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Tendon disorders are a major problem in both sports and occupational medicine. Tendons have the highest tensile strength of all connective tissue due to a high proportion of collagen in the fibers and their closely packed parallel arrangement in the direction of force. The individual col- lagen fibrils are arranged into fascicles which contain blood vessels and nerve fibers lymph. Specialized fibrob- lasts, tenocytes, lie within these fascicles, and exhibit high structural organization [1,2].
Tendon healing is classically considered to occur through extrinsic and intrinsic healing. The intrinsic model produces obliteration of the tendon and its tendon sheath. Healing of the defect involves an exudative and a formative phase, which are very similar to those asso- ciated with wound healing [3]. In the extrinsic healing pathway, tenocytes migrate through chemiotaxis into the defect from the ends of the tendon sheath [4]. The process can be divided into 3 phases: inflammation, repair, and organization or remodeling. In the inflammatory phase, occurring 3 to 7 days after the injury, cells migrate from the extrinsic peritendinous tissue such as the tendon sheath, periosteum, subcutaneous tissue and fascicles, as well as from the epitenon and endotenon [5]. Initially, the extrinsic response is more florid than the intrinsic one with the rapid filling of the defect with granulation tissue, tissue debris and hematoma. The migrating fibroblasts still play a phagocytic role, and are arranged in a radial fashion in relation to the direction of the fibers of the tendon [1]. Biomechanical stability is given by fibrin.
The migrated fibroblasts begin to synthesize collagen around day 5. Initially, these collagen fibers are randomly orientated. Tenocytes become the main cell type, and over the next 5 weeks collagen is synthesized. During the fourth week, a noticeable increase in proliferation of fibroblasts of intrinsic origin, mainly from the endotenon, takes place. These cells take over the main role in the healing process, and both synthesize and reabsorb colla- gen. The newly formed tissue starts to mature, and the collagen fibers are increasingly orientated along the
direction of force through the tendon. This phase of repair continues for approximately 2 months after the initial injury. Final stability is acquired during the remod- eling induced by the normal physiological use of the tendon. This allows further orientation of the fibers into the direction of force. In addition, cross linking between the collagen fibrils increases the tendon tensile strength.
During the repair phase, the mechanically stronger Type I collagen is produced in preference to Type III collagen, thus slightly altering the initial ratio of these fibers to increase the strength of the repair [6].
Despite intensive remodeling over the following months, complete regeneration of the tendon is never achieved, and the tissue replacing the tendon defect remains hypercellular.The diameter of the collagen fibrils remains thinner fibrils, with reduction in the biomechan- ical strength of the tendon when measured per unit of cross sectional area.
In tendinopathic and ruptured Achilles tendons, there is a reduction in the proportion of Type I collagen, and a significant increase in the amount of Type III collagen [7], responsible for the reduced tensile strength of the new tissue due to a reduced number of cross links compared to Type I collagen [8]. Recurring microinjuries lead to the development of hypertrophied biologically inferior tissue replacing the intact tendon.
Cytokinetic Modulation of Tendon Healing
Growth factors and other cytokines play a key role in the embryonic differentiation of tissue and in the healing of tissues [9]. Growth factors stimulate cell proliferation and chemotaxis aid angiogenesis, influencing cell differentia- tion. They regulate cellular synthetic and secretory activ- ity of components of extracellular matrix, and influence the process of soft tissue healing.
29
Optimization of Tendon Healing
Nicola Maffulli and Hans D. Moller
29. Optimization of Tendon Healing 305
In the normal flexor tendon of the dog, the levels of basic fibroblast growth factor (bFGF) are higher than the levels of platelet derived growth factor (PDGF). In injured tendons, the converse is true [10]. Under the influ- ence of PDGF, chemotaxis and the rate of proliferation of fibroblasts and collagen synthesis are increased [11].
Fibroblasts of the patellar tendon show increased prolif- eration in vitro after the administration of bFGF [12].
In addition, an angiogenic effect is evident [13]. During the embryogenesis of tendon, bone morphogenic pro- teins (BMP), especially BMP 12 and 13, cause increased expression of elastin and collagen Type I. Also, animal studies demonstrated that BMP 12 exerts a positive effect on the healing processes of the patellar tendon [14].
The growth factors of the transforming growth factor beta superfamily induce an increase in mRNA expres- sion of Type I collagen and fibronectin in cell culture experiments [15].
As the high expression of collagen Type I is probably essential to achieve faster healing of tendons, there should be a shift from the initial production of collagen Type III to Type I early in the healing process. The afore- mentioned growth factors could potentially be used to influence the processes of regeneration of tendons ther- apeutically. However, it is unlikely that a single growth factor will give a positive clinically relevant result. The interaction of many factors present in the appropriate concentration at the right time for the correct duration will be necessary.
Gene Therapy to Provide Growth Factors
Growth factors have a limited biological half-life. Given the complexity of the healing process of tendons, a single application of growth factors is unlikely to be successful.
As there is no bio-availability of oral proteins, repeated local injections would be necessary to maintain levels in the therapeutic range, which may prove technically diffi- cult in operatively treated tendons. The transfer of genes for the relevant growth factors seems an elegant alterna- tive [16]. After cellular uptake and expression of genes a high level of the mediators can be locally produced and secreted.
To achieve this goal, vectors, either viral or nonviral, would enable the uptake and expression of genes into target cells. Viral vectors are viruses deprived of their ability to replicate into which the required genetic mate- rial can be inserted. They are effective, as the introduc- tion of their genetic material into host cells forms part of their normal life cycle. Nonviral vectors have specific characteristics which enable penetration of the nucleus, e.g. liposomal transport. The genes are released in the vicinity of the target cells without systemic dilution.
Vectors can be transferred using 2 main strategies.
Using in vivo transfer, the vectors are applied directly to the relevant tissue. In vitro transfer involves removal of cells form the body, the gene transfer in vitro and subse- quent culture of these cells before they are reintroduced into the target site. Direct transfer is less invasive and technically easier, and can be started during treatment of the acute phase of the injury. A disadvantage is the non- specific infection of cells during the injection process.
In addition, due to the amount of extracellular matrix present, a vector with high transgenic activity is necessary to be able to transfer the gene to enough cells.
Indirect transfer of genes is safer. The relevant cell type is isolated and genetically modified. Prior to reintroduc- tion into the body, cells can be selected and tested for quality. Due to the work involved in this technique, it would be more suitable for the treatment of degenerative processes instead of the acute injuries. The first studies on the feasibility of this procedure have been conducted using marker genes [17].The main gene used, LacZ, codes for the bacterial beta galactosidase which is not present in eukaryotic cells. The addition of a suitable substrate changes the staining properties of the cells which express the new gene, allowing to ascertain the effectiveness of transmission and the duration of expression of the foreign gene. With the vectors currently available, the gene is expressed for 6 to 8 weeks in tendon tissue [18].
Using this strategy, the transfer and expression of PDGF gene into the patellar tendon of rats lead to an increase in angiogenesis and collagen synthesis in the tendon over 4 weeks. Gene expression of this duration could influence the whole healing process of tendons and could be the start of an optimized healing process.
In summary, even successful tendon healing does not reconstitute a normal tendon, and the result is function- ally satisfactory despite morphological differences and biomechanical weakness compared to a normal tendon.
The therapeutic use of growth factors by gene transfer to produce a new tendon which is biologically, biomechani- cally, biochemically and physiologically more “normal” is promising, but still far from clinical practice.
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