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Extracorporeal Shock Wave Stimulates Angiogenesis and Collagen Production in Facial Soft Tissue
j o u r n a l o f s u r g i c a l r e s e a r c h j a n u a r y 2 0 2 0 ( 2 4 5 ) 4 8 3 e4 9 1
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Extracorporeal Shock Wave Stimulates Angiogenesis and Collagen Production in Facial Soft Tissue Abdulmonem Alshihri, DDS,a,b,c,*,1 Peer W. Ka¨mmerer, MD, DDS, PhD, MA, FEBOMFS,c,d,e,1 Diana Heimes, MD,e Wanting Niu, BEng, PhD,c,d Talal Alnassar, BDS, MDS, FRCD(c), CDT,a and Myron Spector, BS, MS, PhDc,d a
College of Dentistry, King Saud University, Riyadh, Kingdom of Saudi Arabia Harvard School of Dental Medicine, Boston, Massachusetts c VA Boston Healthcare System, Boston, Massachusetts d Department of Orthopedics, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts e Department of Oral, Maxillofacial and Plastic Surgery, University Medical Centre Mainz, Johannes Gutenberg University Mainz, Mainz, Germany b
article info
abstract
Article history:
Background: This study investigated the efficacy of extracorporeal shock wave (ESW)
Received 7 January 2019
application in stimulating dermal thickness, vascularity, and collagen synthesis of facial
Received in revised form
skin in a large animal model.
5 May 2019
Materials and methods: The facial skin of the maxillary and mandibular areas of goats (n ¼ 6
Accepted 19 June 2019
per group) was treated with ESWs of different intensities (0.15 and 0.45 mJ/mm2; 1000
Available online xxx
pulses). After 4 d, histology and immunohistochemistry were used to evaluate the following: dermal thickness, total number and abundance of microvessels, amount of type
Keywords:
1 collagen, and a-smooth muscle actin expression.
Extracorporeal shock wave
Results: Dermal thickness, number and abundance of microvessels, and collagen synthesis
Skin
increased after ESW application at both intensities (each P < 0.05). When comparing ESW
Angiogenesis
groups, the highest collagen abundance was seen after 0.15 mJ/mm2 (P ¼ 0.034), whereas the
Dermal thickness
highest number of microvessels was detected after treatment with 0.45 mJ/mm2 (P ¼ 0.002).
Flap surgery
Conclusions: A single-session application of focused low-energy ESWs to facial skin can increase dermal thickness by stimulating collagen production and local microcirculation. These findings commend the technique for future investigation for pretreatment of local or microvascular skin flaps to enhance tissue healing. ª 2019 Published by Elsevier Inc.
Introduction Perfusion of skin and mucosal flaps is critical in facial and oral surgery, and ischemic necrosis of surgical site is therefore a
major concern. Both macro- and micro-circulation are needed to ensure the viability of flaps. Extracorporeal shock waves (ESWs) are biphasic pressure changes of short duration consisting of a high-magnitude positive wave (40 MPa
* Corresponding author. College of Dentistry, King Saud University, P.O Box 60169, Riyadh 11545, Kingdom of Saudi Arabia. Tel.: þ966 114677325; fax: (þ966) (1) 467-9017. E-mail address: [email protected] (A. Alshihri). 1 These authors contributed equally to this article. 0022-4804/$ e see front matter ª 2019 Published by Elsevier Inc. https://doi.org/10.1016/j.jss.2019.06.077
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compressive phase; w1 ms duration) followed by a lower magnitude negative wave (10 MPa tensile phase; w3 ms duration) wave. This acoustic force is transformed into mechanical energy as it enters the tissue; the biological effect is directly proportional to the difference in impedance of the two adjacent media.1 ESW has been shown to stimulate tissue repair mainly by inducing the collapse of ultrastructural vesicles through negative pressure and causing asymmetrical fluid streams within the tissue.2,3 Mechanical impact is presumed to exert similar beneficial effects, for example, in human osteoblasts,4,5 although the underlying mechanism is poorly understood. Clinical evidence suggests that ESW improves local tissue perfusion in cases of ischemic limb disease6; it was also shown to enhance regional myocardial blood flow in a porcine cardiac ischemia model7 as well as myocardial perfusion and coronary angiogenesis in humans with coronary artery disease.8,9 In critically perfused skin flaps, ESW decreases leukocyte infiltration and tumor necrosis factor-alpha levels.10 ESW application was also found to directly stimulate neovascularization via ESW-induced upregulation of vascular endothelial growth factor (VEGF) messenger RNA and stromal cellederived factor 1 and proliferating cell nuclear antigen (PCNA) protein levels, reflecting chemoattraction and the induction of endothelial progenitor cell proliferation.10-12 Accordingly, a reduction in necrosis via an angioproliferative response to ESW has been reported in several skin flap models.1,13,14 Thus, ESW likely function by inducing inflammation accompanied by long-lasting, nitric oxide (NO)-related capillary recruitment.1 Collectively, these prior studies provide a basis for the investigation of ESW therapy for the stimulation of the vascularity and collagen synthesis in skin responses, which could underlie the treatment of certain dermatologic conditions and enhance the viability of skin flaps for surgical reconstructions. Most studies on angiogenesis and/or soft tissue have been carried out in vitro or else in vivo using small animal models that have not included the facial skin. Experiments on the facial hard and soft tissues of large animals are more relevant to humans. Therefore, the null hypothesis of the present study was that there will be no difference between ESWtreated and nontreated facial skin in terms of dermal thickness, angiogenesis, collagen, and smooth muscle production in an in vivo goat model.
ESW application After endotracheal intubation, animals were placed under general isoflurane anesthesia with 1%-2% O2 and closely monitored for any signs of respiratory distress throughout the procedure. The goats were placed in the prone position, and the hair on areas of the face (maxilla and mandible) where the shock wave applicator would be applied was shaved, and the skin cleaned with iodine. The area was covered with ultrasound coupling gel, and the shock wave applicator (SWISS PIEZOCLAST; E.M.S. Electro Medical Systems S.A., Nyon, Switzerland) was oriented vertically and perpendicular to the target site. Focused ESWs were applied to the facial skin. Each animal received ESWs at 0.15 and 0.45 mJ/mm2, with 1000 pulses to the upper or lower half of one side of the face; the administration of a particular intensity to the upper or lower site was decided in a random fashion from a computergenerated list. The skin on the untreated half of the face served as the control (Fig. 1); thus, there were six samples each in the 0.15 and 0.45 mJ/mm2 groups and 12 negative control samples. Animals were sacrificed on Day 4 after treatment with an overdose of euthanasia solution (1 cc/10 lb; EUTHASOL; Virbac Animal Health, Fort Worth, TX, ANADA #200-071) according to the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (https://www. avma.org/KB/Policies/Pages/Euthanasia-Guidelines.aspx).
Histologic analysis Facial soft tissue samples were removed and immediately fixed in 10% buffered formalin solution, washed in phosphate buffered saline (PBS), stored in 70% ethanol, and embedded in paraffin blocks; these were cut into 3-mm-thick sections that were mounted on slides and stained with hematoxylin and eosin according to standard procedures and then examined by light microscopy. Tissue sections were processed for immunohistochemistry as previously described by our group.15,16 Briefly, after
Materials and methods Animals Six male Spanish Caprine (goats, aged 3-5 y) were used in this study. All procedures were approved by the VA Boston Healthcare System Institutional Animal Care and Use Committee. The study protocol followed the institution’s guidelines and the National Research Council’s criteria for humane care as outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, Revised 1985). Humane treatment of research animals was assured.
Fig. 1 e Application of ESW to the facial soft tissue of an experimental animal. ESWs of different intensities (0.15 and 0.45 mJ/mm2) were administered to two different sites. (Color version of figure is available online.)
alshihri et al shockwave effect on facial skin
deparaffinization in xylene and rehydration in a graded series of alcohol, sections were microwaved (600 W) three times for 5 min each in 10 mM citrate buffer (pH 6) for antigen retrieval. Endogenous peroxidase was quenched with 3% H2O2 in methanol, and the sections were preincubated with 10% normal serum in 2% bovine serum albumin/PBS for 20 min to block nonspecific binding. Cluster of differentiation 31 (CD31) expression was detected using a goat polyclonal antihuman CD31/platelet endothelial cell adhesion molecule primary antibody (R&D Systems, Minneapolis, MN) and polyclonal rabbit antigoat IgG secondary antibody (HAF017; R&D Systems). To detect type 1 collagen expression, rabbit polyclonal antietype 1 collagen primary antibody (ab34710; Abcam, Cambridge, UK) and biotinylated antimouse IgG secondary antibody (KP 50A; Diagnostic BioSystems, Pleasanton, CA) were used. Alpha smooth muscle actin (a-SMA) was detected using a rabbit antiea-SMA primary antibody (ab5694; Abcam) and goat antirabbit secondary antibody (Bio-Rad, Hercules, CA). Peroxidase activity was visualized by applying 2.5% diaminobenzidine chromogen, containing 0.05% hydrogen peroxidase. The sections were washed with PBS, counterstained with hematoxylin, and mounted on plus-gold positive-charged slides using aqueous mounting medium.
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Number of microvessels All sections were screened to identify the areas with the highest number of CD31-positive vessels (hot spots). Two independent examiners (P.W.K. and D.H.) manually counted the number of CD31-positive microvessels in five images (each showing a 1 mm2 area) of hot spots under a light microscope at 100 magnification (Fig. 3). Only signals corresponding to vessels large enough to have a lumen were included in the analysis using ImageJ software (U.S. National Institutes of Health, Bethesda, MD).15 A mean value was determined for each sample.
Immunohistochemistry To quantify microvessel, type 1 collagen and a-SMA expression in skin tissue, immunolabeling was performed using CD31, type 1 collagen, and a-SMA, respectively, as previously described.18,19 Briefly, a digitized image of each slide under 25 magnification was opened in Photoshop v.7 software (Adobe Systems, San Jose, CA). Areas with the same color and a tolerance level of 15 were selected with the Magic Wand tool and quantified using the Histogram command under the Image menu.16 Staining intensity values were multiplied by the number of immunolabeled pixels and were expressed as a percentage.
Evaluated parameters Statistical analyses Dermal thickness Five randomly selected hematoxylin and eosinestained tissue sections in each group from each animal were visualized under a light microscope under 25 magnification.16 Dermal thickness was calculated as previously described,17 and a mean value was determined for each sample (Fig. 2). The numbers of slides that were analyzed were 0.15 mJ/mm2 n ¼ 30, 0.45 mJ/mm2 n ¼ 30, and control n ¼ 60.
Values were expressed as a mean standard deviation. Differences between group means were evaluated by analysis of variance for repeated measures followed by the appropriate post-hoc test, including correction of alpha error according to Bonferroni probabilities. A P value < 0.05 was considered statistically significant. All analyses were carried out using SPSS version 22 for Macintosh (IBM, Armonk, NY).
Fig. 2 e Measurements of dermal thickness at five different sites perpendicular to the skin surface and basal layer (red lines). Examples for all groups are given. (Color version of figure is available online.)
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Fig. 3 e Immunohistochemical analysis of CD31 expression for quantification of microvessel number and abundance (original magnification: 253). Examples for all groups are given. (Color version of figure is available online.)
Results Dermal thickness The mean (standard deviation) dermal thickness in the control group was 530 35.9 mm. ESW administration at 0.15 mJ/mm2 increased the thickness by 17% to 621.5 26.9 mm (P < 0.001) and by 16% to 613.6 44.9 mm (P < 0.001) using a dose of 0.45 mJ/mm2. There was no statistically significant difference observed between ESW groups (Fig. 4; P > 0.05).
Microvessel number and abundance After only 4 d, the mean number of microvessels in ESWtreated groups was significantly higher (þ43% and þ61%) than in the control group: 58.4 5.5 (0.15 mJ/mm2) and 65.7 3.9 (0.45 mJ/mm2) versus 40.9 5.4 (both P < 0.001). There was also a statistically significant difference in the ESW-treated groups in favor of 0.45 mJ/mm2 (þ12%; P ¼ 0.002; Fig. 5). The abundance of microvessels was lower in the control group (1.1 0.37%) than in animals treated with ESW at 0.15 mJ/mm2 (1.9 0.35%) or 0.45 mJ/mm2 (2.13 0.56%). This mean increase of 72% (0.15 mJ/mm2) and 93% (0.15 mJ/mm2)
Fig. 4 e Box plots showing the dermal thickness (micrometer) in the three treatment groups. *Statistically significant differences.
alshihri et al shockwave effect on facial skin
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Fig. 5 e Boxplots showing differences in the number of microvessels between the three groups. *Statistically significant differences; the differences between all groups were significant.
Fig. 6 e Box plots showing the microvessel abundance in tissue samples from the three groups. *Statistically significant differences.
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was significant different (both P < 0.001); however, the difference among the ESW groups was not statistically significant (P ¼ 0.117; Fig. 6).
Type 1 collagen and a-SMA expression in facial skin tissue The abundance of type 1 collagen was 55.3 8.9% in the control group, compared with 68.8 12% in the 0.15 mJ/mm2 group and 62.2 9.5% in the 0.45 mJ/mm2 group,
demonstrating significant 24% and 12% increases, respectively, in collagen production resulting from ESW therapy (control versus 0.15 mJ/mm2, P < 0.001; control versus 0.45 mJ/ mm2, P ¼ 0.008). Also, a significant difference was detected between the ESW groups in favor for 0.15 mJ/mm2 (P ¼ 0.034; Fig. 7A and B). There were no differences in a-SMA expression between the control (8.1 2.16%), 0.15 mJ/mm2 (8.8% 2.3%), and 0.45 mJ/mm2 (8.2% 2.5%) groups (all P > 0.05; Fig. 8A and B).
Fig. 7 e A and B: Box plots showing the abundance of type 1 collagen in tissue samples from the three groups. *Statistically significant differences; the differences between all groups were significant. In panel B, examples for all groups are given. (Color version of figure is available online.)
alshihri et al shockwave effect on facial skin
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Fig. 8 e A and B: Box plots showing the amount of SMA in tissue samples from the three groups. No statistically significant differences were detected between the groups. In panel B, examples for all groups are given. (Color version of figure is available online.)
Discussion This is the first study to examine the potential of ESW application to enhance several soft tissue parameters in facial skin using large animals. It was observed that ESW treatment significantly stimulated dermal thickness, angiogenesis, and collagen production after only 4 d. The absence of an increase in a-SMA after ESW treatment, which is known to play a role in scar contraction, suggests that the dermal response to ESWs might not be complicated by contracture such as shown in a recent publication.20 A connection between the increase
in the number of microvessels and collagen production may lie in work that has demonstrated the potential of endothelial cells to differentiate into fibroblasts through epithelialmesenchymal transition.21 There are several potential clinical applications for an easily applied, noninvasive therapy capable of meaningfully increasing vascularity and collagen production in skin after only 4 d of a one-time application lasting just 2 min (1000 shocks at 8 Hz). For example, local ischemia of facial skin flaps, which mainly results from insufficient vascularity and thrombosis, is a major challenge in reconstructive surgery.
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The energy flux density and number of pulses that were delivered to the skin were selected based on studies in humans that did not require additional anesthesia; higher intensities would likely have caused increased pain or even tissue damage.22-24 ESW treatment (0.15 mJ/mm2, 500 pulses for 5 min) was previously shown to have no adverse effects such as petechiae or hematoma in patients, who also reported Visual Analogue Scale pain scores < 3.23 Because the results of the study at hand did not provide strong evidence that the higher intensity (0.45 mJ/mm2) were more effectively inducing angiogenesis, collagen, and smooth muscle regeneration, a lower intensity of 0.15 mJ/mm2 can be recommended for future applications. A single ESW treatment was shown to be beneficial, whereas two sessions were harmful to flap tissue survival.10 Therefore, a single application of ESW was used in this study only. Samples showed obvious effects of EWS after 4 d; this is consistent with the finding that even when enzymatic upregulation occurred, there was no evidence of angiogenesis after the first 3 d.1 On the other hand, significant differences were detected in rats treated with ESW on postoperative day 3.10 Persistent improvement of microcirculation in ischemic limbs after repeated ESW treatment has also been reported,6,22 and in a rodent model, skin flap survival was enhanced after a single ESW application relative to the untreated group, which was accompanied by greater perfusion, a higher microvessel density, and increased VEGF-to-total protein ratio.13 The beneficial effects of ESW treatment have been attributed to changes in metabolic activities that reduce cell permeability, cause vasodilation, and optimize the cellular and molecular microenvironments.22,25 ESW has been shown to promote angiogenesis in small animal models,10,26,27 improve microcirculation in ischemic limbs,6,22,28 and accelerate wound healing.23 ESW (500 or 1000 pulses at 0.8 mJ/mm2) applied to the dorsal skinfold chambers of mice induced the upregulation of endothelial NO synthase (eNOS) and slight inflammation and increased the number of von Willebrand factorepositive endothelial cells and functional capillary density relative to the baseline and the control group, implying that mechanical force applied via ESW activates the endothelium.1 These results are in accordance to other experimental and clinical studies demonstrating that ESW improves local perfusion via upregulation of VEGF, eNOS, and PCNA,6,10,26,29,30 leading to the increased angiogenesis in facial soft tissue that was observed in the present study. Type 1 collagen is the main component of the dermis and constitutes dermal collagen fibers. Therefore, it is a useful marker for skin tissue repair. In the present study, ESW application (especially at a dose of 0.15 mJ/mm2) stimulated collagen production. This is in accordance with reports that ESWs enhance re-epithelialization in several dermal pathologies.23,30-32 An ESW-induced increase in PCNA expression was observed in fibroblasts and basal layers of the epidermis, indicating an increase in cell proliferation.10 In a randomized clinical trial, a single low dose of ESW (0.1 mJ/mm2; 100 pulses/cm2 with a mean treatment time of 13 min) accelerated skin re-epithelialization and healing,33 possibly as a result of increased angiogenesis and collagen synthesis proven in the present study. NO is mostly generated by eNOS and promotes the proliferation of vascular smooth muscle cells. As such, it was expected that smooth muscle growth would be accelerated by
ESW treatment. However, although others have reported a change in a-SMA expression in mice subjected to ESW treatment,30 it was not observed in the present study. This may be because of the differences in experimental setup and the fact that we did not specifically evaluate wound-healing capacity, but only evaluated histologic and molecular changes in otherwise healthy skin. The results of this study indicate that focused ESW pretreatment before surgery may be a manageable, safe, and noninvasive tool for enhancing facial soft tissue angiogenesis before local or free-flap transfer. In addition, studies are needed to establish an optimal protocol for ESW application and evaluate its effectiveness in a clinical setting.
Conclusion In a goat model, a one-time application of focused ESWs, at doses which would not necessitate anesthesia in human subjects, significantly increases the dermal thickness, number and abundance of microvessels, and the amount of type 1 collagen in facial skin after 4 d. These changes are not accompanied by increases in the expression of a-SMA.
Acknowledgment Authors’ contributions: All authors were involved in the experimental design. A.A., W.N., T.A., and M.S. carried out the conception and design of the experiments. M.S., A.A., and W.N. organized and supervised animal experiments, the measurements, and the collection of the data. A.A. and W.N. performed the animal experiments. P.W.K., A.A., D.H., and W.N. did the measurements of the histopathologic specimens. A.A., P.W.K., D.H., W.N., T.A., and M.S. analyzed the data, did the statistics, and drafted the article. A.A., P.W.K., D.H., W.N., T.A., and M.S. conducted the interpretation of the data. All authors reviewed, edited, and approved the article for submission. The authors gratefully acknowledge the loan of the PiezoClast apparatus from Electro Medical Systems Corporation (EMS), Dallas, TX. The authors would like to thank the College of Dentistry Research Center, the deanship of scientific research, and PN investigator support unit at King Saud University, Riyadh, Saudi Arabia, for their support during the completion of this project. P.W.K. especially thanks the German Research Foundation, Germany (DFG; project number KA 3720/1-1) for their unlimited support.
Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.
references
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alshihri et al shockwave effect on facial skin
2. Gerdesmeyer L, Maier M, Haake M, Schmitz C. [Physicaltechnical principles of extracorporeal shockwave therapy (ESWT)]. Orthopade. 2002;31:610e617. 3. Ogden JA, Toth-Kischkat A, Schultheiss R. Principles of shock wave therapy. Clin Orthop Relat Res. 2001:8e17. 4. Ziebart J, Fan S, Schulze C, et al. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res. 2018;7:187e195. 5. Ka¨mmerer PW, Thiem DGE, Alshihri A, et al. Cellular fluid shear stress on implant surfaces - establishment of a novel experimental set up. Int J Implant Dent. 2017;3:22. 6. De Sanctis MT, Belcaro G, Nicolaides AN, et al. Effects of shock waves on the microcirculation in critical limb ischemia (CLI) (8-week study). Angiology. 2000;51:S69eS78. 7. Nishida T, Shimokawa H, Oi K, et al. Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo. Circulation. 2004;110:3055e3061. 8. Fukumoto Y, Ito A, Uwatoku T, et al. Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease. Coron Artery Dis. 2006;17:63e70. 9. Ito K, Fukumoto Y, Shimokawa H. Extracorporeal shock wave therapy for ischemic cardiovascular disorders. Am J Cardiovasc Drugs. 2011;11:295e302. 10. Kuo YR, Wu WS, Hsieh YL, et al. Extracorporeal shock wave enhanced extended skin flap tissue survival via increase of topical blood perfusion and associated with suppression of tissue pro-inflammation. J Surg Res. 2007;143:385e392. 11. Gutersohn A, Caspari G, Erbel R. Upregulation of vascular endothelial growth factor m-RNA in human umbilical vascular endothelial cells via shock waves. Eur J Heart Fail. 2000;2:42. 12. Aicher A, Heeschen C, Sasaki K, et al. Low-energy shock wave for enhancing recruitment of endothelial progenitor cells: a new modality to increase efficacy of cell therapy in chronic hind limb ischemia. Circulation. 2006;114:2823e2830. 13. Keil H, Mueller W, Herold-Mende C, et al. Preoperative shock wave treatment enhances ischemic tissue survival, blood flow and angiogenesis in a rat skin flap model. Int J Surg. 2011;9:292e296. 14. de Lima Morais TM, Meyer PF, de Vasconcellos LS, et al. Effects of the extracorporeal shock wave therapy on the skin: an experimental study. Lasers Med Sci. 2019;34:389e396. 15. Thiem DGE, Schneider S, Venkatraman NT, et al. Semiquantifiable angiogenesis parameters in association with the malignant transformation of oral leukoplakia. J Oral Pathol Med. 2017;46:710e716. 16. Ka¨mmerer PW, Al-Nawas B, Kalkan S, et al. Angiogenesisrelated prognosis in patients with oral squamous cell carcinoma-role of the VEGF þ936 C/T polymorphism. J Oral Pathol Med. 2015;44:429e436. 17. Hattori N, Mochizuki S, Kishi K, et al. MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in mouse skin wound healing. Am J Pathol. 2009;175:533e546.
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18. Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM. Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem. 1997;45:1559e1565. 19. Heinrich UR, Brieger J, Selivanova O, et al. COX-2 expression in the Guinea pig cochlea is partly altered by moderate sound exposure. Neurosci Lett. 2006;394:121e166. 20. Zhao JC, Zhang BR, Hong L, et al. Extracorporeal shock wave therapy with low-energy flux density inhibits hypertrophic scar formation in an animal model. Int J Mol Med. 2018;41:1931e1938. 21. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420e1428. 22. Belcaro G, Cesarone MR, Dugall M, et al. Effects of shock waves on microcirculation, perfusion, and pain management in critical limb ischemia. Angiology. 2005;56:403e407. 23. Arno A, Garcia O, Hernan I, et al. Extracorporeal shock waves, a new non-surgical method to treat severe burns. Burns. 2010;36:844e849. 24. Klonschinski T, Ament J, Schlereth T, Rompe JD, Birklein F. Application of local anesthesia inhibits effects of low-energy extracorporeal shock wave treatment (ESWT) on nociceptors. Pain Med. 2011;12:1532e1537. 25. Belcaro G, Nicolaides AN, Cesarone MR. Shock waves (SW) noninvasive extracorporeal thrombolysis treatment (NISWT). Angiology. 1999;50:707e713. 26. Wang CJ, Wang FS, Yang KD, et al. Shock wave therapy induces neovascularization at the tendon-bone junction: a study in rabbits. J Orthop Res. 2003;21:984e989. 27. Kuo YR, Wang CT, Wang FS, et al. Extracorporeal shock wave treatment modulates skin fibroblast recruitment and leukocyte infiltration for enhancing extended skin-fiap survival. Wound Repair Regen. 2009;17:80e87. 28. Delius M. Biological effect of shock-wavesdmore than just lithotripsy? Zentralbl Chir. 1995;120:259e273. 29. Huemer GM, Shafighi M, Meirer R, et al. Adenovirus-mediated transforming growth factor-b ameliorates ischemic necrosis of epigastric skin flaps in a rat model. J Surg Res. 2004;121:101e107. 30. Hayashi D, Kawakami K, Ito K, et al. Low-energy extracorporeal shock wave therapy enahnces skin wound healing in diabetic mice: a critical role of endothelial nitric oxide synthase. Wound Repair Regen. 2012;20:887e895. 31. Schaden W, Thiele R, Ko¨lpl C, et al. Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study. J Surg Res. 2007;143:1e12. 32. Sparsa A, Lesaux N, Kessler E, et al. Treatment of cutaneous calcinosis in CREST syndrome by extracorporeal shock wave lithotripsy. J Am Acad Dermatol. 2005;53:S262eS265. 33. Ottomann C, Hartmann B, Tyler J, et al. Prospective randomized trial of accelerated re-epithelization of skin graft donor sites using extracorporeal shock wave therapy. J Am Coll Surg. 2010;211:361e367.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Extracorporeal Shock Wave Stimulates Angiogenesis and Collagen Production in Facial Soft Tissue Abdulmonem Alshihri, DDS,a,b,c,*,1 Peer W. Ka¨mmerer, MD, DDS, PhD, MA, FEBOMFS,c,d,e,1 Diana Heimes, MD,e Wanting Niu, BEng, PhD,c,d Talal Alnassar, BDS, MDS, FRCD(c), CDT,a and Myron Spector, BS, MS, PhDc,d a
College of Dentistry, King Saud University, Riyadh, Kingdom of Saudi Arabia Harvard School of Dental Medicine, Boston, Massachusetts c VA Boston Healthcare System, Boston, Massachusetts d Department of Orthopedics, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts e Department of Oral, Maxillofacial and Plastic Surgery, University Medical Centre Mainz, Johannes Gutenberg University Mainz, Mainz, Germany b
article info
abstract
Article history:
Background: This study investigated the efficacy of extracorporeal shock wave (ESW)
Received 7 January 2019
application in stimulating dermal thickness, vascularity, and collagen synthesis of facial
Received in revised form
skin in a large animal model.
5 May 2019
Materials and methods: The facial skin of the maxillary and mandibular areas of goats (n ¼ 6
Accepted 19 June 2019
per group) was treated with ESWs of different intensities (0.15 and 0.45 mJ/mm2; 1000
Available online xxx
pulses). After 4 d, histology and immunohistochemistry were used to evaluate the following: dermal thickness, total number and abundance of microvessels, amount of type
Keywords:
1 collagen, and a-smooth muscle actin expression.
Extracorporeal shock wave
Results: Dermal thickness, number and abundance of microvessels, and collagen synthesis
Skin
increased after ESW application at both intensities (each P < 0.05). When comparing ESW
Angiogenesis
groups, the highest collagen abundance was seen after 0.15 mJ/mm2 (P ¼ 0.034), whereas the
Dermal thickness
highest number of microvessels was detected after treatment with 0.45 mJ/mm2 (P ¼ 0.002).
Flap surgery
Conclusions: A single-session application of focused low-energy ESWs to facial skin can increase dermal thickness by stimulating collagen production and local microcirculation. These findings commend the technique for future investigation for pretreatment of local or microvascular skin flaps to enhance tissue healing. ª 2019 Published by Elsevier Inc.
Introduction Perfusion of skin and mucosal flaps is critical in facial and oral surgery, and ischemic necrosis of surgical site is therefore a
major concern. Both macro- and micro-circulation are needed to ensure the viability of flaps. Extracorporeal shock waves (ESWs) are biphasic pressure changes of short duration consisting of a high-magnitude positive wave (40 MPa
* Corresponding author. College of Dentistry, King Saud University, P.O Box 60169, Riyadh 11545, Kingdom of Saudi Arabia. Tel.: þ966 114677325; fax: (þ966) (1) 467-9017. E-mail address: [email protected] (A. Alshihri). 1 These authors contributed equally to this article. 0022-4804/$ e see front matter ª 2019 Published by Elsevier Inc. https://doi.org/10.1016/j.jss.2019.06.077
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compressive phase; w1 ms duration) followed by a lower magnitude negative wave (10 MPa tensile phase; w3 ms duration) wave. This acoustic force is transformed into mechanical energy as it enters the tissue; the biological effect is directly proportional to the difference in impedance of the two adjacent media.1 ESW has been shown to stimulate tissue repair mainly by inducing the collapse of ultrastructural vesicles through negative pressure and causing asymmetrical fluid streams within the tissue.2,3 Mechanical impact is presumed to exert similar beneficial effects, for example, in human osteoblasts,4,5 although the underlying mechanism is poorly understood. Clinical evidence suggests that ESW improves local tissue perfusion in cases of ischemic limb disease6; it was also shown to enhance regional myocardial blood flow in a porcine cardiac ischemia model7 as well as myocardial perfusion and coronary angiogenesis in humans with coronary artery disease.8,9 In critically perfused skin flaps, ESW decreases leukocyte infiltration and tumor necrosis factor-alpha levels.10 ESW application was also found to directly stimulate neovascularization via ESW-induced upregulation of vascular endothelial growth factor (VEGF) messenger RNA and stromal cellederived factor 1 and proliferating cell nuclear antigen (PCNA) protein levels, reflecting chemoattraction and the induction of endothelial progenitor cell proliferation.10-12 Accordingly, a reduction in necrosis via an angioproliferative response to ESW has been reported in several skin flap models.1,13,14 Thus, ESW likely function by inducing inflammation accompanied by long-lasting, nitric oxide (NO)-related capillary recruitment.1 Collectively, these prior studies provide a basis for the investigation of ESW therapy for the stimulation of the vascularity and collagen synthesis in skin responses, which could underlie the treatment of certain dermatologic conditions and enhance the viability of skin flaps for surgical reconstructions. Most studies on angiogenesis and/or soft tissue have been carried out in vitro or else in vivo using small animal models that have not included the facial skin. Experiments on the facial hard and soft tissues of large animals are more relevant to humans. Therefore, the null hypothesis of the present study was that there will be no difference between ESWtreated and nontreated facial skin in terms of dermal thickness, angiogenesis, collagen, and smooth muscle production in an in vivo goat model.
ESW application After endotracheal intubation, animals were placed under general isoflurane anesthesia with 1%-2% O2 and closely monitored for any signs of respiratory distress throughout the procedure. The goats were placed in the prone position, and the hair on areas of the face (maxilla and mandible) where the shock wave applicator would be applied was shaved, and the skin cleaned with iodine. The area was covered with ultrasound coupling gel, and the shock wave applicator (SWISS PIEZOCLAST; E.M.S. Electro Medical Systems S.A., Nyon, Switzerland) was oriented vertically and perpendicular to the target site. Focused ESWs were applied to the facial skin. Each animal received ESWs at 0.15 and 0.45 mJ/mm2, with 1000 pulses to the upper or lower half of one side of the face; the administration of a particular intensity to the upper or lower site was decided in a random fashion from a computergenerated list. The skin on the untreated half of the face served as the control (Fig. 1); thus, there were six samples each in the 0.15 and 0.45 mJ/mm2 groups and 12 negative control samples. Animals were sacrificed on Day 4 after treatment with an overdose of euthanasia solution (1 cc/10 lb; EUTHASOL; Virbac Animal Health, Fort Worth, TX, ANADA #200-071) according to the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (https://www. avma.org/KB/Policies/Pages/Euthanasia-Guidelines.aspx).
Histologic analysis Facial soft tissue samples were removed and immediately fixed in 10% buffered formalin solution, washed in phosphate buffered saline (PBS), stored in 70% ethanol, and embedded in paraffin blocks; these were cut into 3-mm-thick sections that were mounted on slides and stained with hematoxylin and eosin according to standard procedures and then examined by light microscopy. Tissue sections were processed for immunohistochemistry as previously described by our group.15,16 Briefly, after
Materials and methods Animals Six male Spanish Caprine (goats, aged 3-5 y) were used in this study. All procedures were approved by the VA Boston Healthcare System Institutional Animal Care and Use Committee. The study protocol followed the institution’s guidelines and the National Research Council’s criteria for humane care as outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, Revised 1985). Humane treatment of research animals was assured.
Fig. 1 e Application of ESW to the facial soft tissue of an experimental animal. ESWs of different intensities (0.15 and 0.45 mJ/mm2) were administered to two different sites. (Color version of figure is available online.)
alshihri et al shockwave effect on facial skin
deparaffinization in xylene and rehydration in a graded series of alcohol, sections were microwaved (600 W) three times for 5 min each in 10 mM citrate buffer (pH 6) for antigen retrieval. Endogenous peroxidase was quenched with 3% H2O2 in methanol, and the sections were preincubated with 10% normal serum in 2% bovine serum albumin/PBS for 20 min to block nonspecific binding. Cluster of differentiation 31 (CD31) expression was detected using a goat polyclonal antihuman CD31/platelet endothelial cell adhesion molecule primary antibody (R&D Systems, Minneapolis, MN) and polyclonal rabbit antigoat IgG secondary antibody (HAF017; R&D Systems). To detect type 1 collagen expression, rabbit polyclonal antietype 1 collagen primary antibody (ab34710; Abcam, Cambridge, UK) and biotinylated antimouse IgG secondary antibody (KP 50A; Diagnostic BioSystems, Pleasanton, CA) were used. Alpha smooth muscle actin (a-SMA) was detected using a rabbit antiea-SMA primary antibody (ab5694; Abcam) and goat antirabbit secondary antibody (Bio-Rad, Hercules, CA). Peroxidase activity was visualized by applying 2.5% diaminobenzidine chromogen, containing 0.05% hydrogen peroxidase. The sections were washed with PBS, counterstained with hematoxylin, and mounted on plus-gold positive-charged slides using aqueous mounting medium.
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Number of microvessels All sections were screened to identify the areas with the highest number of CD31-positive vessels (hot spots). Two independent examiners (P.W.K. and D.H.) manually counted the number of CD31-positive microvessels in five images (each showing a 1 mm2 area) of hot spots under a light microscope at 100 magnification (Fig. 3). Only signals corresponding to vessels large enough to have a lumen were included in the analysis using ImageJ software (U.S. National Institutes of Health, Bethesda, MD).15 A mean value was determined for each sample.
Immunohistochemistry To quantify microvessel, type 1 collagen and a-SMA expression in skin tissue, immunolabeling was performed using CD31, type 1 collagen, and a-SMA, respectively, as previously described.18,19 Briefly, a digitized image of each slide under 25 magnification was opened in Photoshop v.7 software (Adobe Systems, San Jose, CA). Areas with the same color and a tolerance level of 15 were selected with the Magic Wand tool and quantified using the Histogram command under the Image menu.16 Staining intensity values were multiplied by the number of immunolabeled pixels and were expressed as a percentage.
Evaluated parameters Statistical analyses Dermal thickness Five randomly selected hematoxylin and eosinestained tissue sections in each group from each animal were visualized under a light microscope under 25 magnification.16 Dermal thickness was calculated as previously described,17 and a mean value was determined for each sample (Fig. 2). The numbers of slides that were analyzed were 0.15 mJ/mm2 n ¼ 30, 0.45 mJ/mm2 n ¼ 30, and control n ¼ 60.
Values were expressed as a mean standard deviation. Differences between group means were evaluated by analysis of variance for repeated measures followed by the appropriate post-hoc test, including correction of alpha error according to Bonferroni probabilities. A P value < 0.05 was considered statistically significant. All analyses were carried out using SPSS version 22 for Macintosh (IBM, Armonk, NY).
Fig. 2 e Measurements of dermal thickness at five different sites perpendicular to the skin surface and basal layer (red lines). Examples for all groups are given. (Color version of figure is available online.)
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Fig. 3 e Immunohistochemical analysis of CD31 expression for quantification of microvessel number and abundance (original magnification: 253). Examples for all groups are given. (Color version of figure is available online.)
Results Dermal thickness The mean (standard deviation) dermal thickness in the control group was 530 35.9 mm. ESW administration at 0.15 mJ/mm2 increased the thickness by 17% to 621.5 26.9 mm (P < 0.001) and by 16% to 613.6 44.9 mm (P < 0.001) using a dose of 0.45 mJ/mm2. There was no statistically significant difference observed between ESW groups (Fig. 4; P > 0.05).
Microvessel number and abundance After only 4 d, the mean number of microvessels in ESWtreated groups was significantly higher (þ43% and þ61%) than in the control group: 58.4 5.5 (0.15 mJ/mm2) and 65.7 3.9 (0.45 mJ/mm2) versus 40.9 5.4 (both P < 0.001). There was also a statistically significant difference in the ESW-treated groups in favor of 0.45 mJ/mm2 (þ12%; P ¼ 0.002; Fig. 5). The abundance of microvessels was lower in the control group (1.1 0.37%) than in animals treated with ESW at 0.15 mJ/mm2 (1.9 0.35%) or 0.45 mJ/mm2 (2.13 0.56%). This mean increase of 72% (0.15 mJ/mm2) and 93% (0.15 mJ/mm2)
Fig. 4 e Box plots showing the dermal thickness (micrometer) in the three treatment groups. *Statistically significant differences.
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Fig. 5 e Boxplots showing differences in the number of microvessels between the three groups. *Statistically significant differences; the differences between all groups were significant.
Fig. 6 e Box plots showing the microvessel abundance in tissue samples from the three groups. *Statistically significant differences.
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was significant different (both P < 0.001); however, the difference among the ESW groups was not statistically significant (P ¼ 0.117; Fig. 6).
Type 1 collagen and a-SMA expression in facial skin tissue The abundance of type 1 collagen was 55.3 8.9% in the control group, compared with 68.8 12% in the 0.15 mJ/mm2 group and 62.2 9.5% in the 0.45 mJ/mm2 group,
demonstrating significant 24% and 12% increases, respectively, in collagen production resulting from ESW therapy (control versus 0.15 mJ/mm2, P < 0.001; control versus 0.45 mJ/ mm2, P ¼ 0.008). Also, a significant difference was detected between the ESW groups in favor for 0.15 mJ/mm2 (P ¼ 0.034; Fig. 7A and B). There were no differences in a-SMA expression between the control (8.1 2.16%), 0.15 mJ/mm2 (8.8% 2.3%), and 0.45 mJ/mm2 (8.2% 2.5%) groups (all P > 0.05; Fig. 8A and B).
Fig. 7 e A and B: Box plots showing the abundance of type 1 collagen in tissue samples from the three groups. *Statistically significant differences; the differences between all groups were significant. In panel B, examples for all groups are given. (Color version of figure is available online.)
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Fig. 8 e A and B: Box plots showing the amount of SMA in tissue samples from the three groups. No statistically significant differences were detected between the groups. In panel B, examples for all groups are given. (Color version of figure is available online.)
Discussion This is the first study to examine the potential of ESW application to enhance several soft tissue parameters in facial skin using large animals. It was observed that ESW treatment significantly stimulated dermal thickness, angiogenesis, and collagen production after only 4 d. The absence of an increase in a-SMA after ESW treatment, which is known to play a role in scar contraction, suggests that the dermal response to ESWs might not be complicated by contracture such as shown in a recent publication.20 A connection between the increase
in the number of microvessels and collagen production may lie in work that has demonstrated the potential of endothelial cells to differentiate into fibroblasts through epithelialmesenchymal transition.21 There are several potential clinical applications for an easily applied, noninvasive therapy capable of meaningfully increasing vascularity and collagen production in skin after only 4 d of a one-time application lasting just 2 min (1000 shocks at 8 Hz). For example, local ischemia of facial skin flaps, which mainly results from insufficient vascularity and thrombosis, is a major challenge in reconstructive surgery.
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The energy flux density and number of pulses that were delivered to the skin were selected based on studies in humans that did not require additional anesthesia; higher intensities would likely have caused increased pain or even tissue damage.22-24 ESW treatment (0.15 mJ/mm2, 500 pulses for 5 min) was previously shown to have no adverse effects such as petechiae or hematoma in patients, who also reported Visual Analogue Scale pain scores < 3.23 Because the results of the study at hand did not provide strong evidence that the higher intensity (0.45 mJ/mm2) were more effectively inducing angiogenesis, collagen, and smooth muscle regeneration, a lower intensity of 0.15 mJ/mm2 can be recommended for future applications. A single ESW treatment was shown to be beneficial, whereas two sessions were harmful to flap tissue survival.10 Therefore, a single application of ESW was used in this study only. Samples showed obvious effects of EWS after 4 d; this is consistent with the finding that even when enzymatic upregulation occurred, there was no evidence of angiogenesis after the first 3 d.1 On the other hand, significant differences were detected in rats treated with ESW on postoperative day 3.10 Persistent improvement of microcirculation in ischemic limbs after repeated ESW treatment has also been reported,6,22 and in a rodent model, skin flap survival was enhanced after a single ESW application relative to the untreated group, which was accompanied by greater perfusion, a higher microvessel density, and increased VEGF-to-total protein ratio.13 The beneficial effects of ESW treatment have been attributed to changes in metabolic activities that reduce cell permeability, cause vasodilation, and optimize the cellular and molecular microenvironments.22,25 ESW has been shown to promote angiogenesis in small animal models,10,26,27 improve microcirculation in ischemic limbs,6,22,28 and accelerate wound healing.23 ESW (500 or 1000 pulses at 0.8 mJ/mm2) applied to the dorsal skinfold chambers of mice induced the upregulation of endothelial NO synthase (eNOS) and slight inflammation and increased the number of von Willebrand factorepositive endothelial cells and functional capillary density relative to the baseline and the control group, implying that mechanical force applied via ESW activates the endothelium.1 These results are in accordance to other experimental and clinical studies demonstrating that ESW improves local perfusion via upregulation of VEGF, eNOS, and PCNA,6,10,26,29,30 leading to the increased angiogenesis in facial soft tissue that was observed in the present study. Type 1 collagen is the main component of the dermis and constitutes dermal collagen fibers. Therefore, it is a useful marker for skin tissue repair. In the present study, ESW application (especially at a dose of 0.15 mJ/mm2) stimulated collagen production. This is in accordance with reports that ESWs enhance re-epithelialization in several dermal pathologies.23,30-32 An ESW-induced increase in PCNA expression was observed in fibroblasts and basal layers of the epidermis, indicating an increase in cell proliferation.10 In a randomized clinical trial, a single low dose of ESW (0.1 mJ/mm2; 100 pulses/cm2 with a mean treatment time of 13 min) accelerated skin re-epithelialization and healing,33 possibly as a result of increased angiogenesis and collagen synthesis proven in the present study. NO is mostly generated by eNOS and promotes the proliferation of vascular smooth muscle cells. As such, it was expected that smooth muscle growth would be accelerated by
ESW treatment. However, although others have reported a change in a-SMA expression in mice subjected to ESW treatment,30 it was not observed in the present study. This may be because of the differences in experimental setup and the fact that we did not specifically evaluate wound-healing capacity, but only evaluated histologic and molecular changes in otherwise healthy skin. The results of this study indicate that focused ESW pretreatment before surgery may be a manageable, safe, and noninvasive tool for enhancing facial soft tissue angiogenesis before local or free-flap transfer. In addition, studies are needed to establish an optimal protocol for ESW application and evaluate its effectiveness in a clinical setting.
Conclusion In a goat model, a one-time application of focused ESWs, at doses which would not necessitate anesthesia in human subjects, significantly increases the dermal thickness, number and abundance of microvessels, and the amount of type 1 collagen in facial skin after 4 d. These changes are not accompanied by increases in the expression of a-SMA.
Acknowledgment Authors’ contributions: All authors were involved in the experimental design. A.A., W.N., T.A., and M.S. carried out the conception and design of the experiments. M.S., A.A., and W.N. organized and supervised animal experiments, the measurements, and the collection of the data. A.A. and W.N. performed the animal experiments. P.W.K., A.A., D.H., and W.N. did the measurements of the histopathologic specimens. A.A., P.W.K., D.H., W.N., T.A., and M.S. analyzed the data, did the statistics, and drafted the article. A.A., P.W.K., D.H., W.N., T.A., and M.S. conducted the interpretation of the data. All authors reviewed, edited, and approved the article for submission. The authors gratefully acknowledge the loan of the PiezoClast apparatus from Electro Medical Systems Corporation (EMS), Dallas, TX. The authors would like to thank the College of Dentistry Research Center, the deanship of scientific research, and PN investigator support unit at King Saud University, Riyadh, Saudi Arabia, for their support during the completion of this project. P.W.K. especially thanks the German Research Foundation, Germany (DFG; project number KA 3720/1-1) for their unlimited support.
Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.
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