franceschi et al., 2004).

The adenovirus contains double-stranded DNA and has no

enveloping membrane (Fig. 3). A so-called “penton fibre” that is

involved in the attachment of the virus to the host cell isfi

xed on

each apex of the icosahedral particle. Because of its non-enveloped

structure, the adenovirus is initially taken up by receptor mediated

endocytosis by binding to the coxsackie/adenovirus receptor (CAR)

(Fechner et al., 1999) on the cell membrane of regenerating cells,

such as immature osteoblasts (Ito et al., 2003). After endocytosis,

endosomes are formed that encase the virus and become acidic,

which activates the virus to undergo multiple uncoating steps.

Once one of the capsid proteins is released, lysis of the endosome

begins, and the virus is released into the cytosol. The viral core

binds to the nucleus core complex and releases the viral DNA intothe nucleus, which does not integrate into the host DNA, but

is translated using host cell mechanisms. Therefore an adenoviral-

transduced gene is expressed for only a limited period of time

(Tepper and Mehrara, 2002). For the acceleration of regenerative

responses, a process that requires only the transient delivery of 

cytokines or growth factors, the adenoviral vector is a valuable

resource (Asahara et al., 2000).

Adenovirus vectors are rendered unable to replicate through the

deletion of early transcription units (Musgrave et al., 1999). These

vectors contain a promoter of choice driving transgene expression,

often the constitutively active cytomegalovirus promoter.

Recombinant adenoviruses have traditionally been cloned but may

also be constructed through Cre/lox recombination (Hardy et al.,

1997). Adenovirus (Ad) attachment and uptake into cells are

Fig. 3. A and B diagrams and EM-image of an adenovirus demonstrating its capsid and “penton fibres” (Stannard LM,1995, online image [http://web.uct.ac.za/depts/mmi/stannard/

adeno.html]).

Fig. 2. Diagram of a retrovirus (National Health Museum, 2004, online image [ http://www.accexcellence.org/AB/GC/diagram.html]).

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separate but cooperative events that result from the interaction of 

the viral fibre and penton base proteins with specific cell surface

receptors. The fibre knob domain mediates the primary event, cell

attachment of Ad, which projects outward from the virion and

binds to the receptor (Miura et al., 2007).

Although the standard adenoviral vector does not allow stable

integration, in vivo studies of bone regeneration have shown that

adenoviral-mediatedgenescan be expressed forup to6 weeks in bone

and upto 4 weeks in the surrounding tissue (Baltzer et al., 2000). This

is longer than has been achieved with direct delivery of the protein.

Adenovirus-mediated gene transfer has been shown to achieve

a suf ficient dose of BMPs over time in vitro and in vivo, promoting

bone formation at both ectopic and orthotopic sites (Musgrave et al.,

1999; Baltzer et al., 2000; Franceschi et al., 2000; Li et al., 2003).

These studies demonstrated the clinical potential of gene therapy for

the induction bone formation at orthotopic sites, providing a clinical

alternative to BMP protein therapy (Franceschi et al., 2004).

Despite the promising results, the immune response to the

capsid protein reduces the transduction rate (Mahr and Gooding,

1999), resulting in less or no bone formation (Musgrave et al.,

1999; Chen et al., 2002). However, the greatest concern is

the threat to the recipient, as highlighted by the 1999 death of an

18-year-old patient suffering from ornithine transcarbamoylasedeficiency who developed multiple organ failure 4 days after the

systemic administration of a recombinant adenoviral vector

(Ciment, 200 0). Local application may also generate a massive

immune response. Chen et al. (2002) reported that one of 11 rats

died after direct adenoviral mediated gene transfer on the third day

post-injection. Currently, investigators are examining new strate-

gies to encapsulate the viral vector in order to mitigate the

inflammatory response, e.g., encapsulation in an alginate micro-

sphere (Sailaja et al., 2002).

  2.9. AAVs

To overcome the problems arising in immune competent indi-

viduals, the AAV-based vector has been developed, based on a non-pathogenic and replication-defective virus (Muzyczka, 1992) that

retains the advantages of a viral vector for direct gene therapy. The

recombinant AAV vector contains almost no viral-coding genes,

and therefore evades cell-mediated immunity (Monahan and

Samulski, 2000), resulting in low or no immune response (Chen

et al., 2003; Luk et al., 2003). AAV vectors are able to mediate

gene transfer to both dividing and non-dividing cells, and can

promote sustained gene expression in various tissues (Rabinowitz

and Samulski, 1998). In 2003, the AAV vector system was used to

transfer BMP-2 (Chen et al., 2003) and BMP-4 (Luk et al., 2003)

genes directly to the muscles of immune competent rats. Both

studies reported the successful transduction of cells in vitro and in

vivo, and enhanced bone formation compared to previous studies

using adenovirus vectors. The BMPs were expressed by local cellsat the end of the experiment at 8 weeks post-injection, which

is much longer than the transient expression using adenoviral

vectors. In contrast to AV vectors, adeno-associated vectors initi-

ated no detectable immunological responses (Chen et al., 2003).

Because of its safety, longevity, ef ficiency, and the ability to carry

out direct application in immune competent individuals, the AAV 

vector offers a very promising option for gene transfer within the

musculoskeletal system (Luk et al., 2003). Recently, light activated

gene transduction (LAGT) has been developed to increase the

transduction of human bone marrow derived stem cells using

ultraviolet light (Ito et al., 2004). LAGT acts as a safety measure to

confine the expression of the transducted gene to the illuminated

areas, controlling the location and amount of therapeutic gene

expression.

Despite the many advantages of AAV vectors, their poor capacity

to accommodate foreign DNA is a major limitation to their use in

gene therapy. Furthermore, it is dif ficult to produce suf ficient

amounts of the virus for clinical application.

  2.10. Other viral vectors

The retrovirus-mediated transduction of stem cells is restricted

by the quiescent nature and reduced receptor expression of these

cells (Havenga et al., 1997). Lentivirus-based vectors, derived from

another retrovirus subgroup, possess the ability to overcome these

limitations, and represent an alternative to retroviruses (Asahara

et al., 2000). Herpes virus saimiri (HVS)-based vectors were also

shown to infect human bone marrow stromal cells and their

progenitors (Frolova-Jones et al., 2000). In cell culture experiments,

stromal cells were susceptible to HVS-based infection in a dose-

dependent manner.

3. Candidate genes for bone regeneration

A number of genes are involved in the multiple steps of bone

regeneration and repair, and are potential candidates for gene

therapy. Beside the genes that encode for GDFs, such as PDGF, FGF,IGF, BMPs, parathyroid hormone (PTH), TGF-b, and VEGF, other

genes encode for transcription factors that are associated with

bone/cartilage-related gene expression (Runx2/Cbfal, Osterix,

Sox9), extra-cellular matrix molecules that induce or inhibit min-

eralisation (bone sialoprotein, dentin sialophosphoprotein, matrix

Gla protein, osteopontin), and receptor antagonists (soluble inter-

leukin-1 (IL-1), soluble BMP receptor) (Franceschi et al., 2004),all of 

which can be considered for use in therapy.

Because of their potent induction of de novo bone formation

with a single application, rhBMP-2, -4, and -7 are preferred

candidates for local gene therapy for bone regeneration. The

earliest studies proved the feasibility of transferring single BMP

genes both ex vivo and in vivo (Lieberman et al., 1995; Fang et al.,

1996; Lieberman et al., 1998; Musgrave et al., 1999). Later, otherBMPs were sequenced and characterised, such as the novel first-

generation adenoviral constructs encoding for BMP-4 or BMP-6

developed by Jane et al. (2002) for subcutaneous injection with

which ectopic bone formation was observed. At a low vector dose,

intended to diminish the immune response to the adenoviral

vector, AdBMP-4 and -6 demonstrated a more potent induction of 

bone formation when compared to the prototypical AdBMP-2

(Blum et al., 2003). Furthermore, whereas AdBMP-4 produced

ectopic bone in a manner similar to endochondral ossification,

AdBMP-6 seemed to induce both the intramembranous and

endochondral ossification pathways (  Jane et al., 2002). More

recently, Li et al. (2003) used a standardised adenovirus vector to

compare the activity of the BMP-2, -4, -6, -7, and -9 as measured by

an increase in the alkaline phosphatase activity in vitro and boneformation in vivo in athymic rats and immunocompetent Spra-

gueeDawley rats. The prototypical AdBMP-2 construct demon-

strated less bone-inducing activity than AdBMP-4, -6, and -9. The

latterdisplayed the highest osteogenic potential both in vitro and in

immunocompetent animals. Rundle et al. (2003) investigated the

effect of the substitution of a BMP-4 signal with a BMP-2 signal on

the expression of mature BMP-4. The BMP-2/4 hybrid gene induced

enhanced bone formation.

Most of the studies on gene therapy focused exclusively on

the use of a single factor. The significance of the results is,

therefore, limited by the fact that the native processes of bone

regeneration and fracture repair require the cooperation of 

multiple factors in a specific temporal sequence. Fang et al.

(1996) discovered the benefi

t of the combined expression of 

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interacting genes. A gene-activated matrix seeded with the two

plasmids, encoding for BMP-4 and hPTH 1e34, showed acceler-

ated bone formation, as compared to the administration of either

factor alone (Fang et al., 1996). More recently, multiple trans-

duction of two osteoprogenitor cell lines by AdBMP-7 and either

AdBMP-2 or AdBMP-4 resulted in a higher alkaline phosphatase

activity than expected for transduction with a single factor

(Franceschi et al., 2004). Currently, this group is investigating

these synergistic effects on bone induction in vivo. At a molec-

ular level, the BMP heterodimers possess a greater functional

activity than similar homodimers (Kawabata et al., 1998). Peng

et al. (2002) tested the interaction between an angiogenic

(VEGF) and an osteogenic (BMP-4) factor in bone formation and

demonstrated a synergistic effect when muscle-derived stem

cells were transduced with both factors.

Another approach focuses on the use of intracellular compo-

nents such as transcription factors in order to influence the activity

of osteoprogenitor cells. It has been shown that an overexpression

of the transcription factor Runx2 enhances osteoblast-specific gene

expression in mesenchymal cells (Ducy et al., 1997). Recently it has

been reported that a co-transduction of AdBMP-2 transduced cells

with AdRunx2 clearly enhanced the responsiveness to BMP-2 in

vitro and in vivo, resulting in more extensive ossification comparedto a transgene used alone (Yang et al., 2003).

4. In vivo versus ex vivo approach

Both in vivo and ex vivo methods have been successfully applied

in animal models to achieve bone regeneration. The in vivo

procedure involves the direct delivery of genes at the target site,

followed by transduction of target cells in situ (Fig. 4). Using the

appropriate delivery strategies, the in vivo gene transfer has been

shown to be a feasible, practicable, simple, minimally invasive, and

inexpensive strategy (Evans and Robbins, 1995; Bonadio et al.,

1999; Musgrave et al., 1999; Chen et al., 2003; Park et al., 2003;

Rundle et al., 2003). Although all studies have reported that

transfected cells are restricted to the injection site, it remainsdif ficult to target specific cell types for transduction (Musgrave

et al., 1999; Franceschi et al., 2004). Another disadvantage is the

dif ficulty in achieving a high transduction ef ficiency (Bonadio et al.,

1999). The first use of a retroviral vector in fracture healing showed

an adequate transduction rate but also faced the problem of 

undesired extra-periosteal bone formation (Rundle et al., 2003).

The immunogenicity of adenoviral vectors remains an unresolved

problem, whereas AAV vectors appear most promising in terms of 

immune compatibility and sustained gene expression (Chen et al.,

2003, Luk et al., 2003).

The ex vivo approach is a more sophisticated and expensive

technique involving target cell harvesting, expansion in cell culture,

ex vivo genetic manipulation, and (re)-implantation into target

tissue (Fig. 4). A disadvantage of the ex vivo procedure is the

morbidityassociated with cell collection from patients,when a pre-

existing cell line cannot be used (Hollinger et al., 2000). The ex vivo

expansion of cells adds considerably to the time required for the

process, and excludes this approach from the treatment of acute

conditions. However, significant advantages of ex vivo transduction

are the impossibility of uncontrolled diffusion of viral particles or

DNA complexes into the surrounding tissue, and the possibility of 

selecting the target cells (Scaduto and Lieberman, 1999). Ex vivo

transduction is highly ef ficient (Oakes and Lieberman, 2000). The

successfully transduced cells can be selected and precisely

implanted at the target site using a suitable carrier matrix. If the

cells are manipulated to secrete a soluble factor, the factor can

affect other host cells (paracrine effect) or the transduced cells

themselves (autocrine effect) (Franceschi et al., 2004). To make the

most of the autocrine effect, investigators use cells for transduction

that are highly responsive to the secreted factor and can differen-

tiate into the desired tissue.

4.1. Target cells

Various cells such as gingival or dermal fibroblasts (Rutherford

et al., 2002; Hirata et al., 2003; Jin et al., 2003), periosteal cells

(Mason et al., 1998; Breitbart et al., 1999), primary articulated joint

chondroblasts (Musgrave et al., 2000), bone marrow stromal cells/

MSCs (Musgrave et al., 2000; Gysin et al., 2002; Chang et al., 2003 ),

muscle-derived stem cells (Peng et al., 2002), fat-derived stem cells

(Dragoo et al., 2003), osteoblasts, and myoblasts (Musgrave et al.,

2000; Chen et al., 2003, Luk et al., 2003) have been successfully

transduced using in vivo or ex vivo techniques and the different

vector systems.

The transduction of cells has also been shown to be dependent

on the vector dose. Baltzer et al. (2000) found that the transduction

of murine stromal cells with 108 and 109 Ad-BMP-2s increasedalkaline phosphatase activity, while 1010 particles led to cell death,

highlighting the dif ficulty of defining the therapeutic dose. Simi-

larly, Mason et al. (1998) achieved bone regeneration with BMP-7

transfected periosteal, but also showed cytotoxicity at high doses.

Hence, the therapeutic window must be defined for every target

cell type, vector type, and therapeutic gene under consideration.

This is possible for ex vivo strategies but will be exceedingly dif fi-

cult for in vivo strategies where the target cells are not well defined

and may be different depending on the site of treatment. Overall,

these studies highlight the challenges in delivering BMPs, despite

their documented usefulness in gene therapy.

5. Summary 

Many clinical conditions require regeneration or implantation of 

bone, while various problems with bone healing remain. Modern

therapeutic options, such as bonegrafting and protein-based therapy

donot alwaysprovidesatisfactorysolutions tothe problem of massive

bonedefects. Non-traditional approaches for bone-loss repair, suchas

gene therapy, have been investigated as a potential solution. Of the

wide range of methods used for local bone regeneration, osteoin-

ductive approaches now play a central role. They have evolved

significantly since thefirst proteins capable of inducingde novo bone

formation were discovered (Urist, 1965). Since then, various factors

have been identifiedthat influence bone formation beneficially, such

as growth factors like PDGF, FGFs, IGFs, TGF-b, and VEGF, hormones

(e.g., PTH), and bone BMPs (e.g., BMP-2, -4, -6, -7, -9). BMPs have

recently attracted great interest because of their ability to induce de

Fig. 4. Strategies for delivering therapeutic genes to target tissue: in vivo transduction

involves direct gene transfer to target cells. In an ex vivo approach, cells are harvested,

expanded in culture, transduced in vitro, and re-implanted into the target site. Gene

transfer can be achieved by viral or non-viral vectors.

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novo bone formation (Schmidmaier et al., 2007). The ability to clone

andrecombinantly produce human BMPs hasopened a broadfieldfor

further research and clinical application. RhBMPs (2, 4, 6, 7, and 9)

possess a high potential for inducing ectopic and orthotopic bone, as

has been demonstrated in various animal models. Despite promising

results, futureresearchmust investigate optimal dosesand molecular

combinations, as wellas developa structureeactivityrelationship for

the different members of the BMP family in order to provide

predictable and effective clinicalapplications (De Laporte et al., 2006;

De Laporte and Shea, 2007). The biological activity of the soluble

factors that promote bone formation in vivo is transient, and sus-

tained local delivery remains a problem in clinical application.

Multiple materials including hydroxyapatite, tricalcium phosphate,

demineralised bone matrices, poly-lactic acid homo-/heterodimers,

and collagen have been tested as carriers for controlled delivery of 

these factors (De Laporte and Shea, 2007). Unfortunately, many of 

these delivery vehicles show limitations in terms of biodegradability,

inflammatory reaction, immunological rejection, disease trans-

mission, and, most importantly, the inability to provide a sustained

therapeutic factor level.

In response to these limitations, attempts have been made to

develop local gene therapy, in which the genes encoding the desired

proteins are delivered to the target cells rather than the proteinsthemselves. The transfected cells serve as local “bioreactors” as they

secrete the desired proteins in their vicinity. Ef ficient gene transfer,

however, requires an appropriate vector of either viral or non-viral

origin. Viral vectors have the advantage of a high transduction ef fi-

ciency (Vogt et al., 2008), and the disadvantages of a high immuno-

genic potential and, in the case of retroviruses and certain AAVs, the

threat of disturbance to normal gene function through insertional

mutagenesis. Non-viral vectors avoid these problems, but possess

a lower rate of transfection. With regard to ef ficiency and safety,

various studies conclude that adenovirus- and retrovirus-based

vectors are appropriate for an ex vivo approach, whereas AAVs are

a promising source for in vivo transduction. Nevertheless, further

studies are required to confirm the ef ficiency of lipoplexes and pol-

yplexes as non-viral vectors, which have the advantages of lowerimmunological and security risk, simple preparation, and almost

unlimited capacity for the size of genes that can be inserted.

Currently, an attempt has been made to successfully combine

the expression of different secreted factors (e.g., amongst the BMP

family, BMPþVEGF), or secreted and intracellular transcription

factors (Runx2) in order to enhance the responsiveness of target

cells (Li et al., 2009). Future research should focus on improvement

of the control of gene expression, the definition of optimal vector

dosages, and more accurate delivery of genes to the target cells in

vivo. Although promising results have been achieved in animal

defect models, human trials using gene therapy for local bone

regeneration have not yet been reported. A wide range of clinical

surgical applications is expected: correction of non-union fractures,

implant fixation, and reconstruction of skeletal defects. Additionalapplications exist in oral surgery where the successful placement of 

implants often requires bone augmentation of the maxillary sinus

floor or alveolar ridge. In these clinical situations, pre-existing local

bone and surrounding tissue are extremely compromised and are,

incapable of generating an adequate biological response to either

local protein therapy or gene therapy. In these cases, further

investigation in the field of tissue engineering might provide

support for the osteoinductive approach.

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