Gene Therapy and Tissue Engineering in Sports Medicine

Vladimir Martinek, MD; Freddie H. Fu, MD; Johnny Huard, PhD

THE PHYSICIAN AND SPORTSMEDICINE - VOL 28 - NO. 2 - FEBRUARY 2000

In Brief: Treatment of sports injuries has improved through sophisticated rehabilitation programs, novel operative techniques, and advances in biomechanical research during the past two decades. Despite considerable progress, treatments remain limited due to poor healing capacity for anterior or posterior cruciate ligament rupture, central meniscal tear, cartilage lesions, and delayed bone fracture. New biological approaches seek to treat these injuries with growth factors to stimulate and hasten the healing process. Gene therapy using the transfer of defined genes such as those encoding growth factors represents a promising way to deliver therapeutic proteins to the injured tissue. Tissue engineering, which may eventually be combined with gene therapy, offers the potential to create tissues or scaffolds for regeneration of defects occurring from trauma.

The treatment of sports-related injuries has improved continuously during the last two decades. New, minimally invasive operative techniques, especially arthroscopy, novel instruments, modern rehabilitation and medications, as well as increasing knowledge of joint biomechanics and trauma pathophysiology, have enhanced and accelerated therapy. Despite this progress, deficits in injury treatment remain because of the limited healing capacity of certain musculoskeletal system tissues. Ligaments, tendons, menisci, and articular cartilage have a limited blood supply and a slow cell turnover. For this reason, healing is prolonged and often results in the formation of a dysfunctional scar or a tissue defect.

Various growth factors effect musculoskeletal tissue healing (1). These growth factors are small proteins that can be synthesized both by resident cells at the injury site (eg, fibroblasts, endothelial cells, and mesenchymal stem cells) and infiltrating repair or inflammatory cells (eg, platelets, macrophages, and monocytes). The proteins are capable of stimulating cell proliferation, migration, and differentiation as well as matrix synthesis (2,3), and their effects have been shown on different tissues (2-12). Genes encoding most of the known growth factors have been determined. Using recombinant deoxyribonucleic acid (DNA) technology, researchers can produce large quantities of protein for use in treatment (1).

Although direct application of recombinant human proteins has some beneficial effect on healing (4), their relatively short half-lives in vivo often require high doses and repeated injections. Another major limitation of using growth factors to promote healing is the mode of delivery to the injury (13). Many strategies, including polymers, pumps, and heparin, have been tested in attempts to achieve consistent growth factor levels at the injured site (14,15). Despite the improved local persistence of growth factor proteins with various approaches, the results of these delivery techniques remain limited. Among the methods developed for local administration of growth factors, gene transfer techniques have proven the most promising (16).

Tissue defects are another challenge for treatment of injuries. These occur as a direct consequence of trauma (eg, cartilage lesion, osteochondral defect), treatment (eg, partial meniscus resection), or poor or impaired healing capacity (eg, anterior cruciate ligament [ACL] rupture, delayed bone union). Using autogenous or allogeneic grafts is the most common therapeutic approach for treatment of tissue defects such as these (17,18). Both treatments have disadvantages that include additional trauma from graft harvesting (autografts) (19), transmission of infectious diseases such as hepatitis B and HIV and immunorejection (allografts) (20). Tissue engineering, a new technology, focuses on the creation of tissues and scaffolds as a solution to these problems (21). The technology combines the use of adequate scaffolds (shaped polymers that serve as a matrix for tissue growth), selected growth factors, and responsive cells to improve the healing of several musculoskeletal system tissues, especially for treatment of cartilage and bone defects.

Gene Therapy

Gene therapy delivers therapeutic genes into cells and tissues. Originally, it was conceived for the manipulation of germ-line cells for treating heritable genetic disorders, but that application has been greatly limited because of inefficient technology and considerable ethical concern. Gene manipulation of somatic cells has been widely accepted, however. Gene therapy applied to sports medicine includes transfer of defined genes (such as those encoding growth factors or antibiotics) into the target tissue. Thus, successful application of gene therapy would promote production of therapeutic levels of desired proteins by the transformed cells at the site of injury or inflammation.

Vectors. For gene therapy to allow expression of an inserted gene, the DNA must be packaged into a vector and enter the cell nucleus, where it is either integrated into the chromosomes of the host cell (figure 1) or maintained in the nucleus as a separate episome. The inserted gene is then transcribed into messenger RNA (mRNA), and mRNA is transported into the cell cytoplasm, where it serves as the template for the production of the therapeutic protein (eg, growth factors) in the ribosomes. Consequently, the transduced cells become a reservoir of secreting growth factors and cytokines capable of improving healing.

Viral and nonviral vectors can be used to deliver genetic material into cells (table 1). Nonviral gene-transfer systems such as liposomes are usually easier to produce and have relatively low toxicity and immunogenicity, but their efficiency of gene delivery is hindered by a low transfection rate. (Transduction refers to introducing DNA into cells with a virus, while transfection refers to introducing DNA into cells with a plasmid vector.) Despite new approaches in enhancing cell transfection rates and development of new nonviral vectors (eg, new liposomes made with diverse lipids that form different composition aggregates containing the DNA), the efficiency of gene transfer remains low.

TABLE 1. Vectors Used for Gene Delivery Into Cells and Their Characteristics
Gene Delivery	Vector	Characteristics
Nonviral	Liposome DNA gene gun DNA-protein complex Naked DNA	Low efficiency of gene delivery Low immunogenicity Easy to produce
Viral	Adenovirus	Infects mitotic/postmitotic cells Low cytotoxicity Immunorejection common
	Retrovirus	Low toxicity/immunogenicity Infects only mitotically active cells Low capacity for gene insert
	Adeno-associated virus	Low toxicity/immunogenicity High persistence of gene transferred Low capacity for gene insert
	Herpes simplex	Infects mitotic/postmitotic cells Large insert capacity Immune rejection common

Currently, viral gene vectors present a more efficient method for gene transfer (13). Before a virus can be used as a vector in gene therapy, all genes for viral replication and genes for pathogenic proteins must be removed and replaced by the desired gene(s). Transfer relies on native viral ability to enter (infect) the cell (see figure 1). The virus attaches to the cell via a receptor, and the genetic material is transported through the cytoplasm by viral and/or cellular proteins and enters the nucleus. The most commonly used viruses include adenovirus, retrovirus, adeno-associated virus, and herpes simplex virus (see table 1). New mutant viral vectors with reduced cytotoxicity and immunogenicity are currently being developed (12).

Delivery strategies. Various gene-transfer strategies, including systemic and local delivery, can be used for gene transfer to musculoskeletal tissues (figure 2) (13). Systemic delivery consists of injecting the vector into the bloodstream, thus disseminating it to all organs of the body; the technique is preferable when the target tissue cannot be reached directly. This approach also has the advantage of better vector distribution than that of direct, local injection of the vector. Major limitations include low specificity of gene expression and large vector concentration required for therapeutic effects. The lack of blood supply in various tissues (eg, cartilage, meniscus), however, makes systemic delivery inappropriate for most musculoskeletal system injuries.

Two basic strategies for local gene therapy in the musculoskeletal system have been extensively investigated (see figure 2) (13). Vectors can be directly injected in the host tissue (in vivo technique), or the cells from the injured tissue can be removed, genetically altered (transduced [with a virus] or transfected [with a plasmid]) in vitro, and reinjected in the injury site (ex vivo technique). While the direct method is technically more simple, indirect delivery poses less risk because gene manipulation takes place under controlled conditions outside the body. With the ex vivo approach, growth factors can be delivered with endogenous cells capable of responding to stimuli and participating in the healing of the injured tissue. Tissue engineering-based approaches to gene delivery that employ cells from various tissues (eg, mesenchymal stem cells, muscle derived cell, dermal fibroblasts) may offer additional ways to improve healing. Selecting the appropriate gene delivery procedure depends on many factors that include the division rate of the target cells, pathophysiology of the disorder, and accessibility of the target tissues.

Limitations. For treatment of sports injuries, the major concern for using gene therapy is safety. While gene therapy may represent a "last chance" treatment option for severe disorders such as cancer, Duchenne muscular dystrophy, Gaucher's disease, or cystic fibrosis, the risk of side effects may be unacceptable in elective sports medicine. In addition, integration of viral vectors into the host genome carries the risk of insertional mutagenesis (22). Abnormal regulation of cell growth, toxicity from chronic overexpression of the growth factor and cytokines, and malignancy are all theoretically possible, but no cases have been reported. However, there is no guarantee that integrated DNA sequences will not cause mutations or malignancies years later. For this reason, long-term records of all human trials in gene therapy need to be kept and exchanged among the research groups. Most clinical trials of gene therapy are using the ex vivo approach, so the virus is not directly introduced into patients and cells can be extensively tested before implantation.

Loss of expression of the transferred gene after a few weeks is a common and not fully understood phenomenon. However, temporary and self-limiting gene expression could be useful in the treatment of musculoskeletal injuries, in which only transient high levels of growth factors are needed to promote healing response. Present research is also focusing on the development of specific inducible promoters that regulate the mRNA transcription. (Promoters are DNA sequences that are adjacent to the functional genes and are required for expression and regulation of gene transcription.) The inducible promoters could help control expression of the transferred gene; they could modulate implanted genes as well as turn them on and off. Although these systems are very attractive, they remain under extensive investigation in many laboratories and are not yet ready for clinical trial.

Although great strides in gene therapy techniques have taken place, they still have not become established treatments, partly because of the lack of appropriate gene vectors. Many laboratories successfully focus on developing therapeutic viral and nonviral vectors. Consequently, major advances in vector development can be expected in the near future (12).

Tissue Engineering

Tissue engineering is a technology based on developing biological substitutes for the repair, reconstruction, regeneration, or replacement of tissues (figure 3: not shown). Its long-term goal is to construct biomaterials that are biocompatible, biodegradable, and capable of integrating molecules (eg, growth factors) or cells (23,24). Currently, many different ceramics, polymers of lactic and glycolic acid, collagen gels, and other polymers have been tested in vitro and in vivo (24). More recently, genetic modifications have been included in tissue engineering to optimize the healing process (25). Modified cells are transplanted into injured tissue to effect the repair with the introduced gene.

Bone and cartilage are the tissues in which most tissue engineering techniques have been applied. Bone has a high potential for repair. In large defects or when vascularization is impaired, however, augmentation with scaffolds, genetically engineered cells, and/or growth factors and cytokines can accelerate or enhance the healing (5). Recently, autologous muscle tissue has been used as a delivery vehicle for growth factor genes in treatment of bone defects (26), and laboratory-grown skin has been recently approved for use in treatment of wounds (27,28).

In contrast, cartilage has a poor intrinsic capacity for healing and therefore a limited ability to regenerate (23). Intense investigations have focused on finding biomaterials that would be capable of repairing cartilage defects, but no efficient therapeutic approaches have yet been established (29); candidate materials include fibrin, collagen, ceramics, alginate, polymers of lactic and glycolic acid, hyaluronic acid, and synthetic materials.

Besides bone and cartilage substitutes, biological scaffolds have been developed for other tissues of the musculoskeletal system. The collagen meniscus implant is a biological scaffold for meniscus regeneration made from bovine Achilles tendons. Short-term clinical results were promising in 20 patients who had total meniscal loss (21), but long-term data are not yet available. In addition, biomaterials for ACL replacement have been used in trials but have never gained surgical acceptance (30).

Another tissue engineering approach is the use of autologous tissues or cells. Myoblasts become post-mitotic by fusing with existing myofibers or fusing among themselves to form postmitotic myotubes (31). Hence, muscle-derived cells that have been transduced to express a therapeutic protein may differentiate within the host tissues (muscle, ligament, bone, meniscus) and lead to a stable and persistent expression of the desired protein at the injury site (32).

Approaches for Treating Sports Injuries

Skeletal muscle. Depending on the type of sport, the prevalence of muscle injuries varies from 10% to 55% among all sustained injuries (33). While minor muscle injuries such as strains can heal completely, most severe muscle injuries often heal with dense scar tissue formation, impairing muscle function and potentially leading to muscle contractures and chronic pain. Other clinical problems include disparate limb length after injury, disabling contractures from scar tissue, and post-compartment syndrome muscle necrosis.

Growth factors may offer a new avenue to treat muscle injuries. Recently, in vitro and in vivo experiments with growth factors have shown improved healing, especially with basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and insulin-like growth factor type 1 (IGF-1) (4,33,34). Promising gene therapy approaches have already been used in treating inherited disorders such as Duchenne muscular dystrophy (35). Gene therapy techniques are now being investigated to establish an efficient treatment method for improved healing of sports-related muscle injuries (4).

Cartilage. Damage to knee articular cartilage is a common problem following sports injuries. It leads to premature arthritis, causes a considerable decrease in quality of life, and has enormous long-term healthcare costs (29). Regeneration of damaged articular cartilage is very limited because in adults cartilage lacks a blood supply, lymphatic drainage, and innervation. Furthermore, chondrocytes are sheltered from synovial fluid nourishment and reparative recognition by their large extracellular matrix (36).

The common operative techniques for existing therapy of injured articular cartilage are subchondral drilling or microfracture, transplantation of autologous or allogenic chondrocytes, autogenous or allogenic osteochondral transplantation, and the use of scaffolds (37). Despite some promising clinical results (38), new strategies are required to obtain consistently good long-term results. Growth factors, including BMP-2 (6), bFGF, transforming growth factor ß (TGF-beta), epidermal growth factor (EGF), IGF-1 (39), and cartilage-derived morphogenic proteins (CDMP) (39) have demonstrated both in vitro and in vivo positive effects on chondrocyte growth and cartilage healing. Several gene therapy and tissue engineering techniques are currently being investigated for treating cartilage defects, but the most efficient methodology for solving this sophisticated problem has not yet been established (39).

Anterior cruciate ligament. The ACL is the second most frequently injured knee ligament: More than 100,000 ruptures are estimated to occur every year in the United States (40). Although tears of the medial collateral ligament heal spontaneously in most cases, the ACL has a low healing capacity (41). To restore the normal knee function after complete ACL rupture, surgical reconstruction using autograft or allograft tendon is required (41,42). For ACL replacement with autologous material, both the bone-patella tendon-bone (BPTB) graft and hamstring tendon grafts represent the standard choice. Although ACL replacement surgery has been significantly improved in the last decade, there is still a remaining challenge to improve and accelerate healing after ACL reconstruction.

Because ligaments can take up to 3 years to reach full strength (43), the transplanted graft undergoes a period of weakness, and rehabilitation after ACL reconstruction remains slow. Even professional athletes are cautioned not to return to competitive sport until at least 6 months after surgery (see "A Perioperative Rehabilitation Program for Anterior Cruciate Ligament Surgery," January, page 31).

Recently, several studies have shown a positive effect of growth factors (platelet-derived growth factor AB [PDGF-AB], EGF, and bFGF) on ACL fibroblast metabolism (9). The data suggest that these specific growth factors may improve healing of the ACL or "ligamentization" of the ACL graft. Gene therapy holds promise for delivering growth factors to the ligaments (44). The first feasibility studies have demonstrated that ligaments can be transduced with viral vectors (45). The next step is the transduction/transfection of the grafts with vectors expressing appropriate growth factors and cytokines to improve the healing process. Generally, the viral transduction could be performed directly at the time of graft implantation or in vitro prior to the ACL reconstruction (graft preconditioning).

Using the autologous semitendinosus and/or gracilis tendon for ACL reconstruction, surgeons also face disadvantages that stem from the tendon-to-bone healing in the femoral and tibial tunnel, a result that seems inferior to the bone-to-bone fixation of the BPTB graft. The bone morphogenetic protein type 2 (BMP-2) is one of the growth factors that could solve this problem and improve the healing of the tendon in the bone tunnel (46).

Meniscus. Meniscal tears due to twisting or compression forces are common sports injuries. Several repair techniques, including sutures, arrows, and staples, have been developed to preserve the menisci, but only tears in the vascularized peripheral third of the meniscus can heal (47). Meniscal lesions in the avascular central part do not heal and present a critical clinical problem because even partial resection of the meniscus leads over time to cartilage damage and osteoarthritis (48). Experimental studies have shown that healing in the central meniscus might be promoted by some chemotactic or mitogenic stimuli delivered by fibrin clot, synovial tissue, or growth factors (transforming growth factor [TGF] -alpha, bFGF, EGF, and PDGF-AB) (10,49,50). The goals of gene therapy for meniscal healing are to transduce central meniscal tears with vectors directly or transduce indirectly with autologous cells expressing growth factors and cytokines that stimulate cell proliferation and matrix synthesis in meniscal fibroblasts and promote efficient healing.

After a complete loss of the meniscus from an extended injury or repeated resections, rapid impairment of knee function occurs in most of the patients. Without therapy, osteoarthritis develops in most patients in 5 to 10 years, faster than would occur as a consequence of aging (51). The treatment after meniscal depletion is limited. To find a therapeutic solution, meniscal transplantation (allograft) has been performed (17). Clinical studies on this issue are rare and report failures of up to 60% after less than 2 years (52,53). Experimental studies show a slow immune rejection within the transplanted meniscal allografts (54). Pretreatment of meniscus allografts with viral vectors expressing growth factors could lead to acceleration of the graft healing and restructuring, and to suppression of the immunogenicity.

Bone. Bone has sufficient potential to heal, and mechanical fixation is an adequate method for healing most fractures; however, in more than 10% of fractures, delayed unions or non-unions have been described after trauma (55). The non-union presents a severe problem that has to be addressed with sophisticated surgical practices (external fixation and/or bone transplantation, microsurgical tissue or bone transfer, or bone grafting).

Another problem manifested in sports medicine is stress fracture of the lower extremity, which constitutes up to 15% of all injuries in runners (56). The treatment is prolonged and the recovery time often demands 4 to 12 months or more (57).

For treatment of these bone disorders, biological interventions by application of specific growth factors to stimulate bone production have shown promising results (58). Recent in vivo experiments have demonstrated the ability of BMPs, especially BMP-2, IGFs and TGF-beta, to promote bone healing (table 2) (11). Gene therapy may be able to deliver the specific gene to the site of repair followed by continuous production of the desired growth factor (59,60).

TABLE 2. Effect of Growth Factors in Musculoskeletal Tissues
Growth Factor	Skeletal Muscle	Hyaline Cartilage	Meniscus	Ligament	Bone
IGF-1	+	+	+	+	+
bFGF	+	+	+	+	+
NGF	+		-	-
PDGF	-		-	+
EGF	-	+	+	+
TGF-alpha	-		+	-
TGF-beta	+	+	+	+	+
BMP-2		+	+		+
+ = positive effect; - = no or negative effect; blank = not tested IGF-1 = insulin-like growth factor-1; bFGF = basic fibroblast growth factor; NGF = nerve growth factor; PDGF = platelet-derived growth factor; EGF = epidermal growth factor;TGF = transforming growth factor; BMP-2 = bone morphogenetic protein-2

Muscle-derived gene therapy could open another important avenue to treat bone defects (26), because muscle tissue has demonstrated osteogenetic competence in response to osteoinductive stimuli, and muscle-derived cells have been used to deliver therapeutic proteins in nonorthopedic diseases (61).

Future Directions

Although gene therapy is not yet established as an approved therapeutic technique, a great potential exists for the treatment of musculoskeletal injuries in the future (figure 3). Currently, only a few effective therapeutic gene therapy techniques have been tested in human joints (13). At the 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 positive effects of transduced genes (especially BMP-2, IGF-1, TGF-beta in vitro and in vivo. The main obstacle today seems to be the availability of vectors carrying effective genes, and some concern with the safety of viral vectors after the death of a patient in a recent gene therapy trial (62). However, great progress has been noticed in many laboratories working on the engineering of these vectors.

In general, we believe that the combination of gene therapy and tissue engineering will help us develop effective therapies for tissues that has a low healing capacity (ie, cartilage, meniscus, and ligament) and for other disorders such as osseous nonunion and arthritis. With these new technologies, however, a large number of basic science and preclinical studies still need to be performed before the efficiency necessary for orthopedic applications and guaranteed safety are reached (63,64).

Selected Readings*

• Arnoczky SP, Warren RF, Spivak JM: Meniscal repair using an exogenous fibrin clot: an experimental study in dogs. J Bone Joint Surg (Am) 1988;70(8):1209-1217
• Crystal RG: Transfer of genes to humans: early lessons and obstacles to success. Science 1995;270(5235):404-410
• Dhawan J, Pan LC, Pavlath GK, et al: Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 1991;254(5037):1509-1512
• Evans CH, Robbins PD: Possible orthopaedic applications of gene therapy. J Bone Joint Surg (Am) 1995;77(7):1103-1114
• Huard J, Verreault S, Roy R, et al: High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice. J Clin Invest 1994;93(2):586-599
• Mulligan RC: The basic science of gene therapy. Science 1993;260(5110):926-932
• O'Donoghue DH, Frank GR, Jeter GL, et al: Repair and reconstruction of the anterior cruciate ligament in dogs: factors influencing long-term results. J Bone Joint Surg (Am) 1971; 53(4):710-718
• O'Driscoll SW: The healing and regeneration of articular cartilage. J Bone Joint Surg (Am) 1998;80(12):1795-1812
• Sellers RS, Peluso D, Morris EA: The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage. J Bone Joint Surg (Am) 1997;79(10):1452-1463
• Trippel SB: Growth factors as therapeutic agents. Instr Course Lect 1997;46:473-476

*A complete reference list will be available at www.physsportsmed.com beginning in March.

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Dr Martinek is a sports medicine fellow in the department of orthopaedic surgery, and Dr Fu is professor and chair in the department of orthopaedic surgery at the University of Pittsburgh. Dr Huard is an assistant professor in the department of orthopeadic surgery and molecular genetics and biochemistry at the Children's Hospital of Pittsburgh and University of Pittsburgh. Address correspondence to Johnny Huard, PhD, Dept of Orthopaedic Surgery and Molecular Genetics and Biochemistry, Children's Hospital of Pittsburgh and University of Pittsburgh, 3705 Fifth Ave, Pittsburgh, PA 15213-2583; e-mail to jhuard+@pitt.edu.