Growth factor delivery may be useful to accelerate the rate of tendon healing. We studied Platelet Concentrate, which in effect can be regarded as a cocktail of growth factors relevant for tendon healing. In a rat Achilles tendon transection model, one postoperative injection of Platelet Concentrate resulted in increased strength even 3 weeks later. Mechanical stimulation improves the repair of ruptured tendons. We studied the effects of platelets upon Achilles tendon regenerates in rats 3, 5 and 14 days after transection, either unloaded or mechanically stimulated. At 14 days, physical activity and platelets increased repair independently. Unloading decreased the mechanical properties of the repair tissue to less than half of normal. Moreover, the platelets had no effect without loading. Thrombin, which we used for platelet activation, improved healing of the rat Achilles tendon by itself. Conversely, continuous inhibition of thrombin by low molecular weight heparin (LMWH) inhibited tendon repair. However, intermittent inhibition, similar to clinical thromboprophylaxis, had no effect on tendon healing. Cartilage Derived Morphogenetic Protein-2 (CDMP-2) can improve tendon healing in loaded defect models. We now studied unloaded repair in a rabbit patellar tendon model. Two hours postoperative, the rabbits received CDMP-2 injected into the haematoma. The healing tendon became 65 % stronger than controls. We then studied Achilles tendon healing with CDMP-2 injections in sheep, to get a bigger animal model. There was an unexpectedly high variation of repair in these animals, and the study turned out to be underpowered. Spontaneous ruptures in humans have a more variable geometry than in our sheep model, so humans can also be expected to vary a lot in mechanical characteristics of Achilles tendon repair. This accentuates the importance of individualized rehabilitation programs. In conclusion, both platelet concentrate and CDMP-2 injections might be of interest for clinical use as a complement to surgical or conservative treatment of tendon ruptures. Platelet treatment for tendon ruptures should probably be combined with early physiotherapy.

Tendinopathies are painful tendon conditions of presumably multifactorial genesis. In tendinosis, as in Achilles tendinosis, there is apart from pain also morphological changes which are described as degenerative with no signs of inflammation. The exact mechanisms behind these conditions are still, to a large extent, unknown. Pain, being the foremost impairing symptom, leads us to the hypothesis that nerves are deeply involved in the symptoms and processes of Achilles tendinosis. Locally produced nerve signal substances may also be involved in the processes. Knowledge of the innervation patterns within the tendon and knowledge on a possible local nerve signal substance production are therefore of utmost importance. There is a lack of information on these aspects. The specific aims of this thesis were 1) to investigate the innervation patterns regarding general, sensory, cholinergic and sympathetic innervations, and 2) to examine for the possible occurrence of a production of nerve signal substances and a presence of receptors related to these in the tendon cells, the tenocytes. Painfree normal and tendinosis Achilles tendons were examined. Immunohistochemistry, using antibodies against the general nerve marker PGP9.5, the synthesizing enzymes for acetylcholine (choline acetyltransferase; ChAT), and catecholamines (tyrosine hydroxylase; TH), the vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), substance P and calcitonin gene-related peptide, was applied. Immunohistochemistry was also used for the delineation of muscarinic (M2R), adrenergic (α1-AR) and NPY-ergic (Y1 and Y2) receptors. To detect mRNA for TH and ChAT, in situ hybridization was used. In normal Achilles tendons, as well as in the tendinosis tendons, there was a very scanty innervation within the tendon tissue proper, the main general, sensory and sympathetic innervations being found in the paratendinous loose connective tissue. Interestingly, the tenocytes showed immunoreactions for ChAT, VAChT, TH, M2R, α1-AR and Y1R. The reactions were clearly more observable in tendons of tendinosis patients than in those of controls. The tenocytes of tendinosis patients also displayed mRNA reactions for ChAT and TH. Nevertheless, all tenocytes in the tendinosis specimens did not show these reactions. Immunoreactions for α1-AR, M2R and Y1R were also seen for blood vessel walls. The present thesis shows that there is a very limited innervation within tendon tissue proper, whilst there is a substantial innervation in the paratendinous loose connective tissue. It also gives evidence for an occurrence of production of catecholamines and acetylcholine in tenocytes, especially for tendinosis tendons. Furthermore, that ACh, catecholamines and NPY can have effects on these, as well as on blood vessels, via the receptors observed. The observations suggest that Achilles tendon tissue, whilst containing a very scarce innervation, exhibits autocrine/paracrine cholinergic/catecholaminergic/NPY-ergic effects that are upregulated in tendinosis. These findings are of great importance as the results of such effects may mimic processes that are known to occur in tendinosis. That includes effects related to proliferation and angiogenesis, and blood vessel and collagen regulating effects. In conclusion, within the Achilles tendon there is a very scarce innervation, whilst there appears to be a marked local production of nerve signal substances in Achilles tendinosis, namely in the tenocytes, the cells also harbouring receptors for these substances. The observations give a new insight into how the tendon tissue of the Achilles tendon is influenced by signal substances and may give options for new treatments of Achilles tendinosis.

Chronic painful tendinopathies are common, not only in sports and recreationally active people, but also among people with a sedentary lifestyle. Both the lower and upper limbs are affected. There is lack of knowledge about the etiology and pathogenesis to tendinopathy, and many different treatments options have been presented. Unfortunately, most treatments have not been tested in scientific studies. Conservative (non-surgical) treatment has since long shown unsatisfactory results and surgical treatment is known to give unpredictable results. The aim of this thesis was to evaluate new models of painful eccentric training for the conservative treatment of different chronic tendinopathies. After promising results in a pilot study, using painful eccentric calf muscle training in patients with chronic mid-portion Achilles tendinopathy, we investigated if these results could be reproduced in a larger group of patients with both mid-portion and insertional Achilles tendinopathy (study I). After 12 weeks, 89% of the patients with pain from the mid-portion were satisfied and back in previous activities. In the group with insertional Achilles tendinopathy the results were poor. A new model for eccentric training was designed for patients with insertional Achilles tendinopathy. The eccentric calf muscle training was done from tip-toe to floor level (study II). With this new regimen 67% of the patients were satisfied and back in previous activities. The next step was to investigate the effects of painful eccentric quadriceps training on patients with jumper´s knee/patellar tendinopathy (study III). Two different training protocols were used. Eccentric training performed on a 250 decline board showed promising results with reduced pain and a return to previous activities, while eccentric training without the decline board had poor results. In a following prospective study, patients with jumper´s knee/patellar tendinopathy were randomised to either concentric or eccentric painful quadriceps training on a 250 decline board (study IV). After 12 weeks of training, there were significantly better results in the group that did eccentric training. In a pilot study (study V), we investigated painful eccentric deltoideus and supraspinatus muscle training on a small group of patients on the waiting list for surgical treatment of subacromial impingement syndrome. After 12 weeks of training, 5 out of 9 patients were satisfied with the results of treatment and withdrew from the waiting list for surgery. In conclusion, the present studies showed good clinical results with low risks of side effects and low costs. Thus, we suggest that painful eccentric training should be tried in patients with Achilles and patellar tendinopathy before intratendinous injections and surgery are considered. For patients with chronic painful impingement syndrome, the results of our small pilot study are interesting, and stimulates to randomised studies on larger materials.

Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.

Background: Achilles tendinopathy is a common clinical syndrome of chronic Achilles tendon pain combined with thickening of the tendon and impaired tendon function. Tendinopathy is often, but not always, induced by mechanical overload, and is frequently accompanied by abnormalities at the tissue level, such as hypercellularity and angiogenesis, in which case the condition is called tendinosis. In tendinosis, there are no signs of intratendinous inflammation, but occasionally increased apoptosis is observed. Tendinosis is often hard to treat and its pathogenesis is still not clear. Recently, a new hypothesis has gained support, suggesting a biochemical model based on the presence of a non-neuronal production of classically neuronal signal substances by the primary tendon cells (tenocytes) in tendinosis. The possible functional importance of these signal substances in tendons is unknown and needs to be studied. In particular, the neuropeptide substance P (SP) and catecholamines are of interest in this regard, since these substances have been found to be up-regulated in tendinosis. As both SP and catecholamines are known to exert effects in other tissues resulting in changes similar to those characteristic of tendinosis, it is possible that they have a role in tendinosis development. It is furthermore unknown what elicits the increased intratendinous neuropeptide production in tendinosis, but given that tendon overload is a prominent riskfactor, it is possible that mechanical stimuli are involved. The hypothesis of this thesis work was that intratendinous production of SP is up-regulated in response to load of Achilles tendons/tenocytes, and thatstimulation of the preferred SP receptor, the neurokinin-1 receptor (NK-1 R), aswell as stimulation of the catecholamine α2 adrenoreceptors, contribute to the hypercellularity seen in tendinosis, via increased proliferation and/or decreased apoptosis, and that SP stimulates tendon angiogenesis. The purpose of the studies was to test this hypothesis. To achieve this, two model systems were used: One in vivo (rabbit Achilles tendon overload model of tendinosis) and one in vitro (human primary Achilles tendon cell culture model). Results: In the rabbit Achilles tendon tissue, SP and NK-1 R expression was extensive in the blood vessel walls, but also to some extent seen in the tenocytes. Quantification of endogenously produced SP in vivo confirmed intratendinous production of the peptide. The production of SP by human tendon cells in vitro was furthermore demonstrated. The catecholamine synthesizing enzyme tyrosine hydroxylase (TH), as well as the α2A adrenoreceptor (α2A AR), were detected in the tenocytes, both in vivo in the rabbit tissue and in vitro in the human tendon cells. As a response to mechanical loading in the in vivo model, the intratendinous levels of SP increased, and this elevation was found to precede distinct tendinosis changes. The in vitro model demonstrated the same response to load, i.e. an increased SP expression, but in this case also a decrease in the NK-1 R expression. In the in vivo model, exogenously administered SP, as well as clonidine (an α2 AR agonist), accelerated tenocyte hypercellularity, an effect that was not seen when administrating a specific α2A AR antagonist. Exogenous administration of SP also resulted in intratendinous angiogenesis and paratendinous inflammation. In the in vitro model, both SP and clonidine had proliferative effects on the human tenocytes, specifically mediated via NK-1R and α2A AR, respectively; both of which in turn involved activation/phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Exogenously administered SP, in Anti-Fas induced apoptosis of the tenocytes in vitro, confirmed SP to have an anti-apoptotic effect on these cells. This effect was specifically mediated via NK-1 R and the known anti-apoptotic Akt pathway. Conclusions: In summary, this thesis concludes that stimulation of NK-1 R and α2A AR on tenocytes, both in vitro and in vivo, mediates significant cell signalling effects leading to processes known to occur in tendinosis, including hypercellularity. The pathological role of the hypercellularity in tendinosis is still unclear, but it is likely to affect collagen metabolism/turnover and arrangement, and thereby indirectly tendon biomechanical function. Additional evidence is here provided showing that SP not only causes tenocyte proliferation, but also contributes to anti-apoptotic events. Furthermore, it was concluded that SP may be involved in the development of tendinosis, since its production is increased in response to load, preceding tendinosis, and since SP accelerates tendinosis changes, through some mechanistic pathways here delineated. These findings suggest that inhibition of SP, and possibly also catecholamines, could be beneficial in the reconstitution/normalization of tendon structure in tendinosis.

Augmented Soft Tissue Mobilization, a massage therapy which uses a solid instrument rather than human fingers to treat musculoskeletal injuries, has been successful in treating tendinitis. Davidson et al. studied the functional and morphological affects of ASTM on collagenase induced Achilles tendinitis in Sprague-Dawley rats. Morphological observations showed a significant increase in the number and activation of fibroblasts in the ASTM treated Groups. The authors suggested that the physical force of ASTM may promote tendon healing via increased fibroblast recruitment. An important, but unexplained, question is how ASTM would affect the fibroblasts of native, noncollagenase injured, tendons. Studies have shown that mechanical forces can alter cellular functions. The purpose of this study was to examine the morphological changes in native Sprague-Dawley rat Achilles tendons after ASTM therapy using different application pressures.Three animal Groups were randomly established: A) control Group with no ASTM; B) light ASTM with 1 Newton of pressure; and C) heavy ASTM with 3 Newtons of pressure. Upon completion of the therapy, the Achilles tendons of each Group were examined with light and electron microscopy techniques to assess fibroblast number, tendon morphology, and the presence of type I and type III collagen. Fibroblast counts from each Group were compared using a two-way ANOVA, multiple regression, and curvilinear regression analysis. Morphological differences were shown between the three Groups, especially between the non force Group and the two force Groups. The ASTM Group treated with one Newton demonstrated the greatest mean fibroblast count (165.1+/-55.8&160.7+/-49.8). Electron microscopy revealed the presence of activated fibroblasts in the tendons of the two force Groups, ASTM Groups. Polarizing microscopy showed a dramatic increase in the amount of Type III collagen in the two force Groups compared to the non force Group.Ball State UniversityMuncie, IN 47306

Thesis (M.S.)--Mississippi State University. College of Veterinary Medicine. / Title from title screen. Includes bibliographical references.

No description available.

Augmented Soft Tissue Mobilization (ASTM) is a new noninvasive technique utilized to treat chronic musculoskeletal injuries. The purpose of this study was to trace the morphological events in collagenase injured rat Achilles tendons during ASTM treatment and to observe passive occurrence of post treatment remodeling in tendons. Twelve groups of rats were divided into the following categories, A) control, B) sham surgery, C) ASTM only, D) ASTM/sham , E) tendinitis, F) ASTM/tendinitis 1 week, G) ASTM/tendinits 2 weeks H) ASTM/tendinits 3 weeks I) ASTM/tendinits 4 weeks, J) Post ASTM 5 weeks, K) Post ASTM 10 weeks, L) Post ASTM 15 weeks. One week after the last designated treatment, the Achilles tendons were harvested and then prepared for light microscopy, electron microscopy, and bifringence polarizing microscopy. An increase in fibroblast activation and proliferation was noted with the tendinitis, ASTM/tendinitis, and post groups. Ossification occurred in the core of the Achilles tendon in all of the ASTM groups. The presence of inflammatory cells was observed in the tendons and longitudinal remodeling of the collagen fibers did not occur. / Department of Biology

Tenocytes are the principal cells of the human Achilles tendon. In tendinosis, changes in the metabolism and morphology of these cells occur. Neurotrophins are growth factors essential for the development of the nervous system. Tumour necrosis factor alpha (TNF-α) has been found to kill sarcomas but has destructive effects in several major diseases. The two systems have interaction effects and are associated with apoptosis, proliferation, and pain signalling in various diseases. Whether these systems are present in the Achilles tendon and in Achilles tendinosis is unknown. The hypothesis is that the tenocytes produce substances belonging to these systems. In Studies I–III, we show that the potent effects of these substances are also likely to occur in the Achilles tendon. We found tenocyte immunoreactions for the neurotrophins brain-derived neurotrophic factor (BDNF), the nerve growth factor (NGF), the neurotrophin receptor p75, and for TNF-α and both of its receptors, TNFR1 and TNFR2. This occurred in both subjects with painful mid-portion Achilles tendinosis, and in controls. Furthermore, we found mRNA expression for BDNF and TNF-α in tenocytes, which proves that these cells produce these substances. TNFR1 mRNA was also detected for the tenocytes, and TNFR1 immunoreactions were upregulated in tendinosis tendons. This might explain why tenocytes in tendinosis undergo apoptosis more often than in normal tendons. Total physical activity (TPA) level and blood concentration of both soluble TNFR1 and BDNF were measured in Study IV. The results showed that the blood concentration of both factors were similar in subjects with tendinosis and in controls. Nevertheless, the TPA level was related to the blood concentration of sTNFR1 in tendinosis, but not in controls. This relationship should be studied further. The findings of this doctoral thesis show that neurotrophin and TNF-α systems are expressed in the Achilles tendon. We believe that the functions include tissue remodelling, proliferation and apoptosis.