Modulation of peritendinous adhesions using hyaluronic acid and autogenous synovia: an eexperimental study in a rabbit model.
Abstract: Peritendinous adhesions following tendon repair or tendon grafting resulting in restricting of movements and contracture of the finger joints are frequently seen in human being. In draught animals this may also crippled the animal following limiting movements of the joints. We evaluated the efficacy of hyaluronic acid (HA) and autogenous synovia (AS) in modulation of peritendinous adhesions following tendon injury in a rabbit model invivo. The injured tendon tissues were examined at 7, 14, 30 and 60 days after operation using gross, histopathologic, histochemical and scanning electron microscopic methods. Observations revealed that proliferative and inflammatory responses were significantly reduced in HA and AS groups as well as early restoration of normal arrangements of collagen fibres, enhancement of neovascularization and the healing. Our finding suggests that the administration of HA and AS prevent the formation of peritendinous adhesions and improve the gliding function of the tendon.
Article Type: Report
Subject: Adhesions (Research)
Hyaluronic acid (Research)
Tendons (Surgery)
Tendons (Health aspects)
Authors: Kumar, N.
Kumar, Naveen
Sharma, A.K.
Maiti, S.K.
Gangwar, A.K.
Gupta, O.P.
Kumar, Satish
Singh, Rajendra
Pub Date: 05/01/2009
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: May-August, 2009 Source Volume: 23 Source Issue: 1
Topic: Event Code: 310 Science & research
Product: Product Code: 2831981 Hyaluronic Acid NAICS Code: 325414 Biological Product (except Diagnostic) Manufacturing SIC Code: 2833 Medicinals and botanicals
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 204419298
Full Text: Introduction

Tendons are essential component of locomotor system and are prone to injury due to their superficial location in the body [1]. Tendon repair has got great limitation in the form of peritendinous adhesions. Due to formation of these adhesions the tendon can no longer glide freely and may impair the function of limbs seriously [2]. The results of even the first tendon surgery and repair are frequently compromised by adhesions that restrict gliding motion, decrease gliding function and often lead to permanent deformations [3]. However, adhesions formation is often regarded as an undesirable but essential part of tendon healing process. Tendon injuries are liable to heal with dense adhesions and poor functional results [4].

Efforts have been made to elucidate the cellular and molecular mechanisms which lead to the formation of adhesive tissue [5]. Different attempts have been made to prevent peritendinous adhesions after tendon surgery in the form of mechanical barriers around the repaired sites of tendon like cellophane, stainless steel tubes, silastic tubes, polytetrafluroethylene (PTFE), silicon sheath, dacron tube have all been used with disappointing results. Drugs like antihistamines, steroids, penicillamine, dimethyl sulfoxide and inhibitors of collagen synthesis have also been tried but with complicating side effects.

The kinematics of tendon gliding is extremely sophisticated and restoring of gliding is influenced by many factors. An ideal reconstruction of a tendon injury is one which is reinforced by the administration of a lubricant which does not interfere with the healing of the tendon but will allow low resistance gliding. Even after the continued investigation in the field of tendon surgery only a few number of agents have become available to surgeons to assist in reducing peritendinous adhesions and still there is a need to find out an ideal agent with 100% efficacy to prevent peritendinous adhesions. It has been reported that intrasynovial tendons may be better source for the graft [6]. Hyaluronic acid (HA) is a long polysaccharide chain consisting of repeating disaccharide units of N-actyl-glucosamine and glucuronic acid, which occurs naturally in synovial fluid and gives it viscoleasticity [7]. It has been reported that hyaluronic acid treated tendons has less adhesions and better gliding properties than non treated control [8]. Role of administration of autogenous synovia (synovial fluid collected from the contra lateral joint) at the reconstructive site of tendon demonstrated its efficacy in reduction of peritendinous adhesions [9-11]. In the present study we analyze the tissue using clinical, radiology, gross, histopathology, histochemistry and scanning electron microscopy to evaluate the efficacy of hyaluronic acid and autogenous synovia separately for the prevention of peritendinous adhesions.

Materials and Methods

We used twenty-four adult New Zealand white rabbits of either sex weighing between 2.5 and 3.0 kg. The animals were randomly divided into 3 equal groups of 8 animals each. Under general anaesthesia a 2 cm area of left tendoachillis was crushed with toothed forceps for creation of peritendinous adhesions. The surgical wound was closed in a routine manner. Animals of group I were treated with 0.5 ml of 1% solution of high molecular weight hyaluronic acid (HA) (Fluka, Lab Instruments and Chemicals, Ambala, Harayana, INDIA) whereas, animals of group II were treated with 0.25 ml of autogenous synovia (AS) collected from the contra lateral limb. The hyaluronic acid and autogenous synovia was injected at the site on day 1, 3, 5 and 7 postoperatively. In group III no treatment was given after surgical intervention and the group acted as control. After the operation, all rabbits were allowed free activity in the cage. The experimental design and treatment of these animals were approved by the Animal Care Committee of our Institute

Gross observations. We recorded the gross observations at the site on days 7, 14, 30 and 60 post-operatively after euthanising the animals. The adhesions at the operated site were graded as no adhesions (0), thin filamentous easily separable adhesions (1), thick adhesions in a limited area (2) and wide spread thick adhesions (3).

Histopathological observations. We collected the tendon specimen en bloc from the site on days 7, 14, 30 and 60 post-operatively and were placed in a fixative solution of 10% formalin. The sections were stained with haemtoxylin and eosin for routine histopathological examination. Special staining techniques were used to demonstrate collagen fibres (Masson's trichrome), reticular fibres (Silver staining), elastic fibres (Verhoeff's staining) and mucopolysaccharide (Periodic Acid Schift--Alcian blue, PAS-AB) as per Luna [12].

Histochemical observations. The sections were stained by Gomori's method for alkaline phosphatase activity [13]. Reaction of alkaline phosphatase and its distribution in the healing tissue was graded and compared with the normal healthy tissue. The grading was done as no alkaline phosphatase activity (0), mild alkaline phosphatase activity (+), moderate alkaline phosphatase activity (++) and intense alkaline phosphatase activity (+++).

Scanning electron microscopic observations. The collected tendon samples were fixed in 2.5% glutaraldehyde in a phosphate buffer at pH 7.2 for 24 hours at room temperature. Hexamethyl disilizane technique (HMDS) was used for drying of the specimens [14]. We studied the micro structural properties of the tendon using JEOL scanning electron microscope (JEOL Ltd., Tokyo, Japan) operating at 5kV (15 WD, working distance) using different magnifications as needed for the desired observations.

Results

Gross observations. On day 7 in HA and AS groups vascularity was prominent at the site. The healing of cutaneous incision was not complete. Loose and filmy connective tissue adhesions between skin and injured site were observed. Strands of connective tissue were extending between the injured tendon and the skin. Separation of adhesions resulted into minor bleeding. The tendon was swollen at the injured site. There was complete absence of gliding movements. On day 14, the cutaneous incision had completely healed. The injured site of tendon was less swollen. The partial gliding movements of tendon were present. On day 30, gliding movements were near normal. On day 60, tendon had shiny glistening surface, a characteristic of normal tendon. No adhesions around the injured site of tendon were observed. Normal gliding movement of tendon was seen.

In control group on day 7, firm, thick and continues connective tissue adhesions extending between the injured tendon and skin were observed. Small blood clots were also located in the grooves of underlying tissue. Injured achilles tendon was swollen. There was complete absence of gliding movements. On day 14, the surrounding peritendinous tissue was fibrous in nature and comparatively stronger and firmer than on day 7. Injured tendon was adhered with skin and there was slight reduction in swelling of injured tendon. On day 30, there was decrease in swelling. However, adhesions between tendon and skin were still present. On day 60 thin filamentous adhesions between skin and tendon were became apparent.

[FIGURE 1 OMITTED]

Histopathological observations. On day 7 in HA group the tendon sheath showed granulation reaction characterized by large number of intermingled plump shaped fibroblasts aligned concentrically around the tendon fascicles (Fig.1). Plenty of erythrocytes and fibrin were seen in the matrix along with perpendicularly newly formed capillaries. Abundance of fine reticular fibres arranged linearly in the granulation tissue of sheath adjacent to tendon and around the capillaries (Fig.2). On day 14 the fibrous connective tissue adhesions with skin sub cutis were more mature. Normal concentrically arranged thin parallel collagen fibres were seen in the matrix (Fig.3). By day 30, thickness of tendon sheath was very much reduced as compared to day 7 and 14 and consisted of loosely arranged collagen fibres with few fibrocytes. These changes were found further improved on day 60, having apparently no adhesions between tendon and the sheath (Fig.4). The blood vessels were arranged in longitudinal fashion of tendon. In AS group the microscopic picture was more or less similar to HA group. On day 30 thinly populated parallel collagen and fine wavy reticular fibres along with mesenchymal cells and fibroblasts were present. On day 60 there was extensive absorption of the collagen fibres at the adhesion site of sheath and tendon (Fig.5) as compared with day 30.

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In control group on day 7 adhered tendon sheath showing extensive exudation with small number of fibroblasts and mesenchymal cells (Fig.6). Core of the tendon showed stellate shaped fibroblasts arranged parallel within collagen bundles. On day 14 tendon was tightly adhered with the peritendinous tissue (Fig.7). Capillaries network was prominent with perivascular infiltration of neutrophils at the site. The collagen fibers and fibroblasts were progressively becoming more organized along the long axis of tendon. On day 30, there were no areas of necrosis, firbil lyses or signs of acute inflammation. Peritendinous adhesions showed loose and breaking fine strands of fibrous tissue extended in the direction of the gliding (Fig.8). Thick collagen fibers at injured tendon site were concentrically arranged along with mature fibroblasts. On day 60, the adhesions were loose as compared to day 30 and rarified towards the tendon surface.

[FIGURE 5 OMITTED]

Histochemical observations. The intensity and the reaction of alkaline phosphatase at adhesions site between the tendon and its sheath, including the adjacent subcutaneous fascia was compared with that of control animals. On day 7 the alkaline phosphatase reaction was mild (+) (Fig.9), moderate (++) (Fig.10) and intense (+++) (Fig.11) in groups HA, AS and control, respectively. On day 14, the activity was mild (+) in HA and AS treated groups and moderate (++) in control group. On subsequent time intervals no reaction of alkaline phosphatase was observed.

[FIGURE 6 OMITTED]

Scanning electron microscopic observations. On day 7, slight adhesions were seen between tendon and peritendinous tissue in HA group (Fig.12). On higher magnification the peritendinous tissue was relatively more vascular and had increased cellularity. Empty spaces between tendon and peritendinous structure ranged from 2 to 5 micron were visible (Fig.13). The scanning electron microscopic picture of AS group was more or less same in animals of HA group (Fig.14). Animals of control group showed complete adherence with tendon and tendon sheath and was highly vascular. Peritendinous sheath showed irregularly and loosely arranged collagen fibres (Fig.15). On day 14 in HA and AS groups there was further widening of the space between tendon and tendon sheath and ranged from 12 to 15 micron (Fig.16 and Fig.17). Tendon sheath was not completely adhered to tendon and presence of gliding movements was indicative of loosening of adhesions. On higher magnification in AS group there was thin filamentous adhesion of tendon sheath and tendon showed wavy parallel arranged collagen fibres. On day 30 the space between tendon and tendon sheath was further widened. Tendon sheath showed loosely arranged collagen fibrils in AS group (Fig.18). Whereas, in control group the tendon sheath was found adhered with tendon. The scanning electron microscopic observations in HA and AS groups on day 60 were more or less the same. No adhesions were seen between tendon proper and sheath and there was widening of space between tendon and tendon sheath. Wavy and parallel arrangement of collagen fibres was observed (Fig.19).

[FIGURE 7 OMITTED]

Discussion

Surgical wounds in all the animals healed by first intention and none of the surgical wounds exhibited infection. Rabbit as an animal model to study various aspects of tendon healing has been used previously [15-17]. The minimum gliding movements of tendon was seen up to day 10 postoperatively in different groups of animals. Histopathological observations on day 7 and 14 also revealed extensive exudation and increased number of fibroblasts at the site. The degree of tendon gliding movements varied directly with the degree of adhesions formation. More the adhesions, lesser the gliding movement of tendon [18,19]. Earlier improvement in gliding movements in test groups may be due to administration of HA and AS. Improved weight bearing following administration of AS and early restoration of gliding movements of tendon was reported following plasma preserved tendon allografts [10]. This may be due to anti-inflammatory action of HA, the main ingredient of AS. Synovial transplantation provided early rehabilitations following flexor tendon grafting with carbon fibres in calves [11].

[FIGURE 8 OMITTED]

[FIGURE 10 OMITTED]

Animals of control group showed severe adhesions all around the injured tendon. Presence of adhesions was characterized by foamy and reticulated appearance of soft tissue density [20.21]. Histopathological observations also revealed adherence of peritendinous tissue with tendon proper. These adhesions indicated proliferative fibrous tissue growth from the surrounding tissues [22,23]. Peacock Jr. [24] stated that the development of the fibrous tissue adhesions was a normal process of tendon healing, which indicated the cellular phase of tendon or general wound healing. The cells which initiate the reparative process were derived from the outer surface of tendon, the tendon sheath or from the paratenon [1]. The fibrous tissue growth from the surrounding tissue was essential for organization of connective tissue, which in turn allowed the gliding movement of the tendon (24). Progressive organization and regression of adhesions was earlier in HA and AS groups. The fibrous tissue growth from the surrounding tissue was organized and presence of negative contrast around tendon was suggestive of loosening of adhesions [1,25].

[FIGURE 9 OMITTED]

[FIGURE 11 OMITTED]

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Peritendinous adhesions formation has been proved as an undesirable but necessary part of the tendon healing process. Fibrous ingrowths were mostly oriented from skin to injury site of tendon. Haemorrhagic loose connective tissues around the injured tendon have also been observed by other workers [2]. Swelling at the injured site of the tendon may be due to haematoma and postoperative oedema that develops after injury. This haematoma and oedema restrict tendon gliding long before the collagenous adhesions are formed. The scar tissue was primarily comprised of poorly organized fibrous tissue which, limited the normal gliding function [26]. Increased vascularity at the injured site is the normal response to injury and is essential for absorption, removal of clot and dead cells and in the laying down of fibrous tissue. Further, the developing cells require more energy for their growth, which is met by increased vascularity at the site [27]. The adhesions are necessary for tendon healing as they bring fibroblasts and angioblasts from the peritendinous structures [28]. The synthesis of collagen by fibroblasts which is responsible for the constitution of tendon following injury has also been a fundamental part of formation of peritendinous adhesions [29]. Loose and filmy connective tissue adhesions were seen in HA and AS group on day 7 and 14 whereas the peritendinous adhesions were firm and thick in control group. Peritendinous adhesions observed in HA and AS groups were comparatively lesser than control group. Various workers due to its anti-inflammatory action [3,30-32] and viscoelsticity properties [7] have reported hyaluronic acid useful in reducing adhesions. Synovia having hyaluronic acid has also been reported in reduction of peritendinuous adhesions [33]. Adhesions at the injured site became comparatively loose and rarified on day 30 and onwards in HA and AS groups and the gliding movements were near normal. Control group animals still showed relatively firm adhesions although these adhesion strands were very few and easily breakable.

[FIGURE 16 OMITTED]

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[FIGURE 19 OMITTED]

Histopathological examination on day 7, showed extensive exudation at the injury site in control group whereas, it was moderate in HA and AS groups. Presence of exudation within a week after injury has been reported by various workers during tendon healing [2,24,34]. Areas of hemorrhages and damaged collagen fibrils infiltrated with lymphocytes, neutrophils and proliferating small blood capillaries were seen in both the treated groups. No improvement in the arrangement and orientation of collagen fibrils and plump fibroblasts up to day 7 following the repair of injured tendon tissue has also been reported by previous workers [34].

Along with other parameters we also used histological parameters to assess the efficacy of hyaluronic acid and autogenous synovia to tendon healing. The histological changes such as the increase in the number of fibroblasts [29], collagen organization [35], the increase in capillaries, thickening of the epitenon [35] and formation of adhesions has been observed in healing tendon. Potenza [35] demonstrated that proliferation of fibroblasts in both the synovial layer and paratenon led to healing. A number of studies have demonstrated an increase in the number of fibroblasts in tendon healing [2,35]. Potenza [36] showed that tendon healing after injury begins with the infiltration of inflammatory cells and fibroblasts from surrounding tissue. An increase in the number of fibroblasts seen during the initial 3 weeks after injury. The development of adhesions between healing tendon and surrounding structures correlates with the intensity, duration of the inflammatory reaction and degree of mobilization of the tendon during the healing period. Various other workers have reported that pleuripotent primitive mesenchymal cells in the peritendinous tissue are the main source of new fibrous tissue [24,37,38]. Bunnel [39] also reported paratenon to be a greatest source of fibroblast, therefore, the paratenon and surrounding connective tissue played the dominant role in the tendon healing process. Gelberman et al. [40] also reported this phenomenon to be initiated from the tendinous sheath and the surrounding tissue. The proliferative changes initiated from the paratenon and surrounding tissues resulted in adhesion formation [18,37,38].

Cellular reactions and histomorphology on day 14 showed marked improvement in HA and AS groups which were treated with hyaluronic acid and autogenous synovia, respectively. This may be due to anti-inflammatory property of hyaluronic acid and have been the main ingredient of synovia [3,9-11]. The tendon sheath was tightly adhered with tendon and subcutaneous tissue up to day 14 in animals of control group where as, it was comparatively looser and less thick in animals of HA and AS. Improvement in tendon injury cases with the passage of time has been reported by different authors [2,20,23]. Difference in thickness of sheath and its adhesions with the tendon was more markedly different among animals of group HA and AS on day 30 and 60 postoperatively.

The Masson's trichrome stain for selective demonstration of collagen fibres has also been used to study different aspects of tendon healing [9,41]. Some elastin fibres at the adhesion site and injured site of tendon were visible only upto day 7. Later on elastin fibres were seen particularly in blood vessels and the areas where blood vessels entered the tendon. These fibres may be necessary for the protection and maintenance of the vessels, which are subjected to unusual stress because of the mobility of the tendon [42]. Reticular fibres (Gridley's silver staining) were observed only up to day 7 and day 14 intervals. Thereafter no reticular fibres were observed. The disappearance of reticular fibres after day 14 might be due to their maturation and transformation into collagen fibres [43]. Periodic Acid Schiff staining for distribution and concentration revealed only mild activity up to day 7 and further no acitivity indicated the more concentration of mucopolysaccharides at earlier phase of injury, which later diminished out.

The formation of scars and granulation tissue is suppressed by HA [30] which also prevent the formation of adhesions after repair of the tendon without interfering with healing [44]. The decrease in the formation of adhesions may be the result of a decrease in the formation of new extracellular matrix due to the inhibition of mononuclear phagocytosis and lymphocytes. HA suppresses the release of fibronectin which plays a crucial role in the adhesion, proliferation and differentiation of various cells [45]. It also regulates the activity of polylorphonuclear leucocytes. AS having main ingredient as HA may also behave more or less in a same manner as HA.

On day 7, in group III (control) the intensity of alkaline phosphatase reaction was found intense (+++). Increased levels of alkaline phosphatase in the traumatised area have been reported to help in the proliferation of fibroblasts [46]. Further, this enzyme also appeared to be associated with the metabolic process concerning collagen formation [47]. In HA and AS treated groups mild to moderate activity of alkaline phosphatase as compared to control group may be attributed to the action of different agents/drugs used.

The scanning electron microscopy was found to be of immense value in assessing the interaction between tendon and tendon sheath [9]. Spaces observed between the tendon and the peritendinous structures were indicative of loosening of adhesions and presence of gliding movement. The scanning electron microscopic picture of both the groups at different time intervals was more or less the same. This indicated the effectiveness of both the therapies (HA and AS) in reducing peritendinous adhesions. The ultra structural analysis was found helpful to study the tendon healing and in the process of restoration of gliding movements. Ultra structural information allowed studying the events occurring during tendon injury and healing [37,41].

Nishida [48] evaluated the effects of HA on the excursion resistance of tendon grafts in-vitro. They recommended that the clinical advantage may be obtained in rehabilitation after tendon grafting utilizing HA in-vivo. In the present study we are able to demonstrate that HA and AS can be used for modulation of tendon adhesions.

Conclusion

The results of our study have shown that treatment with HA and AS may be used clinically for modulation of tendon adhesions. The administration of HA and AS on alternate day on 4 occasion after tendon injury will reduced the formation of peritendinous adhesions. This appears to be a simple and inexpensive means of refined treatment for the management of peritendinous adhesions.

Acknowledgement

We would like to express our deepest gratitude to Dr B. Singh for his help in statistical analysis. We are also grateful for the assistance received from Central Photography Laboratory, Communication Centre of Indian Veterinary Research Institute, Izatnagar and from Mr Anirudh Jagannath, photographer of Division of Pathology. Two of the authors (N. Kumar and A. K. Gangwar) are thankful to the Indian Council of Agricultural Research, New Delhi for the award of Junior Research Fellowship to them.

Received 8 December 2008; Accepted 15 June 2009; published online 25 June 2009

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N. Kumar, Naveen Kumar *, A. K. Sharma, S. K. Maiti, A. K. Gangwar, O. P. Gupta, Satish Kumar and Rajendra Singh

Division of Surgery, Indian Veterinary Research Institute Izatnagar-243 122 (Uttar Pradesh)

* Corresponding author naveen@ivri.up.nic.in (Naveen Kumar)
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