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Therapeutic effects of high molecular weight hyaluronan injections for tendinopathy in a rat model
J Orthop Sci (2015) 20:186–195 DOI 10.1007/s00776-014-0650-z
ORIGINAL ARTICLE
Therapeutic effects of high molecular weight hyaluronan injections for tendinopathy in a rat model Mamoru Yoshida · Hiroki Funasaki · Makoto Kubota · Keishi Marumo
Received: 1 April 2014 / Accepted: 1 September 2014 / Published online: 25 September 2014 © The Japanese Orthopaedic Association 2014
Abstract Background Tendinopathy is the most common tendon disorder. The etiology is still uncertain, and the disorder poses many therapeutic problems. In a few clinical studies, analgesic effects of high molecular weight hyaluronan (HMW HA) injections were observed, but the underlying mechanisms were not elucidated. In the present study, we analyzed the therapeutic effects of hyaluronan injections for tendinopathy in an animal model. Methods We made the tendinopathy rat model using a rodent treadmill machine. Rats with tendinopathy were injected with HMW HA (HA group), normal saline (NS group), or nothing (control group) into the space between the patellar tendon and the fat pad bilaterally, or were injected with HMW HA into the right knees and with saline to the left knees (HA/NS group), 5 times every 4 days. To assess the pain-relieving effect of HA, the spontaneous locomotor activities at night (12 h) and weight bearing of hind paws were measured every day. Histological sections of the patellar tendon stained with hematoxylin–eosin or prepared by TdT-mediated dUTP nick end labeling were microscopically analyzed. Results The number of spontaneous locomotor activities in the HA group was significantly larger than those in NS or control groups, and in the HA group they recovered up to a healthy level. The percent weight distribution of the right hind paws was significantly increased along with the number of injections. On histologic examinations, the numbers of microtears, laminations, or apoptotic cells in the
M. Yoshida (*) · H. Funasaki · M. Kubota · K. Marumo Department of Orthopaedic Surgery, The Jikei University School of Medicine, 3‑25‑8 Nishi‑Shinbashi, Minato‑ku, Tokyo 105‑8461, Japan e-mail: [email protected]
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patellar tendons in the HA group were significantly lower than those in the NS or the control groups. Conclusions The injections of HMW HA were effective for pain relief and for partial restoration of the patellar tendon in our tendinopathy rat model, and thus may become an effective therapeutic modality for the disease. Abbreviations HMW HA High molecular weight hyaluronan NS Normal saline EDTA Ethylenediamine tetra-acetic acid H&E Hematoxylin–eosin TUNEL TdT-mediated dUTP nick end labeling
Introduction Tendinopathy (often called tendinitis, tendinosis, or enthesopathy) is the most common tendon disorder characterized by activity-related pain, focal tendon tenderness and decreased strength and movement in the affected area. Tendinopathy can occur in almost any tendon, and if developed, it impairs active performance in athletes and workers of many occupations that involve repetitive movements. Some of the most common tendinopathies are tennis elbow, jumper’s knee, and Achilles tendinitis. Increased production or activation of matrix metalloproteinases and stimulated regeneration of free nerve endings around vessels in affected tendons is found by histological or biochemical studies [1]. Moreover, the finding of apoptosis of tendon cells is a the specific feature of the tendinopathy [1]. It has been suggested that oxidative stress facilitated by high levels of cyclical strain and subsequent apoptosis are involved in the pathogenesis of tendinopathy [1]; however, the detailed pathophysiological mechanisms
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are not understood, and therefore, an effective therapy for tendinopathy has not yet been established. Muneta et al. recently reported that injections of high molecular weight hyaluronan (HMW HA) into or near tendinopathy-affected tendons produce relief from pain; the treated tendons were the patellar tendon, biceps tendon of the thigh, medial hamstring tendon, lateral gastrocnemius tendon, ilio-tibial band, or the Achilles tendon (reported in a Japanese journal). A similar effect of HMW HA injections for tendinopathy of the plantar fascia was also observed by Higashiyama et al. [2]. In addition, it has been recently reported that peri-articular HA treatment for tennis elbow produced significantly better pain relief at rest and after maximal grip testing than a control therapy [3]. Although a few clinical reports such as those described above have been published, there has been no study scientifically evaluating the pain-relieving effect of HMW HA for tendinopathy or that has histopathologically analyzed tendon tissues. In order to prove the therapeutic effects of HMW HA injections for tendinopathy, we created an experimental animal model of patellar tendinopathy and scientifically examined the pain-relieving effect of HMW HA and histologically analyzed the tendinopathy-affected tendons. The rat model was prepared using a repetitive running exercise on a rodent treadmill machine. The pain-relieving effect was assessed by measurement of rats’ spontaneous locomotor activities for 12 h following the exercise and by evaluation of weight-bearing percent distribution in hind paws injected with HMW HA. Furthermore, histological examinations were performed to evaluate the effects of HA injections on suppression and acceleration of tendon tissue-repair.
Materials and methods Reagents and materials Suvenyl® (SVE), a preparation of HMW HA for injections into tendons was purchased from Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. The concentration of HMW HA was 10 mg/mL with an average molecular weight of 2700 kDa (viscosity average molecular weight was 1900 kDa). The normal saline for injections was from Otsuka Pharmaceutical Factory, Inc., Naruto, Japan. A rodent treadmill, KN-73, was purchased from Natsume Seisakusho, Co., Ltd., Tokyo, Japan. The Supermex system for measurement of spontaneous locomotor activities of rats was from Muromachi Kikai, Tokyo, Japan. The Incapacitance Tester for measurement of weight bearing distributions in hind paws in rats was from Linton Instrumentation, Norfolk, England. The ApopTag Peroxidase In Situ
Apoptosis Detection Kit was from EMD Millipore Corporation, Billerica, MA, USA. Animals Male Wistar rats (16–18 weeks of age, weighing 350– 400 g) were purchased from Nippon SLC, Hamamatsu, Shizuoka, Japan, and served as subjects in the study. Rats were housed in an environmentally controlled animal facility on a 12:12 light/dark cycle with food and water available ad libitum in the laboratory animal section. The animal model All experiments were conducted in accordance with the institutional guidelines for the care and use of experimental animals after an institutional approval for the study was obtained. A rat model of patellar tendinopathy was made according to the method of Flatow and colleagues [4–6]. A running exercise was performed on a rodent treadmill with 10 % inclination of the running protocol. The rats were acclimated to the treadmill by gradually increasing running speed and time for 2 weeks before the running exercise. Running distance reached up to 40 km during 40 days and the mean running speed was 0.5 km/h. The spontaneous locomotor activities at night were measured for 12 h with the Supermex system on the day before the running exercise and on the day after exercise. The reduction in spontaneous locomotor activity after exercise was different among rats, and it was 26 ± 18 % (3–36 %) for 43 rats. The rats showing >25 % reduction (25–36 %) in spontaneous locomotor activity after exercise were then selected for later study as the tendinopathy rats. Approximately 85 % of the rats ran the whole distance and approximately 90 % were selected for further analyses. Histological sections from the patellar tendons or rotator cuffs in the shoulders of the tendinopathy rats were prepared. Tendinopathy-specific histopathological changes [1] were confirmed with light microscopy at the insertion site of the patellar tendon, while less tendinopathyspecific histopathological changes were observed at the insertion site of the rotator cuffs in shoulders. Group division The rats with tendinopathy were divided into four groups. The rats in the HA group, the normal saline (NS) group, or the control group were injected with HMW HA, normal saline, or nothing, respectively, into the patellar tendons bilaterally. Each group consisted of 10 rats. Rats in the fourth, HA/NS group, were injected with HMW HA into the right patellar tendon and with normal saline into the left patellar tendon at the same time and in the same manner. The HA/NS group consisted of 6 rats.
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Fig. 1 The study protocol
Observation of general status and body weight measurements The general status was observed once a day from the day before the running exercise started to the end of the study. Body weight was measured at every 7 days during the study period. Injections of HMW HA and normal saline into hind paws After general anesthesia with 2.5 % isoflurane, the hair at the knee joints was shaved and 30 μL of HMW HA or normal saline was injected into the space between the patellar tendon and the fat pad tissue at the distal area of the patellar tendon using a 0.5 mL injection syringe and a 27G injection needle from the lateral or medial side of the knees, respectively, in the HA group or the NS group. It was experimentally confirmed that 30 μL of HA or saline containing the indigo carmine dye provided a maximum injectate volume without leakage into the joint cavity. In the control group, the injection needle was inserted between the patellar tendon and the fat pad in the same manner after same general anesthesia and hair shaving procedure as in other groups. The injections or needle pricks were performed once every 4 days, a toa total of five times, from the day following completion of the running exercise (Fig. 1). In the HA/NS group, the same doses of HMW HA and saline were injected into the right and left knees, respectively, to the same animal, at the same time and in the same manner as in the HA, NS, or control groups. All injections were performed in the morning (Fig. 1). It was observed that no injected hyaluronan mixed with dye was leaked into the joint cavity of the knee joints after five injections in 4-day intervals. Measurements of spontaneous locomotor activities Spontaneous motor activity of each rat was examined using the Supermex system for 12 h each night for 27 days after
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the day running exercise had finished and during the injection protocol period in all groups. Details of spontaneous locomotor activity measurement have been described previously [7–10]. Briefly, spontaneous locomotor activities were determined by detecting the movement of infrared radiation emitted from each animal. The activities were measured as a single count when an animal moved from one region of the measurement area divided optically by multiple lenses, to a neighboring region. Total counts were calculated by summing up all counts of 10-min periods. The infrared filter with 25 % signal reduction was used to remove the noise of the grooming motion. The relative number of motor activities in each group was calculated and the mean of the relative changes was plotted on a graph in order to evaluate the relative changes for motor activities of rats during the experiment period, since the absolute values for motor activities were different among rats, which were 9787 ± 1982 (6703–14191) counts for 50 healthy rats. A statistical analysis was performed to examine the outcome differences between each group. Measurements of the weight‑bearing distributions in the hind paws Weight-bearing distributions of the hind paws were measured with the Incapacitance Tester, which independently measured the weight bearing of each hind paw, at 15:00 hours every day for 27 days in the HA/NS group. Measurements were performed three times and the percent weight distributions of the hind paws of the right legs injected with HMW HA were calculated by the following formula: the percent weight distribution of right hind paw = [right weight/(right weight + left weight)] × 100. The percent weight distribution value for the right hind paw per measurement was defined as the mean of three calculations of the percent weight distributions of right hind paws. The relative changes of the percent weight distributions of right hind paws were calculated and the mean of the
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relative changes was plotted on a graph. In order to acclimatize the rats to the Incapacitance Tester, affected rats in the HA/NS group were placed in the holder and immobilized for 5 s daily for 7 days before commencement of the injection protocol [11]. A statistical analysis was performed to examine the significance for the percent weight distribution value for the right hind paw after the fourth injections compared to the values at day 1 in order to assess a painrelieving effect of HMW HA.
Statistical analysis Statistical analysis was done using the Wilcoxon’s matched-pairs signed-rank test. A probability value of <0.05 was considered statistically significant.
Results General status and body weight
Histological examinations The knee tissue samples were obtained from 3 healthy normal rats and 5 tendinopathy rats at day 1 (controls), and from 5 rats in the HA, the NS, or the control groups, respectively, at day 28. Following inhalation with 5 % isoflurane, the rats were killed by immediate blood draw. Whole knee joints were fixed in 4 % paraformaldehyde at pH 7.4 for 3 days, and decalcified in 20 % EDTA solution for 21 days at 4 °C, and then embedded in paraffin wax. Whole knee joints were sectioned in the sagittal plane with a section width of 5 μm. The sections were stained with hematoxylin–eosin (H&E) for histology. They were assessed with a histopathological score determined by counting the number of microtears and laminations located at the insertion site of the patellar tendon as a point in high-power (magnification 200×) fields in each five sections of tendinopathy rats or HA, NS, or control groups. A point in the score was doubled in cases where the length of tears or laminations were longer than half the diameter of the visual high-power field (magnification 200×) on the microscope. The data values were expressed as the mean of the differences among groups after statistical analysis. The histological sections were also prepared by TdT-mediated dUTP nick end labeling using the ApopTag Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer’s instructions. Tendon cells for apoptosis were quantified by counting the number of TUNEL-positive cells in high-power (magnification 400×) fields at the joint side of the tibial insertion site for the patellar tendon in each of five sections of tendinopathy rats or HA, NS, or control groups. The data values were expressed as the mean of the differences among groups after statistical analysis.
There were no abnormalities observed in any of the groups during the experiment period. The change of body weight during the injection period is shown in Table 1. All groups showed a similar trend of body weight changes compared to the control group. Spontaneous locomotor activities The counts for the spontaneous locomotor activities in HA group increased along with the number of HA injections up to approximate 1.5-fold at day 13 after the fourth HMW HA injection and at day 17 after the fifth injection. Spontaneous motor activities in the HA group recovered up to the original level as before commencement of the running exercise. There was an increasing trend of the motor activities count the next day or 2 days after HA injection and then the counts decreased by approximate 10 % on day 3 after the injection. The counts of spontaneous locomotor activities in the HA group were maintained, not decreased, for 10 days after the fifth injection of hyaluronan until the end of the study at day 27 without any additional injections. There were significantly more counts for the spontaneous locomotor activities in the HA group than in the NS group or the control group during 27 days of the experimental period except at day 3 or 4 (p < 0.05). The counts of the locomotor activities in the NS group and the control group did not increased and remained in the range from 80 to 120 % of the initial activities. No significant differences were observed for the counts of locomotor activities between the NS group and the control group (Fig. 2).
Table 1 Body weight gain Group
n
Days after running exercise 0
7
14
21
28
Control HA
10 10
420 ± 19 422 ± 21
431 ± 21 433 ± 22
437 ± 23 439 ± 22
448 ± 25 450 ± 25
455 ± 26 458 ± 26
NS
10
421 ± 18
428 ± 20
436 ± 21
446 ± 24
453 ± 25
Body weight (g) was measured at indicated days. The data indicate the mean ± SD of ten rats. The HA group or the NS group was compared with the control group using unpaired t test
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Relative values for spontaneous locomotor activities (%)
190
180 *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
160
140
120
100
80
Control group NS group HA group
60
40 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Days Injections with hyaluronan (times) Fig. 2 Relative values for spontaneous locomotor activities in the HA, NS, or control groups. The spontaneous locomotor activities in rats were examined using a Supermex system at each night for 12 h, every day for 27 days, after completion of the running exercise and after commencement of the injection protocol. The relative number of
locomotor activities in the each group was calculated and the means or the SD were plotted on a graph. *Significant difference for the mean of the motor activities between the HA group and the NS or control groups (p < 0.05)
Percent weight distributions of right hind paws
(Table 2), indicating that the reactions of tendon repair in the HA group were progressed compared to those in the NS or control groups. The TUNEL-positive tendon cells were observed at the joint side of the tibial insertion site for the patellar tendon in all tendinopathy rats. The number of TUNEL-positive cells at the same area in five rats of the HA group was significantly lower than those in five rats of the NS group or the control group after the fifth injections of hyaluronan at day 28 (p < 0.05) (Table 3; Fig. 6).
The mean of the relative changes of weight bearing of right hind paws injected with HMW HA increased along with the times of injections for 21 days after the fifth injection (Fig. 3). After 21 days, the relative changes plateaued until the end of the study at day 27 without any additional injections. The relative changes after the fourth injections were significantly greater than those at day 1 (p < 0.05). Histological findings Two major pathological findings were found at the tibial insertion sites of the patellar tendons in tendinopathy rats in all the histological sections stained with hematoxylin– eosin. They were microtears in the tendon body and lamination, i.e., several longitudinal disruptions between the fiber bundle layers (Fig. 4). The same pathological findings were found at the tibial insertion sites of the patellar tendon at day 28 in the HA, NS, or control groups in the histological knee sections stained with hematoxylin–eosin (Fig. 5). However, the histopathological scores determined y the number and length of the microtears and laminations in five rats of the HA group were significantly lower than those in five rats of the NS or control groups (p < 0.05)
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Discussion Several different forms of the tendinopathy have been described; it has been shown that the anatomical location of a given lesion within the tendon plays some role in determining the type of tendinopathy. Chronic tendon abnormalities consistent with tendinopathy can be found in the main body of the tendon (called tendinitis or tendinosis), its insertion site on bone (the enthesis), and the structures surrounding the tendon. Insertional tendinopathy (enthesopathy or enthesiopathy) is one of the most common forms of tendinopathy and is commonly seen in the patellar tendon (jumper’s knee).
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Relative values for weight bearings of hind paws injected with hyaluronan
1.3 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Days Injections with hyaluronan (times) Fig. 3 Relative values for weight-bearing distribution of the hind paws injected with hyaluronan. The percent distribution of weight bearing in the right hind paws were measured with the Incapacitance Tester, which independently measured the weight bearing in each hind paw, at 15:00 hours every day for 27 days in the HA/NS group. The measurement was performed three times and the percent weight distribution of the right hind paws were calculated by the following formula: the percent weight distribution of the right hind
paw = [right weight/(right weight + left weight)] × 100. The value of percent weight distribution of the right hind paw per measurement was defined as the mean of three calculations. The relative changes of percent weight distribution of the right hind paws were calculated and the means or SD were plotted on a graph. *Significant difference for the mean of the relative values for weight-bearing distribution of the hind paws injected with hyaluronan after the fourth injections compared to those at day 1 (p < 0.05)
Fig. 4 Histological sections of the tibial insertion sites of the patellar tendons stained with H&E in a healthy rat (a) or in the tendinopathy rat (b) (×100). Two major pathological findings were found with light microscopy at the tibial insertion sites of the patellar tendons in
the tendinopathy rats: (1) microtears in the body of the tendon and (2) several longitudinal disruptions between the layers of the fiber bundles of the tendon body, so-called laminations
In the present study, we created an insertional tendinopathy model of the patellar tendon in rats and examined the therapeutic effects of HMW HA injections. The following findings were obtained: (1) the injections of HMW HA into the space between the patellar tendon and the fat pad tissue significantly increased the number of spontaneous locomotor activities in the tendinopathy rats proportionally to
the number of injections compared to those in the normal saline or control groups; (2) the spontaneous locomotor activities in the HA group after the fourth or fifth injections of HMW HA recovered up to the original healthy level and were then maintained, not decreased, for the following 10 days without any additional injections of hyaluronan; (3) the weight-bearing distributions of the hind paws
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Fig. 5 Histological sections of the tibial insertion sites of the patellar tendons stained with H&E in the HA group (a), in the NS group (b), or in the control group (c) (×100). The number of microtears or lami-
nations found in 5 rats of the HA group was lower than those in 5 rats of the NS or control group
Table 2 Histopathological score for the histological sections located at the tibial insertion site of the patellar tendon
injections; (4) the number of microtears or laminations observed in histological specimens at the tibial insertion site of the patellar tendon in tendinopathy rats decreased in the HA group compared to those in the NS or control group; (5) the number of TUNEL-positive cells at the joint side of the tibial insertion site of the patellar tendon in the HA group was lower than those in the NS or control groups after the fifth injections of hyaluronan at day 28. The present study shows that spontaneous locomotor activities in tendinopathy rats recovered up to a healthy level after five injections of hyaluronan and that the percent weight-bearing distribution in the hind paws injected with HMW HA also increased along with the number of hyaluronan injections. Moreover, the motor activities increased at 1 or 2 days after injections of HA, subsequently decreased after the following injections, and then gradually increased following five injections of hyaluronan. No remarkable tendinopathy-specific histological changes were observed in the rotator cuffs of the shoulders in tendinopathy rats. It has been suggested that HMW HA exerts a biological function of pain reduction by masking free nerve endings or organelles involved in nociception [12–14]. Therefore, our findings indicate that HMW HA injections into the space between the patellar tendon and the fat pad tissue are an effective analgesic method in tendinopathy rats. There was a report that the fluorescein-labeled HMW HA, of which the molecular size was
Group Tendinopathy Control NS HA
Histopathological score 17 ± 6 16 ± 5 15 ± 6 6±2
∗
∗
∗
* p < 0.05 Table 3 Cell number for apoptosis at the joint side of the tibial insertion site for the patellar tendon
Group Tendinopathy Control NS HA
Apoptosis cell 26 ± 11 28 ± 12 25 ± 11 7±3
∗
∗
∗
* p < 0.05
injected with HMW HA significantly increased along with the number of the injections and stabilized, after the fifth injection of hyaluronan for 10 days without any additional
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(a)
(b) cartilage
cartilage
tendon
(c)
tendon
(d)
cartilage
cartilage
tendon tendon
Fig. 6 Histological sections of the tibial insertion sites of the patellar tendons prepared by TdT-mediated dUTP nick end labeling in the tendinopathy rats (a), in the HA group (b), in the NS group (c), or in the control group (d) (×220). The number of TUNEL-positive ten-
don cells in the HA group was lower than those in the NS or control groups at the joint side of the tibial insertion site of the patellar tendon
the same as the one used in the present study, penetrated synovium from the synovial lining cell layer to the interstitium in subsynovial fat tissue (deep layer) at 3–24 h after injection into the joint cavity of the rabbit knee; and HMW HA remained in the synovial tissue for 3–7 days [15]. This indicates that injected HMW HA infiltrated into the tendon tissue at day 1 and masked free nerve endings or organelles, and that the analgesic effect gradually increased along with the hyaluronan accumulation following each consecutive injection of HMW HA, since accessibility of HMW HA to the tendon might be similar to the one into the synovium. Consequently, our findings support the proposed mechanism of pain relief by HMW HA injection, i.e., masking free nerve endings or organelles involved in nociceptive stimulus production and peripheral sensitization. The present study also shows that spontaneous locomotor activity in the HA group recovered up to the original healthy level after the fourth or fifth injection of HA and was then maintained, not decreased, for 10 days after the last, fifth injection of HA. This finding suggests that pain in the HA group almost disappeared, and the pain-free conditions were maintained at least for 10 days after the fifth injections of hyaluronan. From these data, we think that the consecutive 4 or 5 injections of HMW HA are suitable for relief of pain caused by tendinopathy.
The space between the patellar tendon and the fat pad tissue was chosen for injection of HMW HA because pooling of the HMW HA molecules close to the patellar tendon without diffusion or leakage into the joint cavity was necessary, and to expose biologically the patellar tendon tissue containing cells, nerves, and vessels to HMW HA. Such a space suitable for the storage of hyaluronan molecules in the patellar tendon body was not available. Moreover, there was a possibility that the injected HA expands the tendon tear or worsens lamination if injected directly into the tendon body, since the viscosity of HMW HA is very high compared to that of the normal saline. In order to avoid exposure of HMW HA to the synovium or cartilage in the articular space, it was necessary to block any possible leakage of hyaluronan into the joint cavity. A suitable volume of HMW HA for injection without producing leakage was decided by injecting the dye mixed with hyaluronan into the space between the patellar tendon and the fat pad tissue. In addition, there was a report that the joint side of the tendon may be a main site of lesion in tendinopathy [16]; this finding was also taken into consideration when choosing the injection site in the present study. Apoptosis of tendon cells was examined at the joint side of the patellar tendon at the tibial insertion site in tendinopathy rats by TdT-mediated dUTP nick end
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labeling. On setting the experiment, we presumed that tendon cell apoptosis is induced by repetitive mechanical stress against the insertion site of the patellar tendon or by the oxidative stress at the insertion site of the tendon by the daily running exercise. In fact, the number of apoptotic cells located at the joint side of the tibial insertion site of the patellar tendon in the HA group was lower than those in the NS or control groups after 5 injections of hyaluronan. This finding indicated that injected HMW HA suppressed occurrence of apoptosis, and it was in agreement with some reports that HMW HA exerts a biological function of suppressing apoptosis or oxidative stress that eventually leads to apoptosis [17–19]. In addition, apoptosis suppression might correlate with pain-relief in tendinopathy, since enzymes breaking the extra-cellular matrix are usually released from apoptotic cells and broken or damaged tissues around those cells acting as nociceptive stimuli. It has been reported that HMW HA stimulates chemotaxis and proliferation of fibroblasts [20–22], or promotes fibroblast-induced matrix biosynthesis [23–25]. Migration and proliferation of fibroblasts and promotion of matrix biosynthesis generally accelerate the repair reaction of damaged tissues. Moreover, it has been known that HMW HA inhibits production of matrix metalloproteinases, which induce matrix degradation and release of cytokines from fibroblasts [26–28]. These effects participate in the structure modifying effect. It was observed that the histopathological score determined by the number and length of microtears and laminations in the HA group was lower than those in the NS or control groups. This indicated that the repair reactions in the HA group were initiated by HMW HA injection and progressed; they most probably were either not initiated or stay dormant in the NS and control groups. It was, therefore, speculated that injected HMW HA accelerated tendon repair at the insertion site by exerting its biological functions.
Conclusion Five injections of HMW HA into the space between the patellar tendon and fat pad effectively relieved pain and initiated partial restoration of the patellar tendon in the tendinopathy rat model. The initiated changes were supported by histopathological findings, suggesting that several injections of HMW HA should become a clinically effective modality in treatment of tendinopathy. Acknowledgments We are grateful to all members of the Department of Orthopaedic Surgery of the Jikei University School of Medicine for the partial support of the present study, and appreciate Chugai Pharmaceutical Co., Ltd. for their financial support.
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Hyaluronan injection for tendinopathy 20. Yagishita K, Sekiya I, Sakaguchi Y, Shinomiya K, Muneta T. The effect of hyaluronan on tendon healing in rabbits. Arthroscopy. 2005;21:1330–6. 21. Mast AB, Diegelmann FR, Krummel MT, Cohen KI. Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts. Matrix. 1993;13:441–6. 22. Greco MR, Iocono AJ, Ehrlich PH. Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix. J Cell Physiol. 1998;177:465–73. 23. Kikuchi T, Shimmei M. Effects of hyaluronan on proteoglycan metabolism of rabbit articular chondrocytes in culture. Jpn J Rheumatol. 1994;3:207–15. 24. Kawasaki K, Ochi M, Uchio Y, Adachi N, Matsusaki M. Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels. J Cell Physiol. 1999;179:142–8.
195 25. Smith MM, Ghosh P. The synthesis of hyaluronic acid by human synovial fibroblast is influenced by the nature of the hyaluronate in the extracellular environment. Rheumatol Int. 1987;7:113–22. 26. Shimizu M, Yasuda T, Nakagawa T, Yamashita E, Julovi SM, Hiramitsu T, Nakamura T. Hyaluronan inhibits matrix metalloproteinase-1 production by rheumatoid synovial fibroblasts stimulated by proinflammatory cytokines. J Rheumatol. 2003;30:1164–72. 27. Takahashi K, Goomer RS, Harwood F, Kubo T, Hirasawa Y, Amiel D. The effect of hyaluronan on matrix metalloproteinase-3, interleukin-1 beta, and tissue inhibitor of metalloproteinase-1 gene expression during the development of osteoarthritis. Osteoarthr Cartil. 1999;7:182–90. 28. Tanaka M, Masuko-Hongo K, Kato T, Nishioka K, Nakamura H. Suppressive effects of hyaluronan on MMP-1 and RANTES production from chondrocytes. Rheumatol Int. 2006;26:185–90.
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ORIGINAL ARTICLE
Therapeutic effects of high molecular weight hyaluronan injections for tendinopathy in a rat model Mamoru Yoshida · Hiroki Funasaki · Makoto Kubota · Keishi Marumo
Received: 1 April 2014 / Accepted: 1 September 2014 / Published online: 25 September 2014 © The Japanese Orthopaedic Association 2014
Abstract Background Tendinopathy is the most common tendon disorder. The etiology is still uncertain, and the disorder poses many therapeutic problems. In a few clinical studies, analgesic effects of high molecular weight hyaluronan (HMW HA) injections were observed, but the underlying mechanisms were not elucidated. In the present study, we analyzed the therapeutic effects of hyaluronan injections for tendinopathy in an animal model. Methods We made the tendinopathy rat model using a rodent treadmill machine. Rats with tendinopathy were injected with HMW HA (HA group), normal saline (NS group), or nothing (control group) into the space between the patellar tendon and the fat pad bilaterally, or were injected with HMW HA into the right knees and with saline to the left knees (HA/NS group), 5 times every 4 days. To assess the pain-relieving effect of HA, the spontaneous locomotor activities at night (12 h) and weight bearing of hind paws were measured every day. Histological sections of the patellar tendon stained with hematoxylin–eosin or prepared by TdT-mediated dUTP nick end labeling were microscopically analyzed. Results The number of spontaneous locomotor activities in the HA group was significantly larger than those in NS or control groups, and in the HA group they recovered up to a healthy level. The percent weight distribution of the right hind paws was significantly increased along with the number of injections. On histologic examinations, the numbers of microtears, laminations, or apoptotic cells in the
M. Yoshida (*) · H. Funasaki · M. Kubota · K. Marumo Department of Orthopaedic Surgery, The Jikei University School of Medicine, 3‑25‑8 Nishi‑Shinbashi, Minato‑ku, Tokyo 105‑8461, Japan e-mail: [email protected]
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patellar tendons in the HA group were significantly lower than those in the NS or the control groups. Conclusions The injections of HMW HA were effective for pain relief and for partial restoration of the patellar tendon in our tendinopathy rat model, and thus may become an effective therapeutic modality for the disease. Abbreviations HMW HA High molecular weight hyaluronan NS Normal saline EDTA Ethylenediamine tetra-acetic acid H&E Hematoxylin–eosin TUNEL TdT-mediated dUTP nick end labeling
Introduction Tendinopathy (often called tendinitis, tendinosis, or enthesopathy) is the most common tendon disorder characterized by activity-related pain, focal tendon tenderness and decreased strength and movement in the affected area. Tendinopathy can occur in almost any tendon, and if developed, it impairs active performance in athletes and workers of many occupations that involve repetitive movements. Some of the most common tendinopathies are tennis elbow, jumper’s knee, and Achilles tendinitis. Increased production or activation of matrix metalloproteinases and stimulated regeneration of free nerve endings around vessels in affected tendons is found by histological or biochemical studies [1]. Moreover, the finding of apoptosis of tendon cells is a the specific feature of the tendinopathy [1]. It has been suggested that oxidative stress facilitated by high levels of cyclical strain and subsequent apoptosis are involved in the pathogenesis of tendinopathy [1]; however, the detailed pathophysiological mechanisms
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are not understood, and therefore, an effective therapy for tendinopathy has not yet been established. Muneta et al. recently reported that injections of high molecular weight hyaluronan (HMW HA) into or near tendinopathy-affected tendons produce relief from pain; the treated tendons were the patellar tendon, biceps tendon of the thigh, medial hamstring tendon, lateral gastrocnemius tendon, ilio-tibial band, or the Achilles tendon (reported in a Japanese journal). A similar effect of HMW HA injections for tendinopathy of the plantar fascia was also observed by Higashiyama et al. [2]. In addition, it has been recently reported that peri-articular HA treatment for tennis elbow produced significantly better pain relief at rest and after maximal grip testing than a control therapy [3]. Although a few clinical reports such as those described above have been published, there has been no study scientifically evaluating the pain-relieving effect of HMW HA for tendinopathy or that has histopathologically analyzed tendon tissues. In order to prove the therapeutic effects of HMW HA injections for tendinopathy, we created an experimental animal model of patellar tendinopathy and scientifically examined the pain-relieving effect of HMW HA and histologically analyzed the tendinopathy-affected tendons. The rat model was prepared using a repetitive running exercise on a rodent treadmill machine. The pain-relieving effect was assessed by measurement of rats’ spontaneous locomotor activities for 12 h following the exercise and by evaluation of weight-bearing percent distribution in hind paws injected with HMW HA. Furthermore, histological examinations were performed to evaluate the effects of HA injections on suppression and acceleration of tendon tissue-repair.
Materials and methods Reagents and materials Suvenyl® (SVE), a preparation of HMW HA for injections into tendons was purchased from Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. The concentration of HMW HA was 10 mg/mL with an average molecular weight of 2700 kDa (viscosity average molecular weight was 1900 kDa). The normal saline for injections was from Otsuka Pharmaceutical Factory, Inc., Naruto, Japan. A rodent treadmill, KN-73, was purchased from Natsume Seisakusho, Co., Ltd., Tokyo, Japan. The Supermex system for measurement of spontaneous locomotor activities of rats was from Muromachi Kikai, Tokyo, Japan. The Incapacitance Tester for measurement of weight bearing distributions in hind paws in rats was from Linton Instrumentation, Norfolk, England. The ApopTag Peroxidase In Situ
Apoptosis Detection Kit was from EMD Millipore Corporation, Billerica, MA, USA. Animals Male Wistar rats (16–18 weeks of age, weighing 350– 400 g) were purchased from Nippon SLC, Hamamatsu, Shizuoka, Japan, and served as subjects in the study. Rats were housed in an environmentally controlled animal facility on a 12:12 light/dark cycle with food and water available ad libitum in the laboratory animal section. The animal model All experiments were conducted in accordance with the institutional guidelines for the care and use of experimental animals after an institutional approval for the study was obtained. A rat model of patellar tendinopathy was made according to the method of Flatow and colleagues [4–6]. A running exercise was performed on a rodent treadmill with 10 % inclination of the running protocol. The rats were acclimated to the treadmill by gradually increasing running speed and time for 2 weeks before the running exercise. Running distance reached up to 40 km during 40 days and the mean running speed was 0.5 km/h. The spontaneous locomotor activities at night were measured for 12 h with the Supermex system on the day before the running exercise and on the day after exercise. The reduction in spontaneous locomotor activity after exercise was different among rats, and it was 26 ± 18 % (3–36 %) for 43 rats. The rats showing >25 % reduction (25–36 %) in spontaneous locomotor activity after exercise were then selected for later study as the tendinopathy rats. Approximately 85 % of the rats ran the whole distance and approximately 90 % were selected for further analyses. Histological sections from the patellar tendons or rotator cuffs in the shoulders of the tendinopathy rats were prepared. Tendinopathy-specific histopathological changes [1] were confirmed with light microscopy at the insertion site of the patellar tendon, while less tendinopathyspecific histopathological changes were observed at the insertion site of the rotator cuffs in shoulders. Group division The rats with tendinopathy were divided into four groups. The rats in the HA group, the normal saline (NS) group, or the control group were injected with HMW HA, normal saline, or nothing, respectively, into the patellar tendons bilaterally. Each group consisted of 10 rats. Rats in the fourth, HA/NS group, were injected with HMW HA into the right patellar tendon and with normal saline into the left patellar tendon at the same time and in the same manner. The HA/NS group consisted of 6 rats.
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Fig. 1 The study protocol
Observation of general status and body weight measurements The general status was observed once a day from the day before the running exercise started to the end of the study. Body weight was measured at every 7 days during the study period. Injections of HMW HA and normal saline into hind paws After general anesthesia with 2.5 % isoflurane, the hair at the knee joints was shaved and 30 μL of HMW HA or normal saline was injected into the space between the patellar tendon and the fat pad tissue at the distal area of the patellar tendon using a 0.5 mL injection syringe and a 27G injection needle from the lateral or medial side of the knees, respectively, in the HA group or the NS group. It was experimentally confirmed that 30 μL of HA or saline containing the indigo carmine dye provided a maximum injectate volume without leakage into the joint cavity. In the control group, the injection needle was inserted between the patellar tendon and the fat pad in the same manner after same general anesthesia and hair shaving procedure as in other groups. The injections or needle pricks were performed once every 4 days, a toa total of five times, from the day following completion of the running exercise (Fig. 1). In the HA/NS group, the same doses of HMW HA and saline were injected into the right and left knees, respectively, to the same animal, at the same time and in the same manner as in the HA, NS, or control groups. All injections were performed in the morning (Fig. 1). It was observed that no injected hyaluronan mixed with dye was leaked into the joint cavity of the knee joints after five injections in 4-day intervals. Measurements of spontaneous locomotor activities Spontaneous motor activity of each rat was examined using the Supermex system for 12 h each night for 27 days after
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the day running exercise had finished and during the injection protocol period in all groups. Details of spontaneous locomotor activity measurement have been described previously [7–10]. Briefly, spontaneous locomotor activities were determined by detecting the movement of infrared radiation emitted from each animal. The activities were measured as a single count when an animal moved from one region of the measurement area divided optically by multiple lenses, to a neighboring region. Total counts were calculated by summing up all counts of 10-min periods. The infrared filter with 25 % signal reduction was used to remove the noise of the grooming motion. The relative number of motor activities in each group was calculated and the mean of the relative changes was plotted on a graph in order to evaluate the relative changes for motor activities of rats during the experiment period, since the absolute values for motor activities were different among rats, which were 9787 ± 1982 (6703–14191) counts for 50 healthy rats. A statistical analysis was performed to examine the outcome differences between each group. Measurements of the weight‑bearing distributions in the hind paws Weight-bearing distributions of the hind paws were measured with the Incapacitance Tester, which independently measured the weight bearing of each hind paw, at 15:00 hours every day for 27 days in the HA/NS group. Measurements were performed three times and the percent weight distributions of the hind paws of the right legs injected with HMW HA were calculated by the following formula: the percent weight distribution of right hind paw = [right weight/(right weight + left weight)] × 100. The percent weight distribution value for the right hind paw per measurement was defined as the mean of three calculations of the percent weight distributions of right hind paws. The relative changes of the percent weight distributions of right hind paws were calculated and the mean of the
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relative changes was plotted on a graph. In order to acclimatize the rats to the Incapacitance Tester, affected rats in the HA/NS group were placed in the holder and immobilized for 5 s daily for 7 days before commencement of the injection protocol [11]. A statistical analysis was performed to examine the significance for the percent weight distribution value for the right hind paw after the fourth injections compared to the values at day 1 in order to assess a painrelieving effect of HMW HA.
Statistical analysis Statistical analysis was done using the Wilcoxon’s matched-pairs signed-rank test. A probability value of <0.05 was considered statistically significant.
Results General status and body weight
Histological examinations The knee tissue samples were obtained from 3 healthy normal rats and 5 tendinopathy rats at day 1 (controls), and from 5 rats in the HA, the NS, or the control groups, respectively, at day 28. Following inhalation with 5 % isoflurane, the rats were killed by immediate blood draw. Whole knee joints were fixed in 4 % paraformaldehyde at pH 7.4 for 3 days, and decalcified in 20 % EDTA solution for 21 days at 4 °C, and then embedded in paraffin wax. Whole knee joints were sectioned in the sagittal plane with a section width of 5 μm. The sections were stained with hematoxylin–eosin (H&E) for histology. They were assessed with a histopathological score determined by counting the number of microtears and laminations located at the insertion site of the patellar tendon as a point in high-power (magnification 200×) fields in each five sections of tendinopathy rats or HA, NS, or control groups. A point in the score was doubled in cases where the length of tears or laminations were longer than half the diameter of the visual high-power field (magnification 200×) on the microscope. The data values were expressed as the mean of the differences among groups after statistical analysis. The histological sections were also prepared by TdT-mediated dUTP nick end labeling using the ApopTag Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer’s instructions. Tendon cells for apoptosis were quantified by counting the number of TUNEL-positive cells in high-power (magnification 400×) fields at the joint side of the tibial insertion site for the patellar tendon in each of five sections of tendinopathy rats or HA, NS, or control groups. The data values were expressed as the mean of the differences among groups after statistical analysis.
There were no abnormalities observed in any of the groups during the experiment period. The change of body weight during the injection period is shown in Table 1. All groups showed a similar trend of body weight changes compared to the control group. Spontaneous locomotor activities The counts for the spontaneous locomotor activities in HA group increased along with the number of HA injections up to approximate 1.5-fold at day 13 after the fourth HMW HA injection and at day 17 after the fifth injection. Spontaneous motor activities in the HA group recovered up to the original level as before commencement of the running exercise. There was an increasing trend of the motor activities count the next day or 2 days after HA injection and then the counts decreased by approximate 10 % on day 3 after the injection. The counts of spontaneous locomotor activities in the HA group were maintained, not decreased, for 10 days after the fifth injection of hyaluronan until the end of the study at day 27 without any additional injections. There were significantly more counts for the spontaneous locomotor activities in the HA group than in the NS group or the control group during 27 days of the experimental period except at day 3 or 4 (p < 0.05). The counts of the locomotor activities in the NS group and the control group did not increased and remained in the range from 80 to 120 % of the initial activities. No significant differences were observed for the counts of locomotor activities between the NS group and the control group (Fig. 2).
Table 1 Body weight gain Group
n
Days after running exercise 0
7
14
21
28
Control HA
10 10
420 ± 19 422 ± 21
431 ± 21 433 ± 22
437 ± 23 439 ± 22
448 ± 25 450 ± 25
455 ± 26 458 ± 26
NS
10
421 ± 18
428 ± 20
436 ± 21
446 ± 24
453 ± 25
Body weight (g) was measured at indicated days. The data indicate the mean ± SD of ten rats. The HA group or the NS group was compared with the control group using unpaired t test
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Relative values for spontaneous locomotor activities (%)
190
180 *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
160
140
120
100
80
Control group NS group HA group
60
40 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Days Injections with hyaluronan (times) Fig. 2 Relative values for spontaneous locomotor activities in the HA, NS, or control groups. The spontaneous locomotor activities in rats were examined using a Supermex system at each night for 12 h, every day for 27 days, after completion of the running exercise and after commencement of the injection protocol. The relative number of
locomotor activities in the each group was calculated and the means or the SD were plotted on a graph. *Significant difference for the mean of the motor activities between the HA group and the NS or control groups (p < 0.05)
Percent weight distributions of right hind paws
(Table 2), indicating that the reactions of tendon repair in the HA group were progressed compared to those in the NS or control groups. The TUNEL-positive tendon cells were observed at the joint side of the tibial insertion site for the patellar tendon in all tendinopathy rats. The number of TUNEL-positive cells at the same area in five rats of the HA group was significantly lower than those in five rats of the NS group or the control group after the fifth injections of hyaluronan at day 28 (p < 0.05) (Table 3; Fig. 6).
The mean of the relative changes of weight bearing of right hind paws injected with HMW HA increased along with the times of injections for 21 days after the fifth injection (Fig. 3). After 21 days, the relative changes plateaued until the end of the study at day 27 without any additional injections. The relative changes after the fourth injections were significantly greater than those at day 1 (p < 0.05). Histological findings Two major pathological findings were found at the tibial insertion sites of the patellar tendons in tendinopathy rats in all the histological sections stained with hematoxylin– eosin. They were microtears in the tendon body and lamination, i.e., several longitudinal disruptions between the fiber bundle layers (Fig. 4). The same pathological findings were found at the tibial insertion sites of the patellar tendon at day 28 in the HA, NS, or control groups in the histological knee sections stained with hematoxylin–eosin (Fig. 5). However, the histopathological scores determined y the number and length of the microtears and laminations in five rats of the HA group were significantly lower than those in five rats of the NS or control groups (p < 0.05)
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Discussion Several different forms of the tendinopathy have been described; it has been shown that the anatomical location of a given lesion within the tendon plays some role in determining the type of tendinopathy. Chronic tendon abnormalities consistent with tendinopathy can be found in the main body of the tendon (called tendinitis or tendinosis), its insertion site on bone (the enthesis), and the structures surrounding the tendon. Insertional tendinopathy (enthesopathy or enthesiopathy) is one of the most common forms of tendinopathy and is commonly seen in the patellar tendon (jumper’s knee).
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Relative values for weight bearings of hind paws injected with hyaluronan
1.3 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Days Injections with hyaluronan (times) Fig. 3 Relative values for weight-bearing distribution of the hind paws injected with hyaluronan. The percent distribution of weight bearing in the right hind paws were measured with the Incapacitance Tester, which independently measured the weight bearing in each hind paw, at 15:00 hours every day for 27 days in the HA/NS group. The measurement was performed three times and the percent weight distribution of the right hind paws were calculated by the following formula: the percent weight distribution of the right hind
paw = [right weight/(right weight + left weight)] × 100. The value of percent weight distribution of the right hind paw per measurement was defined as the mean of three calculations. The relative changes of percent weight distribution of the right hind paws were calculated and the means or SD were plotted on a graph. *Significant difference for the mean of the relative values for weight-bearing distribution of the hind paws injected with hyaluronan after the fourth injections compared to those at day 1 (p < 0.05)
Fig. 4 Histological sections of the tibial insertion sites of the patellar tendons stained with H&E in a healthy rat (a) or in the tendinopathy rat (b) (×100). Two major pathological findings were found with light microscopy at the tibial insertion sites of the patellar tendons in
the tendinopathy rats: (1) microtears in the body of the tendon and (2) several longitudinal disruptions between the layers of the fiber bundles of the tendon body, so-called laminations
In the present study, we created an insertional tendinopathy model of the patellar tendon in rats and examined the therapeutic effects of HMW HA injections. The following findings were obtained: (1) the injections of HMW HA into the space between the patellar tendon and the fat pad tissue significantly increased the number of spontaneous locomotor activities in the tendinopathy rats proportionally to
the number of injections compared to those in the normal saline or control groups; (2) the spontaneous locomotor activities in the HA group after the fourth or fifth injections of HMW HA recovered up to the original healthy level and were then maintained, not decreased, for the following 10 days without any additional injections of hyaluronan; (3) the weight-bearing distributions of the hind paws
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Fig. 5 Histological sections of the tibial insertion sites of the patellar tendons stained with H&E in the HA group (a), in the NS group (b), or in the control group (c) (×100). The number of microtears or lami-
nations found in 5 rats of the HA group was lower than those in 5 rats of the NS or control group
Table 2 Histopathological score for the histological sections located at the tibial insertion site of the patellar tendon
injections; (4) the number of microtears or laminations observed in histological specimens at the tibial insertion site of the patellar tendon in tendinopathy rats decreased in the HA group compared to those in the NS or control group; (5) the number of TUNEL-positive cells at the joint side of the tibial insertion site of the patellar tendon in the HA group was lower than those in the NS or control groups after the fifth injections of hyaluronan at day 28. The present study shows that spontaneous locomotor activities in tendinopathy rats recovered up to a healthy level after five injections of hyaluronan and that the percent weight-bearing distribution in the hind paws injected with HMW HA also increased along with the number of hyaluronan injections. Moreover, the motor activities increased at 1 or 2 days after injections of HA, subsequently decreased after the following injections, and then gradually increased following five injections of hyaluronan. No remarkable tendinopathy-specific histological changes were observed in the rotator cuffs of the shoulders in tendinopathy rats. It has been suggested that HMW HA exerts a biological function of pain reduction by masking free nerve endings or organelles involved in nociception [12–14]. Therefore, our findings indicate that HMW HA injections into the space between the patellar tendon and the fat pad tissue are an effective analgesic method in tendinopathy rats. There was a report that the fluorescein-labeled HMW HA, of which the molecular size was
Group Tendinopathy Control NS HA
Histopathological score 17 ± 6 16 ± 5 15 ± 6 6±2
∗
∗
∗
* p < 0.05 Table 3 Cell number for apoptosis at the joint side of the tibial insertion site for the patellar tendon
Group Tendinopathy Control NS HA
Apoptosis cell 26 ± 11 28 ± 12 25 ± 11 7±3
∗
∗
∗
* p < 0.05
injected with HMW HA significantly increased along with the number of the injections and stabilized, after the fifth injection of hyaluronan for 10 days without any additional
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(a)
(b) cartilage
cartilage
tendon
(c)
tendon
(d)
cartilage
cartilage
tendon tendon
Fig. 6 Histological sections of the tibial insertion sites of the patellar tendons prepared by TdT-mediated dUTP nick end labeling in the tendinopathy rats (a), in the HA group (b), in the NS group (c), or in the control group (d) (×220). The number of TUNEL-positive ten-
don cells in the HA group was lower than those in the NS or control groups at the joint side of the tibial insertion site of the patellar tendon
the same as the one used in the present study, penetrated synovium from the synovial lining cell layer to the interstitium in subsynovial fat tissue (deep layer) at 3–24 h after injection into the joint cavity of the rabbit knee; and HMW HA remained in the synovial tissue for 3–7 days [15]. This indicates that injected HMW HA infiltrated into the tendon tissue at day 1 and masked free nerve endings or organelles, and that the analgesic effect gradually increased along with the hyaluronan accumulation following each consecutive injection of HMW HA, since accessibility of HMW HA to the tendon might be similar to the one into the synovium. Consequently, our findings support the proposed mechanism of pain relief by HMW HA injection, i.e., masking free nerve endings or organelles involved in nociceptive stimulus production and peripheral sensitization. The present study also shows that spontaneous locomotor activity in the HA group recovered up to the original healthy level after the fourth or fifth injection of HA and was then maintained, not decreased, for 10 days after the last, fifth injection of HA. This finding suggests that pain in the HA group almost disappeared, and the pain-free conditions were maintained at least for 10 days after the fifth injections of hyaluronan. From these data, we think that the consecutive 4 or 5 injections of HMW HA are suitable for relief of pain caused by tendinopathy.
The space between the patellar tendon and the fat pad tissue was chosen for injection of HMW HA because pooling of the HMW HA molecules close to the patellar tendon without diffusion or leakage into the joint cavity was necessary, and to expose biologically the patellar tendon tissue containing cells, nerves, and vessels to HMW HA. Such a space suitable for the storage of hyaluronan molecules in the patellar tendon body was not available. Moreover, there was a possibility that the injected HA expands the tendon tear or worsens lamination if injected directly into the tendon body, since the viscosity of HMW HA is very high compared to that of the normal saline. In order to avoid exposure of HMW HA to the synovium or cartilage in the articular space, it was necessary to block any possible leakage of hyaluronan into the joint cavity. A suitable volume of HMW HA for injection without producing leakage was decided by injecting the dye mixed with hyaluronan into the space between the patellar tendon and the fat pad tissue. In addition, there was a report that the joint side of the tendon may be a main site of lesion in tendinopathy [16]; this finding was also taken into consideration when choosing the injection site in the present study. Apoptosis of tendon cells was examined at the joint side of the patellar tendon at the tibial insertion site in tendinopathy rats by TdT-mediated dUTP nick end
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labeling. On setting the experiment, we presumed that tendon cell apoptosis is induced by repetitive mechanical stress against the insertion site of the patellar tendon or by the oxidative stress at the insertion site of the tendon by the daily running exercise. In fact, the number of apoptotic cells located at the joint side of the tibial insertion site of the patellar tendon in the HA group was lower than those in the NS or control groups after 5 injections of hyaluronan. This finding indicated that injected HMW HA suppressed occurrence of apoptosis, and it was in agreement with some reports that HMW HA exerts a biological function of suppressing apoptosis or oxidative stress that eventually leads to apoptosis [17–19]. In addition, apoptosis suppression might correlate with pain-relief in tendinopathy, since enzymes breaking the extra-cellular matrix are usually released from apoptotic cells and broken or damaged tissues around those cells acting as nociceptive stimuli. It has been reported that HMW HA stimulates chemotaxis and proliferation of fibroblasts [20–22], or promotes fibroblast-induced matrix biosynthesis [23–25]. Migration and proliferation of fibroblasts and promotion of matrix biosynthesis generally accelerate the repair reaction of damaged tissues. Moreover, it has been known that HMW HA inhibits production of matrix metalloproteinases, which induce matrix degradation and release of cytokines from fibroblasts [26–28]. These effects participate in the structure modifying effect. It was observed that the histopathological score determined by the number and length of microtears and laminations in the HA group was lower than those in the NS or control groups. This indicated that the repair reactions in the HA group were initiated by HMW HA injection and progressed; they most probably were either not initiated or stay dormant in the NS and control groups. It was, therefore, speculated that injected HMW HA accelerated tendon repair at the insertion site by exerting its biological functions.
Conclusion Five injections of HMW HA into the space between the patellar tendon and fat pad effectively relieved pain and initiated partial restoration of the patellar tendon in the tendinopathy rat model. The initiated changes were supported by histopathological findings, suggesting that several injections of HMW HA should become a clinically effective modality in treatment of tendinopathy. Acknowledgments We are grateful to all members of the Department of Orthopaedic Surgery of the Jikei University School of Medicine for the partial support of the present study, and appreciate Chugai Pharmaceutical Co., Ltd. for their financial support.
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