Introduction
The treatment of musculoskeletal disorders has improved during the last 20 years due to enormous progress in the understanding of basic biology and biomechanical principles. Based on this knowledge, new conservative methods, minimally invasive operative techniques, modern rehabilitation programs, and innova- tive approaches were developed and successfully applied for treatment. Despite this progress, there are still limi- tations in the therapy of musculoskeletal tissues with a limited healing capacity [1].
Tendon tissue has a low blood supply, cell turnover, and metabolism, similar to menisci or articular cartilage. For this reason, the healing of tendon injuries is prolonged and often results in the formation of inferior scars or remaining lesions [2]. In orthopedics, both the healing of acute tendon ruptures and degenerative changes of the tendons represent a serious problem for the clinician.
New approaches have been investigated to improve the healing of tendon tissue and to develop new, biological therapies for tendon ailments [3].
Growth Factors
Growth factors, or cytokines, have been identified as sub- stances that are capable of improving the healing process in tissues [4]. They are peptides that can be generated both by the resident cells at the injury site (e.g. fibrob- lasts, endothelial cells, mesenchymal stem cells) and by invading reparatory cells (e.g. macrophages, monocytes, platelets). Growth factors can stimulate cell proliferation, migration and differentiation as well as the matrix synthesis in different tissues can induce certain healing responses in the injured tissue [3]. Meanwhile, the genes encoding for these growth factors have been identified.
Growth Factor Effecting Tendon Healing In Vitro Using recombinant DNA technology, we are now able to produce growth factors in large quantities [5]. To deter- mine which growth factors can promote the healing of tendon tissue, the role of different cytokines has been investigated in a large number of in vitro experiments [3].
Despite major limitations of studying the effects of growth factors on cultured tendon cells, several cytokines have been identified as potent promoters of fibroblast or endothelial proliferation, synthesis of extracellular matrix proteins such as collagen and proteoglycans, cell migration, and differentiation (Table 30-1). From a review of the literature, the proliferation of fibroblasts is most powerfully stimulated by platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF), while transforming growth factor b (TGFb) and IGF-1 are capable of increasing the matrix synthesis of tendon cells [2,3].
Cytokines such as endothelial growth factor (EGF), hepatocyte growth factor (HGF), bone morphogenetic protein 2 (BMP-2), PDGF, and interleukin IL-1) also showed positive effects on fibroblast migration across collagen coated membranes [6].
Growth Factor Facilitated Tendon Healing In Vivo Two strategies are applied for the determination of specific growth factors improving the healing of tendons and ligaments [2]. The first strategy, which involves the detection of cytokine receptors and endogenous growth factors within the tendon tissue following injury, defines specific growth factors participating in the physiological process of tendon healing. Regarding this method, immunohistochemical staining of ligament and tendon samples have shown up-regulation of PDGF, TGF-b, bFGF, and EGF by 3 to 7 days following injury [7,8]. The second strategy investigates the effect of direct growth
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Gene Therapy in Tendon Ailments
Vladimir Martinek, Johnny Huard, and Freddie H. Fu
factor application in injured tendons and ligaments.
Exogenous application of PDGF and bFGF at the time of injury increases the strength, stiffness, and breaking energy of healed ligaments. Basic FGF proved also to be a potent angiogenic factor [3,9–11]. However, the need for long-term release or high dosages can result in reduc- tion of their beneficial effect, which is characteristic of the complex relationships associated with many cytokines [9,10].
Limitations of Growth Factor Application
Although the direct injection of these human recombi- nant proteins displays some beneficial effect on the healing process, very high dosages and repeated injec- tions are often required due to the relatively short biological half-life [4]. Another major limitation of using these human recombinant growth factor proteins to promote healing is their delivery to the injured site [12].
In fact, the use of various strategies such as polymers, pumps, and heparin, has been investigated to achieve sus- tained levels of therapeutic proteins. Although these approaches have been capable of improving the local persistence of the growth factor proteins, the success remains limited. Among the different methods developed for local administration of growth factors, gene therapy based on the use of various gene transfer techniques has proven to be the most promising [13].
Gene Therapy
Definition of Gene Therapy
Gene therapy is a technique relying on the alteration of the cellular genetic information. Originally, gene therapy was conceived for the manipulation of germ-line cells for the treatment of inheritable genetic disorders, which is greatly limited by the inefficient technology and consid- erable ethical concerns. Meanwhile, gene manipulation of somatic cells has been widely achieved for many tissues [13]. Gene therapy can be used in orthopedic applications using the transfer of genes encoding for growth factors, cytokines, and antibiotics, for example, into the target tissue.Thus, therapeutic substances can be highly and per- sistently produced directly at the site of injury, with ther- apeutic levels of growth factors, which can improve the healing process.
Vectors
In order to achieve gene expression, the transferred DNA material has to enter the nucleus, where it either inte- grates into the chromosomes of the host cells or remains separated from the host DNA in the form of an episome.
After transcription, the generated mRNA is transported outside the nucleus and serves as a template for the pro- duction of proteins (e.g. growth factors) by ribosomes (Figure 30-1). The cells transduced by the vector can express the growth factors and cytokines of interest and become a reservoir of secreting molecules capable of improving the healing process.
Viral and nonviral vectors can be used for delivery of genetic material into cells (Table 30-2). Nonviral gene transfer systems are usually easier to produce and have a significantly lower cell toxicity and immunogenicity than the viral vectors, but the overall use of the nonviral systems has been hindered by the low transfection effi- ciencies [14]. Different approaches are being investigated to improve non-viral gene transfer, e.g. the use of necrotic agents to increase the permeability of the nonviral vectors and the development of liposomes, however, their success remains limited.
Currently, viral gene vectors present the most efficient method [12]. Before using a virus for gene therapy, all genes encoding for pathogenic proteins must be deleted Table 30-1. Gene delivery vectors
Nonviral Viral
liposomes Adenovirus
DNA gene gun Retrovirus
DNA-protein complexes Adeno-associated virus
naked DNA Herpes simplex virus
virus with growth factor gene growth factor
DNA
growth factors cytoplasm
ribosomes nucleus
cell
Figure 30-1. Principle of viral gene transfer.
and replaced therapeutic gene. In fact, many years of basic research were required to characterize and remove the pathogenic genes from the viral genomes. The native ability of the virus to enter (infect) the cell and express its own genetic material within the infected cell could then be utilized. The most commonly used viruses in gene therapy currently are adenovirus, retrovirus, adeno- associated virus, and herpes simplex virus. Although viral vectors display a high efficiency of transfer to many cell types, their cytotoxicity (herpes simplex virus), immuno- genicity (adenovirus), and inability to tendinosis post- mitotic cells (retrovirus) have limited their general application for gene therapy purposes. Consequently, new viral vectors with reduced cytotoxicity and immuno- genicity are under development [5].
Strategies
Various gene delivery strategies such as direct and sys- temic delivery can be used to achieve gene transfer to the knee and the musculoskeletal system (Figure 30-2). The systemic delivery of the viral vectors consists of injecting the vector in the bloodstream, resulting in the dissemi- nation of the vector to the target tissues. This approach represents a major advantage when the target tissue is difficult to reach by direct injection. Moreover, systemic delivery often displays better distribution of the vectors within the targeted tissues since direct injection of the
vectors often results in a localized expression at the injected site. The major disadvantages of this technology are the high amount of vectors required and the lack of specificity of expression, because the majority of the vectors will be absorbed by the lung and the liver tissue.
Furthermore, the low vascularity renders this approach inefficient for some musculoskeletal injuries including tendon disorders.
Two basic strategies for direct gene therapy to the mus- culoskeletal system have been extensively investigated [12]. The vectors either are directly injected in the host tissues (direct, in vivo), or the cells of the injured tissue are removed, genetically altered (transduced/
transfected) in vitro, and implanted in the tissues (indi- rect or ex vivo). The direct, in vivo method is technically easier, but the ex vivo gene delivery offers more safety as the gene manipulation takes place under controlled conditions outside the body. Furthermore, the ex vivo approach leads to the delivery of growth factors and cytokines as well as endogenous cells capable of respond- ing to the stimuli and participating in the healing process.
Tissue engineering-based approaches which aim at using cells from different tissues (mesenchymal stem cells, muscle derived cells, dermal fibroblast) to deliver genes may give rise to further approaches for improving the healing process of various tissues of the musculoskeletal system. Selecting the appropriate procedure depends on various factors such as the division rate of the target cells, pathophysiology of the disorder, availability of cells from the injured tissues, and the type of vector used.
Gene Transfer in Tendons and Ligaments
In the last few years, several investigators have reported successful gene transfer to tendons and ligaments. A number of investigators have demonstrated the feasibil- ity of transferring marker genes directly or indirectly to uninjured and respectively injured tendons [14–19].
Genetically modified, heterologous fibroblasts were injected into rabbit patella tendon where they migrated from the site of injection and integrated into the crimp pattern of the patellar tendon [15]. In our own study, rabbit semitendinosus tendon explants were transduced ex vivo with adenoviral vectors expressing various marker genes. In this experiment, the transgene lacZ Table 30-2. In vivo therapeutic gene therapy in tendons. Abbreviations: HVJ-
hemagglutinating virus of Japan; PDGF-B-platelet derived growth factor B; HGF/
SF-hepatocyte growth factor/scatter factor; BMP-2-bone morphogenetic protein 2;
ODN-oligodeoxynucleotide
Author/Year Species Strategy Vector Cytokine
Nakamura, 1998 [21] rat/patellar tendon in-vivo HVJ liposome PDGF-B Natsuume, 1998 [22] rat/patellar tendon in-vivo HVJ-liposome HGF/SF
Nakamura, 2000 [23] rabbit/MCL in-vivo HVJ-liposome Anti Decorin ODN Martinek, 2001 [20] rabbit/semitendinosus ex-vivo Adenovirus BMP-2
tendon
Gene delivery
“systermic” “local”
ex-vivo in-vivo
injection cell culture
bloodstream
target tissue in the knee joint Figure 30-2. Gene therapy strategies.
expression on the surface of the tendons in tissue culture and in vivo after osseous implantation of the tendons in rabbit knee joints was demonstrated up to 8 weeks fol- lowing transduction [19] (see Figures 30-3 and 30-4, see color insert). In a subsequent experiment, the rabbit semitendinosus tendon explants were transduced ex vivo with adenoviral vectors expressing the bone morpho- genetic protein 2 (BMP-2). After implantation of the
transduced tendons in the bone tunnel, the tendon-bone interface showed a significantly enhanced osteogenic activity in the adjacent bone and ossifications in the implanted tendons (Figure 30-4, see color insert). This histological appearance was confirmed by significantly improved tensile properties of the implanted tendons during the biomechanical pullout testing [20]. Mean- while, other authors also have demonstrated successfully therapeutic gene transfer to tendon tissue in animal studies [21,22] (see Table 30-3). Direct delivery of a PDGF gene and hepatocyte growth factor/scatter factor (HGF/SF) into the healing rat patellar tendon ligament led to enhanced collagen synthesis and angiogenesis [21,22]. In another approach, down-regulating of the pro- teoglycan decorin, a collagen fibrillogenesis inhibitor, by direct gene transfer of antisense decorin oligodeoxynu- cleotides using an in vivo method of hemagglutinating virus of Japan (HVJ) conjugated liposomes led to an increased development of larger collagen fibrils in early scar and a significant improvement in scar failure strength of medial collateral ligaments of rabbits [23].
Figure 30-3. Transgene expression of lacZ on the surface of adenovirally ex vivo transduced rabbit semitendinosus tendon after 4 weeks in tissue culture (A) and 4 weeks following implantation in the rabbit femoral tunnel (B). (See color insert.)
Figure 30-4. Tendon-bone interface 6 weeks after implantation of ex-vivo adenovirally transduced rabbit semitendinosus tendon in the osseous femoral tunnel, (H/E, 60¥). (See color insert.)
Current Limitations of Gene Therapy
For the treatment of musculoskeletal disorders including tendon injuries, the major concern for the use of gene therapy is safety. While gene therapy may represent a
“last chance” treatment option in cases of malignancies or severe genetic disorders (e.g. Duchenne muscular dys- trophy, Gaucher disease, cystic fibrosis), the risk of many side effects and potential consequences of gene therapy may be unacceptable in elective orthopedics. Virus vectors integrating into the genome of the cells bear the danger of insertional mutagenesis [24]. Possible abnormal regulation of cell growth, toxicity due to chronic overex- pression of the growth factor protein and development of a malignancy are theoretically conceivable, although cases have never been reported. Vectors that do not inte- grate into the native DNA avoid these risks, but remain greatly limited by the lack of persistent expressions of the desired genes in the target tissues. The loss of expression of the transferred gene over a few weeks is a frequent and not fully understood phenomenon, especially for adenoviral vectors. However, a temporary and self- limiting gene expression could be useful in the treatment of musculoskeletal injuries, for which only transient high levels of growth factors might be necessary to promote the healing response.
Gene Therapy in Treatment of Tendon Ailments
Clinically relevant issues from the field of tendon dis- orders for which therapeutic interventions based on gene therapy could have a significant impact are basically rotator cuff tendon tears, flexor tendon injuries, tendinopathy, and the healing of tendon grafts used for cruciate ligament reconstruction [25].
Rotator Cuff Lesions
Rotator cuff lesions represent a significant age-related clinical problem with a complex etiology and prolonged treatment course. A rotator cuff rupture is the conse- quence of a disturbed tendon physiology caused either by extrinsic (surrounding structures) or by intrinsic factors. Possible intrinsic factors, which are fairly difficult to influence, include decreased vascularity and hypoxia, intrinsic degeneration, and fibrocartilaginous degenera- tion [26]. Due to these limiting intrinsic factors, healing of the rotator cuff following injuries and after operative cuff reconstructions is limited. Intro- duction of therapeutic genes into a diseased or injured rotator cuff tendon to release growth factors promo- ting cell proliferation, matrix synthesis or ingrowth of blood vessels represent a new promising treatment alternative.
Tendon Injuries
Among tendon injuries, Achilles tendon ruptures repre- sent a relevant clinical problem with an urgent demand for improvements and therapeutic alternatives. There is not only the difficulty of healing of the tendon proper, but there is also the problem of healing between the tendon and the tendon sheath, which can lead to adhe- sions and a massive loss of function [25]. In most cases, a rupture of the flexor tendon follows a process of tendon degeneration that cannot be treated sufficiently with current methods. For this reason, the main goals of gene therapy after tendon injuries is to prevent the tendon degeneration, to promote the tendon regeneration after injuries, and finally to avoid development of adhesions in the healing tendon.
Tendinopathies
Tendinopathies such as tennis and golfer’s elbow or jumper’s knee are very common repetitive motion symp- toms with a microscopic picture of microtraumatic injury response.Apparently, these chronic conditions are caused by small tears within the tendon tissue without a true inflammatory response. The typical microscopic findings are the alteration of the size of mitochondria as well as alterations in the nuclei of the internal fibroblasts or tenocytes [27]. A local genetic modification of these cells could be helpful to stop the collagen breakdown and to maintain the regeneration of the tendon tissue. Also, the improvement of the blood supply of the involved tendon area e.g. by the transfer of the vascular endothelial growth factor (VEGF) is imaginable.
ACL Tendon Grafts
The current standard treatment for anterior cruciate lig- ament (ACL) or posterior cruciate (PCL) ruptures is the replacement with autologous or allogenous tendon grafts.
The golden standards are the middle third of the patellar tendon with the adjacent bone blocks from the patella and the tibial tuberosity or the quadrupled semitendi- nosus/gracilis tendon graft [28]. After implantation the grafts undergo a process of degradation and replacement by a scarlike tissue, which is mechanically inferior in com- parison to the normal ACL. For this reason, the rehabil- itation after ACL reconstruction is prolonged and the return to sports delayed for at least 6 months. Gene therapy approaches could be effective in regeneration of biologic ACL auto-/allografts and in improvement of tendon-to-bone healing after usage of ACL tendon grafts.
Following gene transfer of appropriate growth factors, the healing of cruciate ligament tears may become feasi- ble and finally replace the current routine of complete ACL replacement.
Future Directions
Although gene therapy is not yet established as an approved therapeutic technique, a great potential exists for the treatment of tendon disorders in the future. Until recently, only a few effective therapeutic gene therapy techniques have been shown in humans [12]. At experimental level, many studies have been performed successfully to prove the feasibility of gene delivery into different tissues of the musculoskeletal system. Beyond this stage, initial experimental studies demonstrated pos- itive effects of transduced genes (especially BMP-2, IGF- 1, TGF-b) in vivo. The main obstacle today seems to be the availability of vectors carrying therapeutic genes, but great progress has been noticed in many laboratories working on the engineering of these vectors [5]. In general, gene therapy will potentially help us to develop efficient therapies for tissues with low healing capacity (cartilage, meniscus, tendons) and for other disorders such as osseous nonunion or arthritis.
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