Shock wave therapy induces neovascularization at the tendon-bone junction A study in rabbits

exonAbstract

Despite the success in clinical application, the exact mechanism of shock wave therapy remains unknown. We hypothesized that shock wave therapy induces the ingrowth of neovascularization and improves blood supply to the tissues. The purpose of this study was to investigate the effect of shock wave therapy on neovascularization at the  tendon-bone junction. Fifty New  Zealand white rabbits with  body  weight ranging from 2.5 to 3.5 kg were used in  this study. The right limb (the study side) received shock  wave therapy to the Achilles tendon near the insertion to bone. The left limb (the control side) received no shock wave therapy. Biopsies of the tendon-bone junction were performed in 0, 1,4, 8 and 12 weeks. The number of neo-vessels was examined microscopically with heniatoxylin-eosin stain.  Neovascularization was  confirmed by the angiogenic markers including vessel endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expressions and endothelial cell proliferation determined by proliferating cell nuclear antigen (PCNA) expression examined microscopically with  immunohistochemical  stains. The results showed that shock wave therapy produced a  significantly higher number of  neo-vessels and angiogenesis-related markers including eNOS, VEGF and PCNA than the control without  shock wave treatment. The eNOS and VEGF began to rise in as early as one week and remained high for 8 weeks, then declined at 12 weeks; whereas the increases of PCNA and neo-vessels began at 4 weeks and persisted for 12 weeks. In conclusion, shock  wave therapy induces the ingrowth of neovascularization associated with early release of angiogenesis- related markers at the Achilles tendon-bone junction in rabbits. The neovascularization may play a  role to improve blood  supply and tissue regeneration at the tendon-bone junction.

Introduction

In clinical application, extracorporeal shock wave therapy has shown effect in the treatment of certain orthopedic   conditions including   non-union   of   long bone fracture [21,30,32,37,39], calcifying tendonitis of the   shoulder [15,27,3 1,33,42], lateral  epicondylitis of the   elbow [ 10,13,24,28,34], proximal plantar fasciitis [6,19,20, 25,291 and Achilles tendonitis [24]. The success of shock wave therapy in our experiences  ranged from 80'%1 for non-unions of long bone fractures [39] to 90'% for tendinopathies of the  shoulder, elbow and heel [6,13,42]. In patients with  calcifying tendonitis of the shoulder, radiographs showed that 58% of the  calcium deposits were  completely eliminated after shock wave therapy [42]. In addition, the short-term results of shock wave therapy for avascular necrosis of the femoral head appeared  encouraging [16]. Shock wave therapy  also showed  a positive effect in promoting  bone healing in animal  experiments.
Despite  the success in clinical application, the exact mechanism of shock wave therapy remains unknown. In fracture  non-unions, it  was  speculated that shock wave therapy  produces micro-fracture which  in turn causes hematoma  formation and subsequent callus formation and eventual fracture healing [7,11,14,22]. For tendino- pathy, it was postulated that shock wave therapy provokes  painful level of stimulation, and relieves pain due to tendinopathy at the tendon-bone junction by hyper-stimulation     analgesia [14,17,22,23,26,28,33,34, 371. However,  there were  insufficient data  to support either theory. The results  of our experiments in dogs demonstrated that shock wave therapy  enhanced neo- vascularization at the tendon-bone junction [41]. Neo- vascularization was reported to be in association with painful  tendinosis and may be responsible for pain  in chronic Achilles tendinosis [3,25]. However, we specu- lated that  the reasons for  the dissolution of calcium deposits after shock wave therapy in  calcifying tendo- nitis of the  shoulder may involve a  molecular  mecha- nism of absorption  due to improved  blood  supply. We further hypothesized that shock wave therapy may  have the potential to induce the  ingrowth of neovasculariza- tion and improvement of blood  supply that lead to tis- sue  regeneration. The purpose of this study was to investigate the effect of shock wave therapy on neovas- cularization at the tendon-bone junction of the Achilles tendons in rabbits.

Materials and methods

animals and shock wave treatment
This  study was approved by the  Institutional Review Board, and was performed under the guidelines and  care of animals in research. Fifty New Zealand white rabbits with body weight ranging from 2.5 to 3.5 kg  were  used  in this  study. The right limb received shock wave therapy to the Achilles tendon near the insertion site to the heel bone, and was designated as the study side. The left limb received no shock wave therapy, and was designated as the control side. The rabbit was anesthetized with ketamine (50 mg/kg) and phe- nobarbital (30 mglkg) for the purpose of receiving shock wave appli- cation. The rabbit was placed in prone position with the right heel up. The shock wave tube was  focused on the Achilles tendon  near  the insertion  site to the heel bone, and the depth of the treatment was determined by the  control guide and confirmed  with C-arm image. Surgical lubricate was applied to the area of skin in contact with the shock wave tube. Each of the  study limb received 500 impulses of shock waves at 14 kV (equivalent to 0.12 mJ1mm') in 20 min to the right Achilles tendon near the insertion site. The shock wave dosage so selected was  based on the experience of our previous  animal  studies [40.41,44]. Immediately after  shock wave therapy, the right limb  was checked for swelling, edema, hemorrhage and motion of the limb. No special protection on  the right limb or restriction of activities was provided. The rabbit was returned to the housing cage and was cared for by a  veterinarian.

Histomorpholigical
assessment Biopsies of the Achilles tendon-bone   units were performed in 0, I, 4. 8 and 12 weeks with 10 rabbits at each time interval. The first biopsy was obtained in 24 h  after  shock wave therapy. The specimens were fixed in 4'%1 buffered paraformaldehyde for 48 h and decalcified in PBS- buffered I 0'%, EDTA. Decalcified specimens were longitudinally cut into 5-pm sections and  transferred to poly-lysine-coated  slides. The number of blood vessels including capillaries and inusculariml vessels were examined microscopically, and quantitatively assessed in six areas of tendon-bone  junction  from  three sections of the biopsy  specimens. A professional pathologist blinded to the treatment regimen performed the measurements on all sections under 40x magnification

Immunohistochemistry
For the purpose of confirming neovascularization.  the angiogenic markers including vessel endothelial growth factor (VEGF) and en- dothelial nitric oxide synthase (eNOS) were examined  microscopically with imniunohistochemistry  stains to reflect the  mediators  for neo- vascularization [5,35]. Detection of proliferating cell antigen (PCNA) was  chosen to reflect endothelial cell replication [ 191. Immuno-reac- tivity  in specimens was demonstrated using horseradish peroxidase (HRP)-3'-, 3'-diaminobenzidine (DAB) cell and tissue staining kil (R&D Systems. Inc.  Minneapolis. MN, USA) in accordance with manufacturer's   instructions.  After blocking endogenous peroxidase and non-specific binding, sections were incubated  overnight with the antibodies at 4 "C. Polyclonal antibodies used for  immunohisto- chemistry   were anti-eNOS, anti-VEGF  and anti-PCNA  (Upstate Biotechnology. Lake Placid, NY, USA). Sections were further incu- bated with  biotinylated anti-goat IgG and then  incubated with strep- tavidin conjugated to HRP. Immuno-reactivity was determined by incubating the sections in a  chromogen  solution containing DAB and 0.1% hydrogen peroxide in the dark, followed by counterstaining with hematoxylin. Dehydrate sections were mounted with mounting me- dium. Those without  primary  antibodies were enrolled as negative controls for the  immunostaining. Vessels showing positive VEGF ex- pression and cells displaying positive PCNA and  eNOS expressions were counted microscopically. The numbers with  positive  expression were quantitatively assessed, and the averages were calculated from six areas of tendon-bone   junction selected from  three  sections of each biopsy  specimen.

statistrical analysis
Data were  presented as mean k standard  error. The numbers of vessels and cells  dispbaying positive angiogenic markers at each  time interval between the study side and  the control side were compared statistically  using  Mann-Whitney   test  with  statistical significance at p < 0.05.

Results

Increase of' neo-vessels after shock wave treutment
The results of neo-vessels of the study and the control sides are summarized in Table 1. In the  study side, a significant increase of  neo-vessels  was noted in 4 weeks, and the increase in  neo-vessels persisted  until 12 weeks, whereas no increase of  neo-vessels was  noted in the control side, and the difference  in the number of  neo- vessels was statistically   significant. The microscopic features of neo-vessels of the study and the control sides are shown in Fig. I. It  appeared that formation  of neo- vessels after shock wave therapy was time dependent starting in 4  weeks after  treatment and it  lasted for 12 weeks or longer.

Increase of ungiogenic markers ujter shock wave therapy
The results of angiogenesis-related markers including eNOS, VEGF and PCNA are summarized in Table 2. In the study side, a significant  increase in eNOS, VEGF and PCNA was noted in as  early  as one week. The in- crease of eNOS and VEGF lasted for 8 weeks before they declined to  normal at 12  weeks, whereas  the in- crease of PCNA lasted until 12  weeks. In the  control side, however, no significant changes were noted in eNOS, VEGF and PCNA, and the differences between the  study and  control sides are statistically  significant. The microscopic features of eNOS, VEGF and PCNA expressions of the study and the control sides are shown in Fig. 2. The changes in eNOS, VEGF  and PCNA expressions at different time intervals are graphically illustrated in Fig. 3. The results  of this  study demon- strated that shock wave therapy induces  early  expres- sions of eNOS, VEGF and PCNA in one week, and subsequent  ingrowth of neo-vessels  in approximately 4 weeks. The increases of eNOS and VEGF were transient and only  lasted for 8 weeks; whereas the stimulation of cell proliferation and the ingrowth of new  vessels per- sisted for 12 weeks and longer. The biological  responses after shock wave therapy appear to be time-dependent.

 Discussion

Many authors had studied the mechanism of shock wave therapy [7-9,11,12,14,17,18,22-24,26,28,33,34,36, 38,40,41,4346]. Some   speculated that shock wave therapy relieves pain due to insertional tendinopathy by hyper-stimulation   analgesia [14,22-24,28,37], while others hypothesized the theoretical  mechanism of shock wave therapy in bone healing  including micro-disrup- tion   of avascular or minimally vascular tissue to encourage  revascularization and the recruitment of ap- propriate stem cells conductive to more  normal  bone tissue healing [ 1 I, 12,14,18,22,4346]. However, there are insufficient data to scientifically substantiate either theory concerning the mechanism of shock wave therapy in musculoskeletal disorders. The etiologies   of tendonitis are multi-factorial in- cluding   degenerative   changes, inflammatory process and metabolic disturbance with  hypo-vascularity,  neu- ronal origin as well as neovascularization  in  tendinosis [14,24,25]. Neovascularization    was found to be in close relation to the area of tendinosis that corre- sponded to the painful area  during Achilles loading. Therefore,  neovascularization, rather than a  “healing response”,  was thought to be responsible for the pain in the area with chronic Achilles tendinosis [3,25]. On the contrary, neovascularization,   a  direct effect of angio- genic factors secondary to extracorporeal shock wave therapy was found to be  responsible for improvement in symptoms of plantar fasciitis  [33,34].  In  calcifying tendonitis of the  shoulder, shock wave therapy   not only  provides   pain relief,   it also eliminates  calcium deposits [15,26,27,42]. The mechanical  forces of shock wave therapy may  theoretically  cause fragmentation, but   not total absorption of the calcium   deposits. Therefore, it is reasonable to assume that a biological process through a  molecular  mechanism of absorption may take place to dissolve the calcium deposits  after shock wave therapy.  Nitric oxide and VEGF had been demonstrated as  important mediators of  angiogenesis [5,35]. We suggest that physical shock waves could raise the  mechanotransduction of the tissue to convert acoustic  shock wave  energy into biological  signals. The results of this study showed that shock wave therapy induces  the ingrowth  of neo-vessels and tissue prolif- eration associated with the early release of angiogene- sis-related factors including eNOS and VEGF at the tendon-bone junction in rabbits.  Therefore,  the mech- anism of shock wave therapy appears to involve the early release of angiogenic growth  factors in one week, and induces cell proliferations  and  formation  of neo- vessels in approximately   four weeks at the tendon- bone junction. The neovascularization  may lead to the improvement of blood supply and play  a role  in  tissue regeneration at the tendon-bone  junction. No adverse effect of neovascularization on rabbits was observed during the course  of this study. In non-unions  of  long  bone  fractures, the fracture sites are generally filled with dense,   hypo-vascular fibrous tissues that prevent  the  fractures  from bony union. Many studies in animal experiments had dem- onstrated that shock wave therapy can promote  bone marrow  stromal cell differentiation toward osteopro- genitors  associated  with induction of TGF-P1 and in- duces membrane  hyperpolarization and Ras activation to act as an early  signal for  the osteogenesis in human bone  marrow   stromal cells [36,4346]. Other studies also showed a  positive effect of shock wave therapy in promoting   bone healing in animal experiments [8,11,12,18]. The results of this study showed that shock wave therapy promotes early release of angio- genic factors, and subsequently induces cell prolifera- tions and ingrowth of neo-vessels that in turn may stimulate the stromal cell growth and differentiation and  promote bone healing. In  conclusion, shock wave therapy induces the in- growth of neovascularization and cell proliferation as- sociated with earlier release of angiogenic growth factors at the tendon-bone junction of the Achilles tendon in rabbits. It appears that the  mechanism of shock wave therapy involves the early release  of angiogenic growth factors (eNOS and VEGF) aiid subsequent  induction of neovascularization and tissue proliferation. The neo- vascularization may play  a role in pain relief of  tendi- nitis and   the repair of chronically inflamed tendon tissues at the tendon-bone junction.

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