This work aimed to investigate the synergetic therapeutic effects of polydeoxyribonucleotides (PDRN) combined with extracorporeal shock waves therapy (ESWT) and the effects of the therapy according to ESWT sequences on a chronic traumatic full-thickness rotator cuff tear (RCT) in rabbit models. For this purpose, thirty-two rabbits were randomly allocated into 4 groups. An excision was made to create a 5-mm sized full-thickness RCT right proximal to the insertion site on the supraspinatus. After 6 weeks, 4 different procedures (normal saline, Group 1; PDRN injection, Group 2; PDRN injection before ESWT, Group 3; PDRN injection after ESWT, Group 4) were performed. PDRN injection and radial type ESWT were each performed 4 times per week. Gross morphology, Immunohistochemistry, and motion analysis were performed at 4 weeks after treatments. All parameters including tear size, Masson’s trichrome (MT) staining, anti-collagen 1 monoclonal antibody immunostaining (COL-1), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), anti-platelet endothelial cell adhesion molecule-1 polyclonal antibody (PECAM-1), and motion analysis, were significantly greater in Group 2, Group 3, and Group 4 than in Group 1. In Group 4, all parameters were significantly greater than in Group 2, and parameters including MT staining, COL-1, PCNA, PECAM-1, and fast walking time were significantly greater than Group 3. There were no significant differences between Group 2 and Group 3. This study demonstrated that PDRN injection alone and combined with ESWT were more effective than control treatment and applying ESWT before PDRN injection was also shown to yield better outcomes in parameters including angiogenesis, cell proliferation, and fast walking time in a rabbit model with chronic traumatic full-thickness RCT. Therefore, the application of ESWT before PDRN injection could be recommended for optimal outcomes of the conservative treatments when the patients with full-thickness RCT are not suitable in surgical treatment.
A rotator cuff tear (RCT) is a pathologic condition that causes weakness, pain, and disability in shoulder joints, which may result in a considerable decrease in quality of life (
Chronic tendon degeneration found after surgical treatment is considered a major pathologic cause of these adverse events (
Biological adjuvants, which contribute to the microenvironment of regeneration, have great potential to improve healing rates and the function of injured rotator cuff tendons (
Extracorporeal shock wave therapy (ESWT) is a non-invasive therapeutic method in which a specific target area is treated with a series of single sound wave pulses generated by a proper generator (
A total of 32 skeletally mature male New Zealand white rabbits (twelve-week-old) were used. The rabbits were separately housed in metal cages with a temperature of 24 ± 2°C, and a humidity of 45 ± 10%. The rabbits were fed with a commercial rabbit diet and provided with free access to tap water. All rabbits were allowed to do daily activities in a cage (65 cm × 45 cm × 30 cm). None of these animals were put on additional exercise. Experiments that include animals were conducted according to internationally accredited guidelines. This study is approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Daegu School of Medicine. Isoflurane (Forane; JW Pharmaceutical) vaporized in oxygen was used to induce general anesthesia, and the rabbits were delivered using a large animal cycling system. With punch biopsy (SFM, Wächtersbach, Germany), 5 mm × 5 mm sized full-thickness RCT was created proximal to the insertion site of the right supraspinatus tendon under general anesthesia. In order to induce chronic full-thickness RCT, the excision wound was immediately closed with a non-absorbable round-shaped silicone Penrose drainage tube (Sewoon Medical Co., Ltd., Cheonan-si, Korea) (
At 6 weeks after the excision procedure, inserted silicone tubes were removed to induce chronic full-thickness supraspinatus tendon tear. After all full-thickness supraspinatus tendon tear was confirmed, the incision site was sutured with nylon monofilament. A total of 32 rabbits were randomly allocated to four treatment groups (n = 8 per group). Group 1 (G1-SAL) was injected with 0.2 mL of normal saline into full-thickness RCT, while group 2 (G2-PDRN) with commercially obtained 0.2 mL of PDRN (Placentex Inj.; Mastelli SRL, San Remo, Italy;
Rabbits were euthanized under general anesthesia. The tear area of the supraspinatus tendon was segmented and then, fixed with neutral-buffered formalin solution for 24 h. The specimens were embedded in paraffin (Paraplast, Oxford, St. Louis, Missouri, USA) and cut sagittally into 5 μm-thick serial sections. These specimens were stained with Masson’s trichrome (MT) and examined by light microscopy.
Immunohistochemical staining of serial tendon sections was performed to observe collagen fibers using mouse anti-collagen 1 monoclonal antibody (COL-1; Abcam, Cambridge, UK). The tendon sections were stained on proliferating cells using mouse anti-proliferating cell nuclear antigen monoclonal antibody (PCNA, PC10; Santa Cruz Biotechnology, CA, USA), with angiogenetic markers of anti-vascular endothelial growth factor polyclonal antibody (VEGF, A-20; Santa Cruz Biotechnology) and anti-platelet endothelial cell adhesion molecule-1 polyclonal antibody (PECAM-1, M-20; Santa Cruz Biotechnology). Then the paraffin-embedded sections were cleared, dehydrated, and washed using phosphate-buffered saline (PBS). Antigen was retrieved with citrate buffer (pH 6.0) at 95°C for 30 min and then was followed by cooling. Pre-incubation for preventing endogenous peroxidases was conducted with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min. These sections were blocked for non-specific protein binding for 30 min using PBS with 10% normal horse serum or normal goat serum or normal rabbit serum (Vector Laboratories), incubated for 2 h with primary antibodies (1:100–1:200) at room temperature, and washed three times with PBS. The sections were incubated at room temperature for one hour with secondary antibody (1:100), biotinylated anti-mouse IgG, anti-rabbit IgG or anti-goat IgG (Vector Laboratories). Washed with PBS three times, the sections were put on exposure to avidin-biotin-peroxidase complex (ABC, Vector Laboratories) for 1 h and washed three times with PBS. 0.05 M Tris-HCl (pH 7.6) containing 0.01% H2O2 and 0.05% 3,3’-diaminobenzidine (DAB; Sigma-Aldrich) was used to induce peroxidase reaction on the sections, which were then counterstained with hematoxylin and mounted. An Axiophot Photomicroscope (Carl Zeiss, Germany) equipped with an AxioCam MRc5 (Carl Zeiss, Germany) was used for examining the slides, which were respectively evaluated according to its intensity of positive immunostaining.
(a) PDRN. (b) Gross photograph of ultrasound-guided injection. (c) The injection was made under ultrasound guidance to full-thickness tendon tears in the right shoulder supraspinatus. Longitudinal images showing the needle (white arrow) in the right supraspinatus tendon of the rabbit. (d) PDRN injected into right full-thickness supraspinatus tendon. Abbreviation: PDRN, polydeoxyribonucleotides.
Normal saline group (0.2 mL; G1-SAL), PDRN group (0.2 mL; G2-PDRN), ESWT after PDRN injection group (0.2 mL; G3-PDRN+ESWT), and ESWT before PDRN injection group (0.2 mL; G4-ESWT+PDRN). The injection was performed into the right supraspinatus full-thickness tears after the lesions were created. Gross morphologic evaluation of tear site, histologic examination, and motion analysis were conducted at 4 weeks after treatments. All rabbits were euthanized using carbon monoxide inhalation at 4 weeks after treatment of each solution. Abbreviations: G1-SAL, normal saline group; G2-PDRN, PDRN group; G3-PDRN+ESWT, ESWT after PDRN injection group; G4-ESWT+PDRN, ESWT before PDRN injection group; SAL, normal saline; PDRN, polydeoxyribonucleotides; ESWT, extracorporeal shockwave therapy.
Gross morphologic examinations were performed after euthanizing each rabbit. Each supraspinatus tendon tear was classified as completely healed, partial-thickness, and full-thickness. Gross morphologic supraspinatus tendon tears were photographed with a solid ruler near the center of the tear site. The size of tendon tears was calculated by tracing the outlined tear edge at pre-treatment and 4 weeks after the treatment using Image J macro software (National Institutes of Health, Bethesda, MD).
AxioCam MRc5 interfaced with Axiophot Photomicroscope (Carl Zeiss, Germany) was used to photograph thirty fields randomly selected in each group. AxioVision SE64 (Carl Zeiss, Germany) program was used for immunohistochemical analysis. A semi-quantitative scoring system was used for cytoplasmic or nuclear markers PCNA, VEGF, and PECAM-1, with consideration of the staining extent and intensity of the area, which is a previously accepted method used in recent studies (
Motion analysis of the rabbits was performed at pre-treatment and 4 weeks after the treatment. Before the analysis, the rabbits were habituated to the open field for more than 30 min. They were placed on a 3 × 3 m-sized flat-file and allowed to freely move around for 5 min. Movements of each animal were assessed with a video-tracking system equipped with a camera (SMART 3.0; Panlab, Barcelona, Spain) recording two-dimensional activities of the animal. Three parameters, fast walking time, five-minute walking distance, and mean walking speed, were measured. This assessment had been used in previous studies to evaluate functional outcomes after treatment of RCT (
Statistical analysis was performed using SPSS for Windows program, version 25.0 (SPSS Inc., Chicago, IL, USA). As well as standard descriptive statistical measures (means and standard deviation), analysis of variance (ANOVA) were used to determine statistical differences among inter- and intra-groups. When ANOVA has shown significant differences between the groups, Tukey’s post-hoc test and Mann–Whitney’s
In gross morphologic evaluation, full-thickness tear was observed in eight (100%) rabbits among G1-SAL. In G2-PDRN, full thickness tear was found in five (62.5%) rabbits, while partial-thickness tear in three (37.5%) at 4 weeks after treatment. In G3- PDRN+ ESWT, full thickness tear was found in four (50.0%) rabbits, while partial-thickness tear in four (50.0%). In G4-ESWT+PDRN, full thickness tear was found in three (37.5%) rabbits, while partial-thickness tear in five (62.5%). Significant differences in gross morphologic changes after the treatment were found in all groups except G1-SAL when compared with baseline evaluation (
In MT staining assessed with light microscopy, no signs of inflammation were found in all groups. Newly regenerated collagen fibers were observed in MT staining, and these regenerated tendon fibers were stained with COL-1 in G2-PDRN, G3-PDRN+ESWT, and G4-ESWT+PDRN (
Groups (Injection regimens) | ||||
---|---|---|---|---|
Tear size | 13.79 ± 1.38 | 12.42 ± 1.63* | 11.71 ± 1.24† | 10.60 + 1.54‡∥ |
MTS | 0.33 ± 0.47 | 2.73 ± 0.14* | 2.83 ± 0.16† | 3.23 ± 0.10‡∥¶ |
Anti-collagen type 1 | 0.4 ± 0.62 | 2.58 ± 0.14* | 2.63 ± 0.11† | 2.98 ± 0.12‡∥¶ |
PCNA | 1.25 ± 1.05 | 2.53 ± 0.95* | 2.53 ± 0.08† | 2.88 ± 0.10‡∥¶ |
VEGF | 1.40 ± 0.91 | 2.06 ± 0.81* | 2.13 ± 0.12† | 2.44 ± 0.10‡ |
PECAM-1 | 1.71 ± 0.85 | 2.24 ± 0.70* | 2.29 ± 0.12† | 2.64 ± 0.10‡∥¶ |
Walking distance (cm) | 4852.75 ± 137.27 | 5514.38 ± 257.25* | 5779.00 ± 301.90† | 5994.38 ± 239.00‡∥ |
Fast walking time (%) | 5.62 ± 1.42 | 7.97 ± 0.82* | 7.87 ± 0.74† | 9.22 ± 0.69‡∥¶ |
Mean walking speed (cm/s) | 6.3 ± 0.57 | 8.21 ± 0.58* | 9.35 ± 1.27† | 10.76 ± 1.54‡∥ |
Values expressed as mean ± standard deviation. The intensity of Masson’s trichrome staining or anti-collagen type-1 immunostaining was classified as 0–3, indicating 0 = negative staining, 1 = slight positive staining, 2 = moderately positive staining, and 3 = strongly positive staining. The proportion of positive cells PCNA, VEGF, PECAM-1 was scored as 0 (no cells stained positive), 1 (between 1% and 10%), 2 (between 11% and 33%), 3 (between 34% and 66%), and 4 (between 67% and 100%). *
In motion analysis, three parameters, walking distance, fast walking time, and mean walking speed were found to be significantly greater in G2-PDRN, G3-PDRN+ESWT, and G4-ESWT+PDRN than in G1-SAL. There were no significant differences in walking distance, fast walking time, and mean walking speed between G2-PDRN and G3-PDRN+ESWT. In motion analysis, walking distance, fast walking time, and mean walking speed were found to be greater in G4-ESWT+PDRN than in the G2-PDRN and G3-PDRN+ESWT (
In this study, local injection of PDRN alone or combined with ESWT application was found to be more effective than normal saline administration in the regeneration of rotator cuff tendon in the rabbit model. In terms of ESWT sequences, it is also shown that the ESWT application may yield better outcomes in several parameters when performed before PDRN injection than later. Therefore, it is suggested that local injection of PDRN alone or combined with ESWT application could improve the microenvironment for regenerative processes of full-thickness RCT without any surgical repair.
PDRN, a mixture of deoxyribonucleic acid polymers, may activate wound healing by stimulating adenosine A2 receptor and thereby enhancing the production of VEGF and angiogenesis (
In this study, 0.2 mL of PDRN was injected into the right full-thickness RCT under US guidance in a rabbit model. There was no established optimal dose of PDRN required for rotator cuff tendon regeneration. In previous clinical studies, 1.5 mL of PDRN was injected three times per week in the patients with plantar fasciitis, while 3 mL of PDRN in the patient with rotator cuff tendinopathy (
We used radial type ESWT to evaluate the synergetic regenerative effects of PDRN. Compared to the commonly used focused type of ESWT, radial type ESWT could be characterized with larger therapeutic areas and simplified applications with the reflection of pathologic zones rather than a point because full-thickness RCT is superficial and larger areas than focal tear (
In our study, we decided to set the energy level of radial type ESWT to 2.5 bars and 2000 pulses, which is, according to a previous study, equivalent to 0.12 mJ/mm2, when converted into Joules (
With ESWT being applied therapeutically for the musculoskeletal system, successful therapeutic effects have been reported in various disorders such as lateral epicondylitis, plantar fasciitis, patellar tendinitis, and calcific tendinitis of the shoulder (
The exact mechanism of the combined effects of ESWT and PDRN still remains unclear. The combination of ESWT and PDRN could be possibly advantageous in that the ESWT hits the tissue and forms cavitation with conflicting pressures (
There are several limitations in this study. First, we created full-thickness RCT just proximal to the insertion site on the supraspinatus tendon, but enthesis healing was not evaluated. Second, the follow-up duration after the treatment was limited to only 4 weeks. Tendon regeneration was identified, but complete healing was not observed. If the observation period were longer than four weeks, more tendon healing or decrement of tear size could have been found. Therefore, further studies on long-term treatment effects are required. Third, motion analysis was used to evaluate rotator cuff function after treatments instead of biomechanical testing. Although motion analysis has not been proven to be better in evaluation than biomechanical testing, it may provide important information distinct from those of histologic examination regarding the therapeutic effect on RCT in both human and animal studies (
In conclusion, PDRN injection alone and application of ESWT combined with PDRN injection were found to be effective in the treatment of rabbit model with chronic traumatic full-thickness RCT. Moreover, applying ESWT before PDRN injection has shown significant improvements in parameters indicating angiogenesis, cell proliferation, and fast walking time when compared with ESWT application after PDRN injection. Therefore, the application of ESWT before PDRN injection could be recommended for optimal outcomes of the conservative treatments when the patients with full-thickness RCT are not suitable in surgical treatment.