Outline

Weak intrinsic healing potential is the central issue underlying difficulties of tendon healing and major clinical problems associated with tendon surgery. Biological augmentation of the healing process has long been sought. In the past decade, growth factor gene therapy emerged as an attractive treatment modality. Our work over the past decade has not only identified appropriate genes as the therapeutic target and vectors as gene-carrying vehicles but also assessed the efficiency of transgene expression and sustained supply of the growth factors in the healing tendon. In testing the efficacy of growth factor gene therapy in vivo, we found that injection of adeno-associated viral vectors harboring basic fibroblast factor or vascular endothelium growth factor cDNAs to the tendon during surgery substantially increases tendon healing strength in a chicken tendon cut and repair model. Transgenes were expressed from postsurgical weeks 2 to 6, covering the critical healing period. In the healing tendons, gene therapies increased the levels of growth factors, promoted synthesis of extracellular matrix components, decreased tenocyte apoptosis, and enhanced cellular proliferation. These evidences show the promise of gene therapies in the arena of tendon repair. Our studies also indicate the possibility of delivery of synthetic micro-RNA in limiting adhesions. These findings may lay the groundwork for establishment of methods to correct the insufficiency in intrinsic healing capacity of the tendon.

Improvement in tendon healing through biological approaches, molecular manipulations in particular,¹^–⁸ is a relatively new area of investigation related to flexor tendon surgery. These biological manipulations are intended to overcome a major obstacle to success in tendon repair—low intrinsic healing potential. The development of molecular treatment of tendon healing has benefited from advances in biotechnology and a recent exponential expansion in available genetic information. We have noted that biological investigations are catching up with mechanical studies, making an increasingly greater contribution to the efforts to improve tendon repairs.⁹^–²⁹ From these biological investigations, we are rapidly accruing information regarding the molecular mechanisms and efficacy of manipulations of tendon healing, which may bear fruit in the future.

Weak healing potential is a common problem in tissues without a rich blood supply. At present, there is a lack of effective treatment to ensure a prompt and strong healing of these tissues. Finding treatments to accelerate healing such tissues is of paramount significance in regenerative medicine, sports medicine, rehabilitation, and in surgery. The treatments not only benefit tendon repair in the hand but also benefit repair and regeneration of tendons, ligaments, and tendon–bone junctions throughout the body. Efforts to accelerate healing of those tissues are expected to bring about significant impacts in a number of medical specialties. Laceration of digital flexor tendon is representative of a wide spectrum of injuries that fail to heal reliably and is an ideal setting for testing novel molecular therapies. Work in this area holds promise for advancing the treatment of tissue injuries due to trauma or sports or in association with degeneration.

Of gene therapy applications in tissue healing, the unique advantage is that this strategy necessitates production of the encoded factors for a fairly short period.³⁰^–³² Gene therapies traditionally aimed at curing tumors or congenital diseases, in contrast, necessitate long-term transgene expression, usually over the entire lifetime. Such prolonged transgene expression is still a difficult goal to reach reliably for chronic conditions, which constitutes an obstacle to ultimate clinical success in curing these diseases. However, for tissue repair, gene therapy can establish a local, endogenous synthesis of the authentically processed materials critical to the healing. Long-term gene expression is neither necessary nor desired.³⁰ The required period of transgene expression for tissue healing exactly fits current possibilities offered by gene therapy.

Innate Weakness in Intrinsic Tendon Healing and “no-Gain” Period in Strength

Tissues nourished with rich blood supplies usually acquire mechanical strength rapidly in the first and second postsurgical weeks. However, the tendon healing process is characterized by a “no-gain” period in strength over the first 3 to 4 weeks (Figure 6-1)^5,³³^–³⁵ due to the weak healing capacity, yielding slow healing responses after injury. The innate weakness in tendon healing relates to the following structural facts: (1) they lack sufficient blood supply, (2) have sparse cellularity, and (3) are mainly composed of tightly packed collagen bundles. The characteristic no-gain period in strength is the central issue underlying the difficulties and clinical problems associated with tendon repairs. The goal of present efforts through biological manipulations is to elicit an earlier increase in strength, helping the tendon “leap over” this lag period. Theoretically, this goal is achievable through targeting major causes of weak tendon healing, either by supplementation of cells involved in healing or by increasing the activity levels of growth factors. Because healing strength is structurally based on collagen connections across the tendon repair site, we expect that addition of specific cells or growth factors will ensure speedy deposition and maturation of collagens at the healing site, resulting in an earlier gain in mechanical strength.

Figure 6-1 The tendon healing process is characterized by a “no-gain” period in strength over the first 3 to 4 weeks. The graph shows our data for the tendon strength obtained from the complete cut and repair model of the FDP tendon in chicken long toes. The tendon was repaired with a modified Kessler method using 5-0 nylon suture. The strength of the healing tendon can be divided into three parts: no gain, swift gain, and further steady gain periods.

Overall Concepts and Methods of Gene Therapy

The central idea of gene therapy is the incorporation of genes of interest into recipient cells to modify levels of expression of the gene, for the purpose of treating disease. The disorders requiring gene therapy are usually refractory to conventional therapeutic approaches. Although the clinical utility of these technologies remains in its infancy, gene therapy has been used with preliminary clinical success in some diseases. More trials are currently under way and may generate evidence for the utility of such techniques over a broader range of medical problems. Gene therapy holds great promise for incorporation into mainstream medicine.

The ideas behind gene therapy date back to the early 1970s. In 1972, Friedmann and Roblin advocated introducing exogenous genes to cure human diseases in an article entitled, “Gene Therapy for Human Genetic Disease?”³⁶ Major advances were made in the technical aspects of gene therapy in the 1990s. Over the past two decades, investigators have taken logical steps of using gene therapy to pursue curing diseases caused by single-gene defects (e.g., cystic fibrosis, hemophilia, muscular dystrophy, and sickle cell anemia) or in the treatment of certain cancers. More recently, clinical work has included a trial for treating Leber congenital amaurosis,³⁷ a disease caused by mutations in the RPE65 gene. The investigators proved the safety of subretinal delivery of recombinant adeno-associated virus (AAV) carrying the RPE65 gene and found a modest improvement in vision of the patients.³⁷ In another study, investigators restored trichromatic vision to squirrel monkeys using gene therapy to cure red-green color blindness.³⁸ Other investigators succeeded in treating adrenoleukodystrophy (ADL), a fetal brain disease, by delivering the gene for the missing enzyme to patients via a lentiviral vector.³⁹

Various techniques have been developed to achieve successful gene transfer,³² including both nonviral methods (e.g., liposomal, electroporation, sonoporation, shots from a “gene gun,” etc.) and viral vectors (e.g., adenoviruses, AAVs, herpes simplex viruses, retroviruses, lentiviruses, etc.).

Gene therapy is commonly considered a possible treatment modality for patients with congenital defects of key metabolic functions or patients with malignancies. Clinical success of gene therapy for congenital diseases and cancers is hindered by problems such as the short-lived function of the transgenes, immune responses, side effects with vectors, the inability to treat multigene disorders, and the risk of insertional mutagenesis. However, these problems rarely constitute a barrier to the use of gene therapy strategies for tissue healing if gene delivery methods are appropriate. The use of gene therapy in tissue repair or regeneration may become “dark horses” in the field of gene therapy.

Biological Methods to Enhance Tendon Healing

Theoretically, tendon healing may be enhanced through biological means other than gene therapy. Nevertheless, none of those methods can be claimed to be of practical value until fully tested. The initial step in the test process involves in vitro studies. The next step is in vivo experimentation, which will generate preclinical data to support the clinical trials, the last step in the test process.

One possible way to augment tendon healing is to provide the tendons with exogenous cells, to ameliorate sparse cellularity. A few such attempts have been made. Direct delivery of bone marrow stem cells to healing rabbit Achilles tendons produced some evidence of increases in tendon strength.⁴⁰ Investigators coated surgical sutures with bone marrow stem cells and delivered those cells to the tendon in vitro,⁶ but the in vivo efficacy of this method has not been reported.

Growth factors, such as insulin-like growth factor-1 (IGF-1),^9,¹⁰ platelet-derived growth factor-B (PDGF-B),²⁰ and basic fibroblast factor (bFGF),^20,^23,²⁵ have been reported to promote tenocyte proliferation or collagen synthesis. Direct supplementation of growth factors to the healing tendons has failed to reliably improve healing strength. Chan and colleagues⁴¹ injected bFGF to rat cut patellar tendon and detected no effects on ultimate strength of the tendon 7 and 14 days after surgery despite increases in cell proliferation and type III collagen production. Surgical sutures can be coated with growth factors such as bFGF or growth differentiation factor-5 (GDF-5).^4,⁴² Surgical repairs using such coated sutures improved the strength of the rabbit flexor tendon only at a single time point (postoperative week 3) but failed to improve strength at other time points.^4,⁴²

Controlled-release drug delivery systems is another way to provide the tendon with a prolonged supply of growth factors.^7,²¹ Growth factors can be incorporated into polymers in vitro, and the impregnated polymers are then implanted into the tissues, which may deliver the growth factors continuously for a prolonged period in a controlled fashion. This system holds promise for use in tendon repairs, but research in this area is just beginning. Growth factors (bFGF and PDGF-BB) have been incorporated into fibrin matrices and the release kinetics has been tested over a 10-day-period in vitro²¹; bFGF and PDGF-BB were found to release gradually from the fibrin matrices and to modulate cell proliferation and extracellular matrix synthesis in vitro. However, this system loaded with PDGF-BB was not found to improve the strength of the healing canine flexor tendons at days 7, 14, and 42 after surgery.⁷ In a canine model, bFGF delivered through fibrin heparin–based controlled-release system stimulated cellular proliferation, promoted neovascularization and inflammation of the flexor digitorum profundus (FDP) tendon in the earliest stages after surgical repair, but it failed to improve mechanical strength of the repair.⁴³ This system remains to be fully tested with respect to its effectiveness to increase tendon strength in vivo.