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Introduction
Tendinopathies present a difficult therapeutic problem for the patient as well as the health care professional because their etiology and management are uncertain [1].
Although many forms of management have been advo- cated, current treatment strategies are not effective because they do not definitively resolve the disease.
Pharmacological management with non-steroidal anti- inflammatory drugs (NSAIDs) or corticosteroids can result in some pain relief but the relief is often tempo- rary. Ideal management should have minimal side effects.
The currently used corticosteroids have frequently been associated with side effect is such as depigmentation, poor wound healing and even complete tendon rupture.
Finally, the ideal treatment should accomplish its goal in a relatively short period of time with little discomfort or disability to the patient. Surgery can definitively treat tendinopathy. However, the recovery is associated with pain and discomfort, and recovery is often protracted.
Many of our current management methods do not fulfill the criteria of ideal treatment, therefore the search for such a treatment continues.
Results of many studies suggest a poor or inadequate healing response in tendinopathies. Part of the search for new treatments has been focused on methods to start or stimulate a healing response. This is fundamentally dif- ferent than the thought behind the use of NSAIDs and corticosteroids. These drugs are aimed at the symptoms that result from the injury or problem rather than the healing response. Symptoms such as pain and swelling can be interpreted as inflammatory aspects of the tendinopathy. Treatment with NSAIDs and corticos- teroids can counteract these responses. On the other hand, the inflammatory response can be viewed as the first physiologic step in the healing of a tendon, followed by cell migration into a wound and matrix maturation.
However, tendinopathies may not evoke a sufficient inflammatory response to elicit an adequate repair response. Inhibition of inflammation with NSAIDs and
corticosteroids will manage some of the symptoms, but may not improve and may even inhibit the eventual healing response. More recent research has been focused on agents that may affect tendinopathies by directly acting on the healing response.
Growth factors such as platelet-derived growth factor (PDGF-AA, BB, or AB), insulin-like growth factor (IGF- I and II), transforming growth factor beta (TGF-b), epi- dermal growth factor (EGF), fibroblast growth factors (FGF 1,2) and bone morphogenetic proteins (BMPs) represent a group of substances that can act in such a manner. Growth factors may be produced locally by cells in areas of injury, growth and repair, or may be delivered by blood. Since their discovery, they have been impli- cated in numerous responses where they modulate cell migration, replication, matrix synthesis, and cell trans- formation. Exogenous supplementation of these factors in failed healing responses, such as in resistant tendinopathies, may lead to a definitive healing response.
The Biology of Growth Factors in Tendons
The existence of factors with stimulatory properties on nerve growth, epidermal cell growth [2] and sulfate incor- poration in cartilage [3] was demonstrated in the 1950s and 1960s. Research in the subsequent decades led to the identification of a significant number of polypeptides that had pronounced effects on cell division, matrix synthesis and many other basic cellular functions. Often these factors were named after their cell or tissue source, or their first observed effects. This led to a variety of names such as platelet-derived growth factors, bone morpho- genetic protein, transforming growth factor-beta, etc.
Only more recent research has led to their biochemical identification of their cDNA and mRNA sequences, primary structure and determination of their effects on cells and tissues.
28
The Use of Growth Factors in the Management of Tendinopathies
Louis C. Almekinders and Albert J. Banes
More recent research has led to the detection of a much more complex system in which these growth factors work. These factors may function in positive or negative feedback loops, have multiple effects depending on dose and act in synergy when given with other factors. Growth factors are responsible for a complex communication system not only from cell to cell but also from exogenous stimuli to cells and as intermediaries between hormones and cytokines. Growth factors can inhibit or stimulate cell migration, division, matrix synthesis and degradation, angiogenesis, mineralization and many more processes.
The effect of a particular growth factor on a cell is depen- dent on the cell type and receptor expression. In some cases, a tenocyte will respond differently than a chon- drocyte, within one cell type a different response to a growth factor can be seen, and young, growing cells may respond differently than mature, nondividing cells.
Finally, differences in the local concentration of growth factor and the presence of other factors may result in pro- foundly different effects. Some factors may be able to amplify others, whereas others may be inhibiting. With this in mind, we are only starting to understand the various effects of growth factors and their complex inter- actions on tendon cells.
Growth factors exert their effects on cells through spe- cific receptors. If these receptors are absent or blocked, the growth factors will be ineffective. For instance, the PDGF receptors must dimerize to signal inwardly (outside-in signaling) to initiate the internal response cascade once the growth factor binds to its receptor. Once the growth factor polypeptide binds to the receptor, this ligand-receptor interaction activates the intracellular domain on the receptor and a biochemical signal is trans- duced into the cell. The signal transduction can take place in various ways. Generally, this signaling pathway appears to be a cascade of phosphorylation of different proteins [4]. At the nuclear level, binding of transcription factors to gene sequences activate gene transcription (see Figure 28-1). This results in the generation of messenger RNA that is transcribed into protein. The protein may have one of many functions such a matrix component, an enzyme that regulates a metabolic event, a promoter or suppressor protein that promotes or suppresses tran- scription of other genes, etc. This is thought to be one of the most common pathways through which growth factors evoke a cellular response. A brief review of the most commonly studied factors and their effects on tendon tissue and cells will follow.
Bone morphogenetic proteins of BMPs are a group of TGF-b superfamily factors that stimulate bone formation (BMP-2) but also stimulate tendon cell mitogenesis (BMP-13). The bone formation is promoted by recruiting precursor cells and stimulating enchondral ossification [5].
Insulin-like growth factor, or IGF, is named after the hypoglycemic effect it has upon intravenous administra-
tion. However, IGF has pronounced effects on mitogen- esis of several cell types [6]. IGF is often subdivided in to classes: IGF-I and IGF-II. IGF-I has direct mitogenic effects, but also appears to mediate the effect of growth hormone. IGF-II is associated with the regulation of fetal growth. The stimulatory effects of IGF-I have been shown in many cell types including cartilage, bone, muscle and tendon cells. Besides a mitogenic effect, it can also stimulate selected components of matrix synthesis.
Recent work shows that it is also produced by tenocytes [7]. It induces tendon cell migration, division, and matrix expression [8,9].
Platelet-derived growth factor, or PDGF, was first iso- lated from platelets, but can be produced by different cells such as smooth muscle cells [10]. Some isoforms of PDGF, such as PDGF-BB, have stimulatory effects on both cell division as well as matrix synthesis. It appears that PDGF hold particular promise in combination with other growth factors. Its effects are noted or even ampli- fied in combination with other factors such as IGF-I.
Tendon cells express the receptor for PDGF but do not normally express PDGF itself [7]. PDGF-BB stimulates robust tendon epitenon and internal fibroblast cell migra- tion, particularly in concert with IGF-I [11]. PDGF also stimulates cell division, particularly when combined with IGF-I [8,9]. These two growth factors act synergistically with cyclic tension to stimulate cell division. Moreover, serum, which contains both PDGF and IGF-I, stimulates cells in whole tendon both mitogenically and matrigeni- cally, and synergistically with cyclic load [12]. This will be discussed in more detail in the following section.
Transforming growth factor beta or TGF-b is a group of polypeptides related to the BMPs. Originally, TGF-b Figure 28-1. Schematic drawing of mechanism of action through which growth factors affect cellular events.
was thought to be related to the cellular transformation to neoplastic growth. More recent research, however, has made clear that TGF-b can have numerous physiologic effects [13]. It appears closely tied to the expression of a differentiated phenotype in many cell lines. Particularly, the mesenchymal precursor can be influenced by TGF-b.
Tendon and ligament formation has been tied directly to factors belonging to the TGF-b superfamily [14].
Proliferation, matrix synthesis and differentiation have also been affected in both chondroblasts and osteoblasts. Whether this is an inhibitory or stimulatory effect depends on the stage of differentiation, presence of other growth factors and assay system used. TGF-b is a weak stimulator of tendon cell migration and mito- genesis but can stimulate robust expression of matrix [15].
Fibroblast growth factors (FGF 1,2) contain a group of heparin-binding proteins. They are named after their mitogenic effects on fibroblasts, and are also found to influence osteoblast precursors and chondrocytes [16].
FGF receptor mutation has been in implicated in a certain form of dwarfism. Two main forms of FGF have been identified: FGF-1 (acidic FGF) and FGF-2 (basic FGF). Both have shown promise in stimulation of new bone formation. FGF 1 and 2 are weak mitogens for tendon cells [15].
Cytokines in Tendon Cells and Tissue Responses
Similar to growth factors, cytokines influence many cel- lular processes. Although they are more commonly asso- ciated with diseased states, they may have physiologic functions and could be considered for therapeutic use if their actions are clearly defined. Results of recent studies indicate that cytokines such as interleukin-1b (IL-1b) and tumor necrosis factor a (TNF-a) can stimulate metallo- proteinase expression in tendon cells (MMP-1,2,3 and 13) [17,18,19]. Inflammatory cytokines such as IL-1b and TNF-a elaborated by lymphocytes and macrophages can stimulate tendon cells to produce interstitial collagenase (MMP-1), gelatinase (MMP-2), stromelysin (MMP-3) as well as MMP-13 which can activate other MMPs to degrade collagens and aggrecans [18]. Moreover, IL-1b and TNF-a can elicit COX-2 expression and PGE2 release from stimulated tendon cells. The latter scenario may involve one of the instigating factors that initiate tendinopathy, although it is not clear why, in overuse tendinopathy, there seems to be no evidence of pro- inflammatory substances in the affected tendon by the time patients come under the care of a physician. Hence, the use of MMP inhibitors and NSAIDs may inhibit the matrix destructive cycle by blocking cyclooxygenase-2 and MMP activity.
The Use of Growth Factors in Tendon Pathology
The use of growth factors in soft tissue problems remains largely experimental and has been restricted to in vitro studies and animal models. In bone research, however, the first clinical studies on the use of bone morphogenetic protein (BMP) have been reported. Encouraging results have been reported with use of BMP in spinal fusion [20]
and lower extremity osteotomy [21].
Several in vitro studies have been performed to deter- mine the effects of growth factors on tendon cells. Gauger et al. tested the effects of epidermal growth factor, insulin and transferrin on avian tendon cells [22].
Both cell division and collagen synthesis were stimu- lated by these factors. The level of stimulation was similar to the effects seen from 10% serum as is commonly used in in vitro studies to maintain cell growth and matrix synthesis.
Banes et al. investigated the effects of PDGF-BB and IGF-I in conjunction with mechanical stimulation on avian tenocytes [9]. The tenocytes were first separated in tendon epitenon surface cells (TSC) and internal teno- cytes. These cell types expressed different markers, had different growth rates and responded differently to growth factors, PGE2 and PTH [12,23]. PDGF-BB clearly stimulated TSC synergistically with mechanical load and IGF-1. PDGF-BB also stimulated TIF in a less dramatic fashion. IGF-1 with mechanical load only modestly stim- ulated both types of tenocytes. In a subsequent experi- ment, Banes et al. found that PDGF-BB was able to induce expression of novel genes in conjunction with load.
IGF and TGF-b were clearly less effective. The stimula- tory effect of PDGF on tenocytes and tissues has been confirmed in several other studies. Spindler et al. docu- mented a mitogenic response of sheep patellar tendon to PDGF-AB. In addition, elevated PDGF levels have been found in healing tendon tissue [24].
FGF also has received some attention in research studies. Basic FGF addition to rat patellar tenocytes resulted in a proliferative response [25]. However, in studies of injured and non-injured tendon, higher levels of bFGF expression were found in normal tendon com- pared to injured tendon [26]. This may suggest that bFGF does not play a major role in the tendon healing response.
This is supported by the fact that Kang et al. were unable to document a significant response from FGF in rabbit flexor tendon culture experiments [27].
Clinical use of growth factors in tendon problems has not yet been reported. One of the issues to be resolved is how to administer the growth factors in a reliable deliv- ery system and assure that they maintain potency at the injection site. Oral or systemic injection of a growth factor are not favored, however, and direct, local admin- istration into the tendon is a logical route. This could be
accomplished in several ways. In a pilot study, we utilized direct administration of growth factors in a patellar tendinopathy lesion. To assure deposit of the factor into the abnormal tissue, real-time ultrasonography was used.
With a sterile ultrasound head in place, a needle was advanced directly into the hypoechogenic area (see Figure 28-2). Once the placement was confirmed by ultra- sound at orthogonal angles the factor was injected. Post- injection, the ultrasound picture confirmed the presence of the injected fluid in the lesion. In a pilot study 6 patients were injected in this manner. In order to obtain injectable factor, venous blood was obtained from each patient prior to the injection. Platelets were isolated from this blood and injected as a 1-milliliter suspension under ultrasound monitoring. Injection of platelets directly in a collagenous structure will result in platelet degranula- tion. PDGF and other growth factors and cytokines are released in the affected area. Two out of 6 patients had complete relief of their symptoms, two had partial relief and two were not improved with 2 to 3 months of treat- ment. No side effects were seen other than some soreness at the injection site. One patient with bilateral problems had a corticosteroid injection in the contralateral tendon.
He obtained immediate relief in the steroid-injected side but pain recurred after six weeks. In the platelet-injected side no immediate relief was noted but symptoms started to resolve at 6 to 8 weeks post-injection and eventually were relieved. A study of pre- and post-injection MRI images revealed complete resolution of the lesion (see Figure 28-3). More controlled studies are needed before definitive conclusions can be drawn from these results.
Another method of growth factor administration would involve the use gene therapy techniques to express
the gene [28]. Plasmid DNA with a sequence coding for a given factor can be expressed in tendon cells. The gene or segment of DNA can be transferred in different ways.
With a direct, in vivo transfer, the DNA is incorporated in a vector, usually a plasmid or nonreplicating virus. The virus or plasmid is administered to the patient and allowed to infect the local cells. The local cells host the plasmid DNA and selected gene sequence and express the growth factor gene. Expression is generally low and transient, but recent data indicate that expression for as long as two months is possible. The in vivo technique is simpler but involves injection of virus particles. The ex- vivo technique is more demanding and requires the harvest and culture of cells from the patient. The cells are infected and then returned to the desired site. This allows better control over the transfected cells and a higher level of expression can be achieved. The initial experience with gene therapy experiments using synovial cells indicates that this may become a viable option in the future.
Many other issues surrounding the clinical use of growth factors need to be resolved before their benefit can be determined on a scientific basis. Timing, dosing and type of growth factor to be used remain largely unre- solved issues. Once the sequence of events relating to growth factors is known, experiments to promote healing with exogenous growth factors can be initiated. One of the major problems in studying the effects of exogenous growth factors in chronic tendon problems is the lack of a reliable animal model. Tendinopathy is difficult to Figure 28-2. Injection of factor in area of the patellar tendon
(T) near the insertion of the patella affected by insertional patellar tendinopathy under ultrasonographic control. Tip the needle (arrow) is advanced into the hypoechogenic area (*).
Figure 28-3. Preinjection MRI (A) and post-injection MRI (B) of patellar tendinopathy with resolution of the tendon lesion (arrow). T represents the patellar tendon and P the patella.
A B
reproduce in a laboratory animal [29,30,31]. If available, the reasons for the slow or absent healing in some of these lesions could be studied and therapeutic interven- tions could be designed. Currently, we rely on patient material, which often does not allow the researcher to get longitudinal or control data. Consequently, we know rel- atively little about the pathologic stages through which these tendon progress. Treatment strategies are limited.
Further research studies will have to address these issues before significant progress can be made in the use of growth factors.
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