Efficacy of light-emitting diode-mediated photobiomodulation in tendon healing in a murine model

Efficacy of light-emitting diode-mediated photobiomodulation in tendon healing in a murine model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of light-emitting diode-mediated photobiomodulation in tendon healing in a murine model Jae Kyung Lim, Jae Ho Kim, Gyu Tae Park, Min Kyung Cho, Seung Hun Woo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4578400/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The application of light-emitting diode (LED)-dependent photobiomodulation (PBM) in promoting post-tendon injury healing has been recently reported. Despite establishing a theoretical basis for ligament restoration through PBM, the lack of empirical evidence deems this therapeutic strategy contentious. Therefore, the aim of this study was to investigate the potency of LED-based PBM in facilitating tendon healing in a murine model. Methods Migration kinetics were analyzed at two specific wavelengths: 630 and 880 nm. The Achilles tendon in the hind limbs of Balb/c mice was severed by Achilles tendon transection. Subsequently, the mice were randomized into LED non-irradiation and LED irradiation groups. Mice with intact tendons were employed as healthy controls. The wounds were LED-irradiated for 20 min daily for two days. Histological properties, tendon healing mediators, and inflammatory mediators were screened on day 14. Results The roundness of the nuclei and fiber structure, indicating the degree of infiltrated inflammatory cells and severity of fiber fragmentation, respectively, were lower in the LED irradiation group than in the LED non-irradiation group. Immunohistochemical analysis depicted an increase in tenocytes (SCX + cells) and a recovery of wounds with reduced fibrosis (lower collagen 3 and TGF-β1) in the LED irradiation group during healing; conversely, the LED non-irradiation group exhibited tissue fibrosis. Overall, the ratio of M2 macrophages to total macrophages in the LED irradiation group was higher than that in the injured group. Conclusion LED-based PBM in the Achilles tendon rupture murine model effectuated a rapid restoration of histological and immunochemical outcomes. These findings suggest that LED-based PBM presents remarkable potential as an adjunct therapeutic for tendon healing and warrants further research to standardize various parameters to advance and establish it as a reliable treatment regime. Photobiomodulation Light-emitting diode Tendon Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In the 1960s, Mester documented the potential applicability of photobiomodulation (PBM) in accelerating wound restoration, following which the National Aeronautics and Space Administration researchers implemented it as a therapeutic regimen to enhance the healing process in space. 1,2) PBM facilitates healing by stimulating the mitochondrial and cell membrane photoreceptor-based synthesis of ATP, which increases cell viability. 3) PBM is performed using lasers or light-emitting diodes (LEDs) that radiate light in the red and near-infrared wavelengths. 4) Numerous studies and trials have been conducted over the past two decades on the clinical application of PBM in the medical and dental fields. PBM is employed in various disciplines of clinical dentistry for post-orthodontic treatment pain alleviation, osseointegration, collagen deposition, and implant stability enhancement. 5,6,7) PBM has demonstrated additional efficacy in various dermatological interventions, including skin rejuvenation, hair growth, and fat reduction. 8,9) Moreover, empirical evidence exhibiting the potential of PBM in promoting fibroblast proliferation, growth factor synthesis, collagen production, and angiogenesis has prompted numerous animal experiments and clinical studies in the field of orthopedic surgery. 3,10,11,12) Nevertheless, specifications on parameters such as wavelength, intensity, and irradiation time associated with PBM therapy have not yet been established, posing limitations on its clinical applicability. Drawing upon prior research, the present investigation assessed the efficacy of PBM on human tendon-derived fibroblasts and revealed over 2-fold cell proliferation and 3-fold cell migration in the PBM-treated group compared to that in the control group. 13) Furthermore, Vinck et al. reported that both LED and laser proliferate fibroblasts in the Achilles tendon of a rat model. 14) Diseases affecting tendons and ligaments constitute a substantial proportion of orthopedic ailments. 15) The prevalence of these diseases is rising annually owing to the ongoing burgeoning of elderly and athletic populations; overall, this trend has been associated with high social costs. 16,17,18) Many treatments are being introduced for these tendon and ligament diseases, including PBM. Although the efficacy of PBM has been established on a theoretical basis, its applicability remains to be validated in a practical or clinical context. Therefore, the aim of this study was to examine the effect of PBM on tendon healing using Achilles tendons in a mouse model. Materials and Methods Animal experiments The animals were housed in an air-conditioned animal room with constant relative humidity and provided with a standard laboratory diet and water as outlined in the Guide for the Care and Use of Laboratory Animals. Animal experiments were performed in adherence to the protocols approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2023-0269). Six-week-old male BALB/c mice weighing 22–24 g were acquired from Koatech (Gyeonggi-do, Korea). To inflict an injury, the mice were administered intraperitoneal anesthesia with 1.25% avertin (2,2,2-tribromoethanol, 250 mg/kg), and the Achilles tendon was exposed through an approximately 10-mm incision on the medial side of the right hind limb. Subsequently, the Achilles tendon was bilaterally lacerated (2 mm in diameter) per prior methodology with slight modifications (Schramme, M. et al., 2010, Vet Comp Orthop Traumatol, 231–9). The mice were randomized into two groups: LED non-irradiated (n = 4) and LED irradiated (n = 4). Mice with intact tendons were employed as healthy controls (n = 4). The mice were euthanized 2 weeks following surgery, and tendon tissues were harvested and stored at − 80℃ or fixed in acetone. LED irradiation The LED irradiant group was anesthetized and subjected to wound irradiation for 20 min daily for 2 weeks, commencing 24 h post-surgery. LED irradiation was applied to the Achilles tendon injury site at wavelengths of 630 nm (10 mW/cm 2 , 100 Hz) and 880 nm (40 mW/cm 2 , 100 Hz). Histological analysis The mice were euthanized, their Achilles tendons excised, and the tissue specimens fixed overnight in acetone at − 20℃ and embedded in an optimum cutting temperature (O.C.T.) compound (Sakura Finetek USA, Inc., Torrance, CA, USA). Tissues were sectioned into 10-µm segments and stained with hematoxylin and eosin (H&E) for histological score analysis. Stained tissue sections were scanned using an Axio Scan.Z1 (Carl Zeiss Microscopy, Germany) at ×200 magnification. The histological outcomes were scored from 0 (best) to 3 (worst) and evaluated on four parameters: cell density, roundness of nuclei, fiber structure, and fiber arrangement. Two blinded pathologists independently graded the histological results from 0 to 3, which subjectively categorized the tissue samples according to the four aforementioned parameters. Three sections were randomly selected from each sample, and the average scores for each group were compared. Immunohistochemistry Tendon sections fixed in acetone and embedded in O.C.T. compound were incubated with anti-tenomodulin (bs-7525R; Bioss, Woburn, MA, USA) and anti-SCX antibodies (MBS9612052; MyBioSource, San Diego, CA, USA) to evaluate tenocyte proliferation. Additionally, the collagen in the specimens was stained with anti-collagen1 (bs-10423R; Bioss) and anti-collagen3 antibodies (ab7778; Abcam, Cambridge, UK). The tendon sections were stained with anti-TGF-β1 antibody (MAB240-100; R&D Systems, Minneapolis, MN, USA) to examine for fibrosis and with anti-CD68 (14-0681-82) and anti-CD163 (ab182422; Abcam) antibodies to identify M2 macrophages. Subsequently, the specimens were incubated with secondary antibodies (Alexa Fluor 488, 568, or 647) for 2 h at room temperature, washed, and mounted with a prolonged gold antifade mounting solution. Stained sections were visualized under a confocal microscope (Zeiss). ImageJ software was used to quantify the number of Tenomodulin + , SCX + , CD68 + , and M2 macrophages (CD68 + CD163 + ) in high-power fields. Western blot analysis Tendon tissues were homogenized and lysed using lysis buffer (pH 7.4; 20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM phenylmethyl sulfonyl fluoride, 1 mM Na 3 VO 4 , 30 mM sodium pyrophosphate, 25 mM β-glycerol phosphate, and 1% Triton X-100) containing protease inhibitors. The protein fractions in the lysates were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Proteins were stained with 0.1% Ponceau S solution (Sigma-Aldrich Co. Ltd., St. Louis, MO, USA) and blocked with 5% nonfat milk. The bound antibodies were visualized using the corresponding horseradish peroxidase-conjugated secondary antibodies. The enhanced chemiluminescence western blotting system (GE-RPN2106) was used for signal detection, and images were captured using an ImageQuant 800 western blot imaging system (GE Healthcare, Chicago, IL, USA). Statistical analysis Results from multiple observations are presented as mean ± SD. A Student’s two-tailed unpaired t -test was used to determine the statistical significance of differences between the two groups. Differences between groups were evaluated using one- or two-way analysis of variance (ANOVA), followed by Scheffé’s post hoc test. Results LED irradiation accelerated the healing of the Achilles tendon injury To investigate the effects of LED irradiation on Achilles tendon injury repair in vivo , we used a rat model. Surgical core lesions of the Achilles tendon were produced in the hind limbs of rats, and the LED was irradiated locally, as described in the preceding Materials and Methods section. As a control, the injured Achilles tendon was not irradiated with LED. H&E staining was performed to examine whether LED irradiation had a therapeutic effect on Achilles tendon injuries. The lesions could be clearly identified by using H&E staining of the injured Achilles tendon. LED irradiation improved tendon healing without any detectable local adverse effects (Fig. 1 A). Next, we analyzed the fiber structure, fiber arrangement, cell density, and roundness of the nuclei in tendon tissues. All four indicators were markedly elevated in the injury group compared to those in the normal group, and fiber structure and arrangement were notably decreased in the LED treatment group compared to those in the injury group (Figs. 1 B, C). However, there were no discernible differences in the cell density or roundness of the nuclei of injured tendons between the control and LED-irradiated mice (Figs. 1 D, E). These results suggest that LED treatment promotes fiber regeneration and arrangement, although the infiltration of inflammatory immune cells in the injured tendons remained unaffected LED irradiation promoted tenocyte proliferation Tenocytes are the principal cellular constituents of the tendon and assume various roles during tendon injury. Tenocytes express Tnmd and SCX, which are well-known tenocyte markers. To explore the effects of LED irradiation on tenocyte proliferation during tendon repair, we determined the effect of LED irradiation on the number of tenocytes in the injured tendons. Immunohistochemistry of tendon tissues revealed overexpression of both genes in the injury group compared to that in the normal group and decreased expression in the LED-treated group (Fig. 2 A). Western blot analysis depicted that the expression levels of Tnmd and SCX were higher in the injury group than in the normal group, whereas they were strikingly lower in the LED group than in the injury group (Fig. 2 B, C). The histological results showed that LED treatment promoted fiber regeneration and arrangement, confirming the restorative effect of LED. Moreover, on day 14, tenocyte markers decreased and were quickly normalized. LED irradiation increased the collagen 1/3 expression To explore the effects of LED irradiation on collagen synthesis, we examined the expression levels of collagens 1 and 3 by using immunocytochemical and western blot analyses. Immunocytochemical analysis exhibited that the expression levels of collagens 1 and 3 were notably higher in the injury group than in the normal group and markedly lower in the LED treatment group than in the injury group (Fig. 3 A). Western blot analysis demonstrated that the protein levels of both collagen 1 and 3 increased in injured tendons compared to those in normal tendons, and the expression levels of collagen 3 were discernibly decreased upon LED treatment in injured tendons (Figs. 3 B–D). The ratio of collagen 1/3 slightly decreased in the injury group compared to that in the normal group but was markedly restored in the LED-treated group to a level comparable to that observed in the normal group (Fig. 3 E). In conjunction with the increased collagen arrangement and structure (Fig. 1 ), these results suggest that LED irradiation stimulates the organization and arrangement of collagen to promote repair, while ensuring reduced inflammatory immune cell infiltration in Achilles tendon injury. LED irradiation reduced the degree of fibrosis As LED irradiation rapidly stabilized the repairing site by regulating collagen synthesis and cross-link formation in the injured Achilles tendon, we examined the expression level of TGF-β1 in the injured tendon using immunocytochemistry and western blot analysis. Immunocytochemical analysis evidenced that TGF-β1 expression was increased in the injured tendon compared to that in the normal tendon, and it was markedly reduced by LED treatment (Fig. 4 A). The expression levels of TGF-β1 and vimentin increased in injured tendon tissue, and the increased expression of TGF-β1 and vimentin was considerably decreased in the LED-treated tendon, suggesting that LED irradiation may reduce fibrosis. (Figs. 4 B–D). LED irradiation prevented the inflammatory macrophage infiltration into the injured site To explore whether LED irradiation affected the activation of macrophages in injured Achilles tendons, tendon tissues were immunostained for CD68, a pan-macrophage marker, and CD163, a M2 macrophage marker (Fig. 5 A). The experimental findings demonstrated that the number of macrophages was higher in the injury group than in the normal group; however, the number of macrophages in the injured tendon decreased in response to LED treatment (Fig. 5 B). The number of CD68 + CD163 + M2 macrophages was higher in the injury group than in the normal group but was not notably affected by LED irradiation (Fig. 5 C). Nonetheless, the ratio of M2 macrophages to total macrophages was higher in the LED irradiation group than in the injured group (Fig. 5 D). This result suggests that LED irradiation modulates inflammatory response to promote the recovery of the injured Achilles tendon. Discussion The results of this study confirmed the initial effect of LED treatment on tendon healing. The histology scores and immunochemistry results exhibited remarkable differences between the LED treatment and control groups. PBM using low-level laser therapy (LLLT) or LED therapy was first introduced by Mester in the early 1960s. PBM accelerates healing by increasing cell activity by stimulating mitochondrial and cell membrane photoreceptor synthesis of ATP. Such modulations in these cells can promote fibroblast proliferation, growth factor synthesis, collagen production, and angiogenesis. Various laboratory studies and animal experiments have been conducted in the field of orthopedic surgery. Silva et al. 19) reported a positive effect of LLLT/LED irradiation on tendon damage. Rosso et al. 20) documented that PBM has beneficial effects on the recovery of nerve lesions. In vitro experimental findings suggest that PBM may facilitate tissue homeostasis, thus stimulating articular tissue components and promoting chondroprotective effects. In our previous study, LED irradiation at 630 nm and 630 nm + 880 nm for 20 min was observed to strikingly affect the proliferation and migration of human tendon fibroblasts. Cell and animal experiments have revealed positive results for PBM in the field of orthopedic surgery, especially in the treatment of ligaments. Future studies are needed to derive clinical results related to this. Ruptured Achilles tendons have been reported to exhibit marked collagen degeneration, disordered arrangement of collagen fibers, augmented cellularity, and an increase in the number of tenocytes with round nuclei. 21) The failure of the intact tendon healing due to fibrotic scar formation after medical and surgical treatment, which can lead to chronic symptoms and reinjury, is a common issue 1–4) . Histologic analysis showed that PBM therapy improved the tendon healing through inhibition of cell density and nuclear circularity. This outcome accelerated fiber regeneration and alignment, and collagen synthesis. The tissue structure in LED-irradiated injured Achilles tendons exhibited greater parallelism, in addition to having a denser deposition of new collagen fibers than that of LED-non-irradiated injured Achilles tendons. These results indicate that LED can shorten the recovery period of the damaged tendon. Collagen 1 is the main ECM component of tendons, whereas collagen 3, which is generally associated with scar tissue and injury, accumulated at injured tendons. The increased content of type 3 collagen can cause thinner collagen fibers, decrease the tensile strength. 22) The ratio of collagen 1 and 3 was markedly restored in the LED treated group to a level similar to the normal group. With histologic results, LED irradiation stimulates the organization and arrangement of collagen to promote healing. Tendon injury is associated with tissue regeneration and fibrosis. TGF-β1 is activated upon tendon injury and is key in tendon healing and fibrosis. 23) The expression of TGF-β1 and vimentin, markers of the presence of myofibroblasts, has been implicated in fibrosis. 24) TGF-β1 and vimentin levels decreased to the normal range in the LED group at the two-week mark. Although our results do not directly reflect changes in collagen level during the early stages of healing, considering the histological and collagen level results, it is thought that the levels normalized as recovery rapidly progress. Macrophages key regulators of the healing of injured tendons. For example, an increase in the concentration of Macrophage has been reported to play a key role in regulating the healing process of injured tendons. 25) The specific function of macrophages depends on their phenotype. While the M1 phenotype macrophage exhibits a phagocytic and proinflammatory function 6–9) , the M2 phenotype macrophage is associated with tissue repair and deposition in inflamed tissue 6, 7, 10) . During tendon healing, increases in the concentration of the M2 macrophage phenotype occur later in the healing process. 25) In this study, the ratio of M2 macrophages to total macrophages increased in the LED irradiation group compared to the injured group. This increase, as driven by LED irradiation, may inhibit abnormal or excessive inflammatory responses. Therefore, LED-facilitated recovery of injured Achilles tendon is controlled through an increased differentiation toward the M2 macrophage phenotype. Our study had several limitations. First, it was an in vivo study and owing to its limited sample size and brief duration, comprehending the cumulative healing process is deemed challenging. Second, the mechanism underlying each stage of the healing process could not be ascertained. Therefore, it is imperative to conduct further investigation into the mechanisms and impacts of each process through long-term experiments. Another major limitation of our study lies in the fact that we were not able to conduct mechanical testing in animals. In general, mechanical testing should be performed in conjunction with histology to accurately illuminate the healing trajectory (or lack thereof) of a tendon. To address this limitation in future studies, we intend to conduct mechanical testing after PBM irradiation to illuminate more accurate dynamics of tendon healing. Conclusions Histological and immunochemical outcomes evidenced the effectiveness of LED-based PBM in stimulating rapid recovery in a murine model of Achilles tendon rupture. These results suggest that LED-mediated PBM possesses considerable potential as an adjunct treatment for tendon healing and warrants further research to standardize various parameters to develop and establish PBM as a reliable treatment regime. Abbreviations light-emitting diode (LED), photobiomodulation (PBM), low-level laser therapy (LLLT) Declarations Ethics approval and consent to participate: Animal experiments were performed in adherence to the protocols approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2023-0269). Consent for publication: Not applicable Availability of data and materials: The datasets used and/or anylsed during the current study are availalbe from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: Not applicable. The authors have nothing to close Authors' contributions: Conceived and desinged the experiments:SWK, JHK. Performed the experiments:JKL, GTP,MKC. Contributed reagents/materials/analysis tools: MKC, Wrote the paper:SWK, JKL. All authors read and approved the final mauscpript. Acknowledgements: This study was supported by Research institue for Convergence of biomedical science and technoloty,Pusan National University Yansan Hospital (30-2022-016) Authors' information; Department of Orthopaedic, Pusan National University Yangsan Hospital, Yangsan, Kore Jae Kyung Lim, Jae Ho Kim, Gyu Tae Park, Min Kyung Cho, Seung Hun Woo & Suk Woong Kang Corresponding author : Suk Woong Kang References Mester A, Mester A. The history of photobiomodulation: endre mester (1903–1984). Photomed Laser Surg. 2017;35:393–4. Whelan HT, Houle JM, Whelan NT, et al. editors. The NASA light-emitting diode medical program—progress in space flight and terrestrial applications. AIP Conference Proceedings; 2000: American Institute of physics. de Freitas LF, Hamblin MR. Proposed mechanisms of photobiomodulation or low-level light therapy. IEEE J Sel Top Quantum Electron. 2016;22(3):348–64. Dompe C, Moncrieff L, Matys J, et al. Photobiomodulation—underlying mechanism and clinical applications. J Clin Med. 2020;9(6):1724. Matys J, Świder K, Grzech-Leśniak K, Dominiak M, Romeo U. Photobiomodulation by a 635 nm Diode Laser on Peri-Implant Bone: Primary and Secondary Stability and Bone Density Analysis—A Randomized Clinical Trial. Biomed Res Int. 2019;2019:2785302. Artés-Ribas M, Arnabat-Dominguez J, Puigdollers A. Analgesic effect of a low-level laser therapy (830 nm) in early orthodontic treatment. Lasers Med Sci. 2013;28:335–41. Genc G, Kocadereli İ, Tasar F, Kilinc K, El S, Sarkarati B. Effect of low-level laser therapy (LLLT) on orthodontic tooth movement. Lasers Med Sci. 2013;28:41–7. Wikramanayake TC, Rodriguez R, Choudhary S, Mauro LM, Nouri K, Schachner LA, Jimenez JJ. Effects of the Lexington LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia areata. Lasers Med Sci. 2012;27:431–6. Barolet D, Roberge CJ, Auger FA, Boucher A, Germain L. Regulation of Skin Collagen Metabolism In Vitro Using a Pulsed 660nm LED Light Source: Clinical Correlation with a Single-Blinded Study. J Invest Dermatol. 2009;129:2751–9. Dompe C, Moncrieff L, Matys J, et al. Photobiomodulation—underlying mechanism and clinical applications. J Clin Med. 2020;9(6):1724. Hosseinpour S, Fekrazad R, Arany PR, Ye Q. Molecular impacts of photobiomodulation on bone regeneration: a systematic review. Prog Biophys Mol Biol. 2019;149:147–59. Rosso MPO, Buchaim DV, Kawano N, Furlanette G, Pomini KT, Buchaim RL. Photobiomodulation therapy (PBMT) in peripheral nerve regeneration: a systematic review. Bioengineering. 2018;5(2):44. Ryu JH, Park J, Kim JW, et al. Exploring the Effects of 630 nm Wavelength of Light-Emitting Diode Irradiation on the Proliferation and Migration Ability of Human Biceps Tendon Fibroblast Cells. Clin Orthop Surg. 2023;12(1):166–74. Vinck EM, et al. Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation. Lasers Med Sci. 2003;18(2):95–9. Clayton RA, Court-Brown CM. The epidemiology of musculoskeletal tendinous and ligamentous injuries. Inj. 2008;39(12):1338–44. James R, Kesturu G, Balian G, Chhabra AB. Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options. J Hand Surg. 2008;33(1):102–12. Mock C, Cherian MN. The global burden of musculoskeletal injuries: challenges and solutions. Clin Orthop Relat Res. 2008;466(10):2306–16. Vitale MA, Vitale MG, Zivin JG, Braman JP, Bigliani LU, Flatow EL. Rotator cuff repair: an analysis of utility scores and cost-effectiveness. J Shoulder Elb Surg. 2007;16(2):181–7. Lopes Silva RSD, Pessoa DR, Mariano RR, Castro ABS, de Oliveira RA, Ferraresi C. Systematic review of photobiomodulation therapy (PBMT) on the experimental calcaneal tendon injury in rats. Photochem Photobiol. 2020;96(5):981–97. Rosso MPO, Buchaim DV, Kawano N, Furlanette G, Pomini KT, Buchaim RL. Photobiomodulation therapy (PBMT) in peripheral nerve regeneration: a systematic review. Bioengineering. 2018;5(2):44. Maffulli N, Barrass V, Ewen S. Light microscopic history of Achilles tendon ruptures. Am J Sports Med. 2000;28(6):857–63. Eriksen HA, Pajala A, Leppilahti J, Risteli J. Increased content of type III collagen at the rupture site of human Achilles tendon. J Orthop Res. 2002;20(6):1352–7. Juneja SC, Schwarz EM, O’Keefe RJ, Awad HA. Cellular and molecular factors in flexor tendon repair and adhesions: A histological and gene expression analysis. Connect Tissue Res. 2013;54(3):218–26. Marconi GD, Fonticoli L, Rajan, et al. Epithelial-mesenchymal transition (EMT): The type-2 EMT in wound healing, tissue regeneration and organ fibrosis. Cells. 2021;10(7):1587. Sunwoo JY, Eliasberg CD, Carballo CB, Rodeo SA. The role of the macrophage in tendinopathy and tendon healing. J Orthop Res. 2020;38(8):1666–75. Additional Declarations No competing interests reported. Supplementary Files Supplementalfigure.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4578400","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322469575,"identity":"1c423ff3-727b-4aba-a7a6-3805d92ee01c","order_by":0,"name":"Jae Kyung Lim","email":"","orcid":"","institution":"School of Medicine, Pusan National University , South Korea","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Kyung","lastName":"Lim","suffix":""},{"id":322469576,"identity":"6f86e382-ff45-416d-b00b-ba54853a332a","order_by":1,"name":"Jae Ho Kim","email":"","orcid":"","institution":"School of Medicine, Pusan National University , South Korea","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Ho","lastName":"Kim","suffix":""},{"id":322469577,"identity":"03565bb0-1341-43c8-bf89-94415e2814bc","order_by":2,"name":"Gyu Tae Park","email":"","orcid":"","institution":"School of Medicine, Pusan National University , South Korea","correspondingAuthor":false,"prefix":"","firstName":"Gyu","middleName":"Tae","lastName":"Park","suffix":""},{"id":322469578,"identity":"21182503-ca94-4320-a30c-e0eaa4861c91","order_by":3,"name":"Min Kyung Cho","email":"","orcid":"","institution":"School of Medicine, Pusan National University , South Korea","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"Kyung","lastName":"Cho","suffix":""},{"id":322469579,"identity":"43b08e83-382a-42b1-90ad-7408226fbdfc","order_by":4,"name":"Seung Hun Woo","email":"","orcid":"","institution":"School of Medicine, Pusan National University , South Korea","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Hun","lastName":"Woo","suffix":""},{"id":322469580,"identity":"1b077f19-9eea-4d4a-816a-740570dc2ee8","order_by":5,"name":"Suk Woong Suk Woong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYHACAyCWYJBgbyBZC88B0rQANUkkEKnevL1564afOyzkJWe+PfiZp+IeA397N37NMmeOld3sPSNhOFs6L1ma50wxg8SZsxvwapGQyDG7wdsmwThPOsdAcmZbAoOBRC4BLfJvzG7+bZOwnyd5xvjnzH/EaJHgMbsNtCVxNpAh8bGBGC08aWW3Zdskkmf25JhZfDiWwEPYL+yHt91821ZnO+P4GeMbCTUJcvztvfi1YAAe0pSPglEwCkbBKMAKAMr8Q4k8jYJ/AAAAAElFTkSuQmCC","orcid":"","institution":"School of Medicine, Pusan National University , South Korea","correspondingAuthor":true,"prefix":"","firstName":"Suk","middleName":"Woong Suk","lastName":"Woong","suffix":""}],"badges":[],"createdAt":"2024-06-13 22:08:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4578400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4578400/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60497676,"identity":"8f332da7-6aef-49ea-a8ec-1b4a70d85ec1","added_by":"auto","created_at":"2024-07-17 11:58:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170864,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LED irradiation on fibrosis\u003cstrong\u003e \u003c/strong\u003ein injured Achilles tendon. (A) H\u0026amp;E staining of the Achilles tendon tissue sections at 2 weeks post-surgery. Scale bar = 100 μm. (B-E) Histological analysis of the representative H\u0026amp;E-stained tissue section of the Achilles tendon. Fiber structure, fiber arrangement, cell density, and roundness of nuclei were quantified. ***p\u0026lt;0.005. Data indicate mean ±SD. (n = 4 per group)\u003c/p\u003e","description":"","filename":"Figure1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/cc1d3d707fdeb75a084a89a4.jpg"},{"id":60496772,"identity":"dcb21050-c8f8-4dd8-9fe1-3cdd8b586f8c","added_by":"auto","created_at":"2024-07-17 11:50:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149478,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LED irradiation on tenocyte proliferation of injured Achilles tendon. (A) Immunostaining of Tnmd and SCX in injured Achilles tendon after 2 weeks post-surgery. Nuclei were stained with DAPI. Scale bar = 50 μm. (B) Representative image (upper panel) of western blot analysis of Tnmd and relative protein levels (lower panel) of Tnmd vs GAPDH expression. (C) Representative image (upper panel) of western blot analysis of SCX and relative protein levels (lower panel) of SCX vs GAPDH expression. *p\u0026lt;0.05, **p\u0026lt;0.01. Data indicate mean ±SD. (n = 4 per group)\u003c/p\u003e","description":"","filename":"Figure2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/2c32c46accd600ae416e4d5b.jpg"},{"id":60496776,"identity":"f12d8f6e-4560-4c88-8fe9-2a873bcb908a","added_by":"auto","created_at":"2024-07-17 11:50:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":141001,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LED irradiation on collagen synthesis in injured Achilles tendon. (A) Immunostaining for collagen 1 and 3 in Achilles tendon tissue at 2 weeks post-surgery. Nuclei were stained with DAPI. Scale bar = 50 μm. (B) Representative images of western blot analysis of collagen 1 and 3. Relative protein levels of collagen 1 (C) and collagen 3 (D) vs GAPDH expression. (E) Ratios of collagen 1/collagen 3. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. Data indicate mean ±SD. (n = 4 per group)\u003c/p\u003e","description":"","filename":"Figure3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/8a9b3e7a9afceb64a8482005.jpg"},{"id":60496771,"identity":"5f3ff0e9-505f-4892-bbe3-c6f5ea99b984","added_by":"auto","created_at":"2024-07-17 11:50:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106720,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LED irradiation on myofibroblast formation of injured Achilles tendon. (A) Immunostaining of TGF-β1 in Achilles tendon tissue at 2 weeks post-surgery. Nuclei were stained with DAPI. Scale bar = 50 μm. (B) Representative image of western blot analysis of TGF-β1 and vimentin. (C-D) Quantification of TGF-β1 and vimentin protein levels. The relative protein levels of TGF-β1 (C) and vimentin (D) vs GAPDH were measured. **p\u0026lt;0.01, ***p\u0026lt;0.005. Data indicate mean ±SD. (n = 4 per group)\u003c/p\u003e","description":"","filename":"Figure4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/a3fa0b0303e6fb2597aa2b96.jpg"},{"id":60496773,"identity":"4b09f43d-e937-4101-b231-0bd71e14bfd6","added_by":"auto","created_at":"2024-07-17 11:50:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":130620,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LED irradiation on macrophage activation in injured Achilles tendon. (A) Immunostaining for CD68 and CD163 expression in Achilles tendon tissue at 2 weeks post-surgery. Nuclei were stained with DAPI. Scale bar = 50 μm. (B) Quantification of CD68\u003csup\u003e+\u003c/sup\u003e macrophages in the Achilles tendon tissues. (C) Quantification of CD68\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e M2 macrophages in the Achilles tendon tissues. (D) Comparison of the ratio of M2 macrophage (CD68\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e) / pan macrophage (CD68\u003csup\u003e+\u003c/sup\u003e). *p\u0026lt;0.05, **p\u0026lt;0.01. Data indicate mean ±SD. (n = 4 per group)\u003c/p\u003e","description":"","filename":"Figure5..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/66aed80e02250e3ddfd3ece4.jpg"},{"id":79448686,"identity":"ebc4b778-ee9e-4471-a0ef-0430f14f127c","added_by":"auto","created_at":"2025-03-28 14:32:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1284080,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/3505b10d-13f6-40e1-8e69-9610e30df667.pdf"},{"id":60496774,"identity":"089e3b32-aa8d-41ca-96bd-0e0de8b0ffa9","added_by":"auto","created_at":"2024-07-17 11:50:08","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5008464,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure.tif","url":"https://assets-eu.researchsquare.com/files/rs-4578400/v1/40066c24d71bd05f20d3e960.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of light-emitting diode-mediated photobiomodulation in tendon healing in a murine model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the 1960s, Mester documented the potential applicability of photobiomodulation (PBM) in accelerating wound restoration, following which the National Aeronautics and Space Administration researchers implemented it as a therapeutic regimen to enhance the healing process in space.\u003csup\u003e1,2)\u003c/sup\u003e PBM facilitates healing by stimulating the mitochondrial and cell membrane photoreceptor-based synthesis of ATP, which increases cell viability.\u003csup\u003e3)\u003c/sup\u003e PBM is performed using lasers or light-emitting diodes (LEDs) that radiate light in the red and near-infrared wavelengths.\u003csup\u003e4)\u003c/sup\u003e Numerous studies and trials have been conducted over the past two decades on the clinical application of PBM in the medical and dental fields. PBM is employed in various disciplines of clinical dentistry for post-orthodontic treatment pain alleviation, osseointegration, collagen deposition, and implant stability enhancement.\u003csup\u003e5,6,7)\u003c/sup\u003e PBM has demonstrated additional efficacy in various dermatological interventions, including skin rejuvenation, hair growth, and fat reduction.\u003csup\u003e8,9)\u003c/sup\u003e Moreover, empirical evidence exhibiting the potential of PBM in promoting fibroblast proliferation, growth factor synthesis, collagen production, and angiogenesis has prompted numerous animal experiments and clinical studies in the field of orthopedic surgery.\u003csup\u003e3,10,11,12)\u003c/sup\u003e Nevertheless, specifications on parameters such as wavelength, intensity, and irradiation time associated with PBM therapy have not yet been established, posing limitations on its clinical applicability. Drawing upon prior research, the present investigation assessed the efficacy of PBM on human tendon-derived fibroblasts and revealed over 2-fold cell proliferation and 3-fold cell migration in the PBM-treated group compared to that in the control group.\u003csup\u003e13)\u003c/sup\u003e Furthermore, Vinck et al. reported that both LED and laser proliferate fibroblasts in the Achilles tendon of a rat model.\u003csup\u003e14)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDiseases affecting tendons and ligaments constitute a substantial proportion of orthopedic ailments.\u003csup\u003e15)\u003c/sup\u003e The prevalence of these diseases is rising annually owing to the ongoing burgeoning of elderly and athletic populations; overall, this trend has been associated with high social costs.\u003csup\u003e16,17,18)\u003c/sup\u003e Many treatments are being introduced for these tendon and ligament diseases, including PBM. Although the efficacy of PBM has been established on a theoretical basis, its applicability remains to be validated in a practical or clinical context. Therefore, the aim of this study was to examine the effect of PBM on tendon healing using Achilles tendons in a mouse model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e The animals were housed in an air-conditioned animal room with constant relative humidity and provided with a standard laboratory diet and water as outlined in the Guide for the Care and Use of Laboratory Animals. Animal experiments were performed in adherence to the protocols approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2023-0269). Six-week-old male BALB/c mice weighing 22\u0026ndash;24 g were acquired from Koatech (Gyeonggi-do, Korea). To inflict an injury, the mice were administered intraperitoneal anesthesia with 1.25% avertin (2,2,2-tribromoethanol, 250 mg/kg), and the Achilles tendon was exposed through an approximately 10-mm incision on the medial side of the right hind limb. Subsequently, the Achilles tendon was bilaterally lacerated (2 mm in diameter) per prior methodology with slight modifications (Schramme, M. et al., 2010, Vet Comp Orthop Traumatol, 231\u0026ndash;9). The mice were randomized into two groups: LED non-irradiated (n\u0026thinsp;=\u0026thinsp;4) and LED irradiated (n\u0026thinsp;=\u0026thinsp;4). Mice with intact tendons were employed as healthy controls (n\u0026thinsp;=\u0026thinsp;4). The mice were euthanized 2 weeks following surgery, and tendon tissues were harvested and stored at \u0026minus;\u0026thinsp;80℃ or fixed in acetone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLED irradiation\u003c/h2\u003e \u003cp\u003eThe LED irradiant group was anesthetized and subjected to wound irradiation for 20 min daily for 2 weeks, commencing 24 h post-surgery. LED irradiation was applied to the Achilles tendon injury site at wavelengths of 630 nm (10 mW/cm\u003csup\u003e2\u003c/sup\u003e, 100 Hz) and 880 nm (40 mW/cm\u003csup\u003e2\u003c/sup\u003e, 100 Hz).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eThe mice were euthanized, their Achilles tendons excised, and the tissue specimens fixed overnight in acetone at \u0026minus;\u0026thinsp;20℃ and embedded in an optimum cutting temperature (O.C.T.) compound (Sakura Finetek USA, Inc., Torrance, CA, USA). Tissues were sectioned into 10-\u0026micro;m segments and stained with hematoxylin and eosin (H\u0026amp;E) for histological score analysis. Stained tissue sections were scanned using an Axio Scan.Z1 (Carl Zeiss Microscopy, Germany) at \u0026times;200 magnification. The histological outcomes were scored from 0 (best) to 3 (worst) and evaluated on four parameters: cell density, roundness of nuclei, fiber structure, and fiber arrangement. Two blinded pathologists independently graded the histological results from 0 to 3, which subjectively categorized the tissue samples according to the four aforementioned parameters. Three sections were randomly selected from each sample, and the average scores for each group were compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTendon sections fixed in acetone and embedded in O.C.T. compound were incubated with anti-tenomodulin (bs-7525R; Bioss, Woburn, MA, USA) and anti-SCX antibodies (MBS9612052; MyBioSource, San Diego, CA, USA) to evaluate tenocyte proliferation. Additionally, the collagen in the specimens was stained with anti-collagen1 (bs-10423R; Bioss) and anti-collagen3 antibodies (ab7778; Abcam, Cambridge, UK). The tendon sections were stained with anti-TGF-β1 antibody (MAB240-100; R\u0026amp;D Systems, Minneapolis, MN, USA) to examine for fibrosis and with anti-CD68 (14-0681-82) and anti-CD163 (ab182422; Abcam) antibodies to identify M2 macrophages. Subsequently, the specimens were incubated with secondary antibodies (Alexa Fluor 488, 568, or 647) for 2 h at room temperature, washed, and mounted with a prolonged gold antifade mounting solution. Stained sections were visualized under a confocal microscope (Zeiss). ImageJ software was used to quantify the number of Tenomodulin\u003csup\u003e+\u003c/sup\u003e, SCX\u003csup\u003e+\u003c/sup\u003e, CD68\u003csup\u003e+\u003c/sup\u003e, and M2 macrophages (CD68\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e) in high-power fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTendon tissues were homogenized and lysed using lysis buffer (pH 7.4; 20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM phenylmethyl sulfonyl fluoride, 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 30 mM sodium pyrophosphate, 25 mM β-glycerol phosphate, and 1% Triton X-100) containing protease inhibitors. The protein fractions in the lysates were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Proteins were stained with 0.1% Ponceau S solution (Sigma-Aldrich Co. Ltd., St. Louis, MO, USA) and blocked with 5% nonfat milk. The bound antibodies were visualized using the corresponding horseradish peroxidase-conjugated secondary antibodies. The enhanced chemiluminescence western blotting system (GE-RPN2106) was used for signal detection, and images were captured using an ImageQuant 800 western blot imaging system (GE Healthcare, Chicago, IL, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults from multiple observations are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. A Student\u0026rsquo;s two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test was used to determine the statistical significance of differences between the two groups. Differences between groups were evaluated using one- or two-way analysis of variance (ANOVA), followed by Scheff\u0026eacute;\u0026rsquo;s \u003cem\u003epost hoc\u003c/em\u003e test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLED irradiation accelerated the healing of the Achilles tendon injury\u003c/h2\u003e \u003cp\u003eTo investigate the effects of LED irradiation on Achilles tendon injury repair \u003cem\u003ein vivo\u003c/em\u003e, we used a rat model. Surgical core lesions of the Achilles tendon were produced in the hind limbs of rats, and the LED was irradiated locally, as described in the preceding \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eMaterials and Methods\u003c/span\u003e section. As a control, the injured Achilles tendon was not irradiated with LED. H\u0026amp;E staining was performed to examine whether LED irradiation had a therapeutic effect on Achilles tendon injuries. The lesions could be clearly identified by using H\u0026amp;E staining of the injured Achilles tendon. LED irradiation improved tendon healing without any detectable local adverse effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Next, we analyzed the fiber structure, fiber arrangement, cell density, and roundness of the nuclei in tendon tissues. All four indicators were markedly elevated in the injury group compared to those in the normal group, and fiber structure and arrangement were notably decreased in the LED treatment group compared to those in the injury group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). However, there were no discernible differences in the cell density or roundness of the nuclei of injured tendons between the control and LED-irradiated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). These results suggest that LED treatment promotes fiber regeneration and arrangement, although the infiltration of inflammatory immune cells in the injured tendons remained unaffected\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLED irradiation promoted tenocyte proliferation\u003c/h2\u003e \u003cp\u003eTenocytes are the principal cellular constituents of the tendon and assume various roles during tendon injury. Tenocytes express Tnmd and SCX, which are well-known tenocyte markers. To explore the effects of LED irradiation on tenocyte proliferation during tendon repair, we determined the effect of LED irradiation on the number of tenocytes in the injured tendons. Immunohistochemistry of tendon tissues revealed overexpression of both genes in the injury group compared to that in the normal group and decreased expression in the LED-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot analysis depicted that the expression levels of Tnmd and SCX were higher in the injury group than in the normal group, whereas they were strikingly lower in the LED group than in the injury group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). The histological results showed that LED treatment promoted fiber regeneration and arrangement, confirming the restorative effect of LED. Moreover, on day 14, tenocyte markers decreased and were quickly normalized.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLED irradiation increased the collagen 1/3 expression\u003c/h2\u003e \u003cp\u003eTo explore the effects of LED irradiation on collagen synthesis, we examined the expression levels of collagens 1 and 3 by using immunocytochemical and western blot analyses. Immunocytochemical analysis exhibited that the expression levels of collagens 1 and 3 were notably higher in the injury group than in the normal group and markedly lower in the LED treatment group than in the injury group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Western blot analysis demonstrated that the protein levels of both collagen 1 and 3 increased in injured tendons compared to those in normal tendons, and the expression levels of collagen 3 were discernibly decreased upon LED treatment in injured tendons (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D). The ratio of collagen 1/3 slightly decreased in the injury group compared to that in the normal group but was markedly restored in the LED-treated group to a level comparable to that observed in the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In conjunction with the increased collagen arrangement and structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), these results suggest that LED irradiation stimulates the organization and arrangement of collagen to promote repair, while ensuring reduced inflammatory immune cell infiltration in Achilles tendon injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLED irradiation reduced the degree of fibrosis\u003c/h2\u003e \u003cp\u003eAs LED irradiation rapidly stabilized the repairing site by regulating collagen synthesis and cross-link formation in the injured Achilles tendon, we examined the expression level of TGF-β1 in the injured tendon using immunocytochemistry and western blot analysis. Immunocytochemical analysis evidenced that TGF-β1 expression was increased in the injured tendon compared to that in the normal tendon, and it was markedly reduced by LED treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The expression levels of TGF-β1 and vimentin increased in injured tendon tissue, and the increased expression of TGF-β1 and vimentin was considerably decreased in the LED-treated tendon, suggesting that LED irradiation may reduce fibrosis. (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLED irradiation prevented the inflammatory macrophage infiltration into the injured site\u003c/h2\u003e \u003cp\u003eTo explore whether LED irradiation affected the activation of macrophages in injured Achilles tendons, tendon tissues were immunostained for CD68, a pan-macrophage marker, and CD163, a M2 macrophage marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The experimental findings demonstrated that the number of macrophages was higher in the injury group than in the normal group; however, the number of macrophages in the injured tendon decreased in response to LED treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The number of CD68\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e M2 macrophages was higher in the injury group than in the normal group but was not notably affected by LED irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Nonetheless, the ratio of M2 macrophages to total macrophages was higher in the LED irradiation group than in the injured group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This result suggests that LED irradiation modulates inflammatory response to promote the recovery of the injured Achilles tendon.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study confirmed the initial effect of LED treatment on tendon healing. The histology scores and immunochemistry results exhibited remarkable differences between the LED treatment and control groups.\u003c/p\u003e \u003cp\u003ePBM using low-level laser therapy (LLLT) or LED therapy was first introduced by Mester in the early 1960s. PBM accelerates healing by increasing cell activity by stimulating mitochondrial and cell membrane photoreceptor synthesis of ATP. Such modulations in these cells can promote fibroblast proliferation, growth factor synthesis, collagen production, and angiogenesis. Various laboratory studies and animal experiments have been conducted in the field of orthopedic surgery. Silva et al.\u003csup\u003e19)\u003c/sup\u003e reported a positive effect of LLLT/LED irradiation on tendon damage. Rosso et al.\u003csup\u003e20)\u003c/sup\u003e documented that PBM has beneficial effects on the recovery of nerve lesions. \u003cem\u003eIn vitro\u003c/em\u003e experimental findings suggest that PBM may facilitate tissue homeostasis, thus stimulating articular tissue components and promoting chondroprotective effects.\u003c/p\u003e \u003cp\u003eIn our previous study, LED irradiation at 630 nm and 630 nm\u0026thinsp;+\u0026thinsp;880 nm for 20 min was observed to strikingly affect the proliferation and migration of human tendon fibroblasts. Cell and animal experiments have revealed positive results for PBM in the field of orthopedic surgery, especially in the treatment of ligaments. Future studies are needed to derive clinical results related to this.\u003c/p\u003e \u003cp\u003eRuptured Achilles tendons have been reported to exhibit marked collagen degeneration, disordered arrangement of collagen fibers, augmented cellularity, and an increase in the number of tenocytes with round nuclei.\u003csup\u003e21)\u003c/sup\u003e The failure of the intact tendon healing due to fibrotic scar formation after medical and surgical treatment, which can lead to chronic symptoms and reinjury, is a common issue\u003csup\u003e1\u0026ndash;4)\u003c/sup\u003e. Histologic analysis showed that PBM therapy improved the tendon healing through inhibition of cell density and nuclear circularity. This outcome accelerated fiber regeneration and alignment, and collagen synthesis. The tissue structure in LED-irradiated injured Achilles tendons exhibited greater parallelism, in addition to having a denser deposition of new collagen fibers than that of LED-non-irradiated injured Achilles tendons. These results indicate that LED can shorten the recovery period of the damaged tendon.\u003c/p\u003e \u003cp\u003eCollagen 1 is the main ECM component of tendons, whereas collagen 3, which is generally associated with scar tissue and injury, accumulated at injured tendons. The increased content of type 3 collagen can cause thinner collagen fibers, decrease the tensile strength. \u003csup\u003e22)\u003c/sup\u003e The ratio of collagen 1 and 3 was markedly restored in the LED treated group to a level similar to the normal group. With histologic results, LED irradiation stimulates the organization and arrangement of collagen to promote healing.\u003c/p\u003e \u003cp\u003eTendon injury is associated with tissue regeneration and fibrosis. TGF-β1 is activated upon tendon injury and is key in tendon healing and fibrosis.\u003csup\u003e23)\u003c/sup\u003e The expression of TGF-β1 and vimentin, markers of the presence of myofibroblasts, has been implicated in fibrosis.\u003csup\u003e24)\u003c/sup\u003e TGF-β1 and vimentin levels decreased to the normal range in the LED group at the two-week mark. Although our results do not directly reflect changes in collagen level during the early stages of healing, considering the histological and collagen level results, it is thought that the levels normalized as recovery rapidly progress.\u003c/p\u003e \u003cp\u003eMacrophages key regulators of the healing of injured tendons. For example, an increase in the concentration of Macrophage has been reported to play a key role in regulating the healing process of injured tendons.\u003csup\u003e25)\u003c/sup\u003e The specific function of macrophages depends on their phenotype. While the M1 phenotype macrophage exhibits a phagocytic and proinflammatory function\u003csup\u003e6\u0026ndash;9)\u003c/sup\u003e, the M2 phenotype macrophage is associated with tissue repair and deposition in inflamed tissue\u003csup\u003e6, 7, 10)\u003c/sup\u003e. During tendon healing, increases in the concentration of the M2 macrophage phenotype occur later in the healing process.\u003csup\u003e25)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, the ratio of M2 macrophages to total macrophages increased in the LED irradiation group compared to the injured group. This increase, as driven by LED irradiation, may inhibit abnormal or excessive inflammatory responses. Therefore, LED-facilitated recovery of injured Achilles tendon is controlled through an increased differentiation toward the M2 macrophage phenotype.\u003c/p\u003e \u003cp\u003eOur study had several limitations. First, it was an \u003cem\u003ein vivo\u003c/em\u003e study and owing to its limited sample size and brief duration, comprehending the cumulative healing process is deemed challenging. Second, the mechanism underlying each stage of the healing process could not be ascertained. Therefore, it is imperative to conduct further investigation into the mechanisms and impacts of each process through long-term experiments. Another major limitation of our study lies in the fact that we were not able to conduct mechanical testing in animals. In general, mechanical testing should be performed in conjunction with histology to accurately illuminate the healing trajectory (or lack thereof) of a tendon. To address this limitation in future studies, we intend to conduct mechanical testing after PBM irradiation to illuminate more accurate dynamics of tendon healing.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHistological and immunochemical outcomes evidenced the effectiveness of LED-based PBM in stimulating rapid recovery in a murine model of Achilles tendon rupture. These results suggest that LED-mediated PBM possesses considerable potential as an adjunct treatment for tendon healing and warrants further research to standardize various parameters to develop and establish PBM as a reliable treatment regime.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003elight-emitting diode (LED), photobiomodulation (PBM), low-level laser therapy (LLLT)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eEthics approval and consent to participate:\u0026nbsp;Animal experiments were performed in adherence to the protocols approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2023-0269).\u003c/li\u003e\n \u003cli\u003eConsent for publication: Not applicable\u003c/li\u003e\n \u003cli\u003eAvailability of data and materials: The datasets used and/or anylsed during the current study are availalbe from the corresponding author on reasonable request.\u003c/li\u003e\n \u003cli\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/li\u003e\n \u003cli\u003eFunding: Not applicable. The authors have nothing to close\u003c/li\u003e\n \u003cli\u003eAuthors\u0026apos; contributions: Conceived and desinged the experiments:SWK, JHK. Performed the experiments:JKL, GTP,MKC. Contributed reagents/materials/analysis tools: MKC, Wrote the paper:SWK, JKL. All authors read and approved the final mauscpript.\u003c/li\u003e\n \u003cli\u003eAcknowledgements: This study was supported by Research institue for Convergence of biomedical science and technoloty,Pusan National University Yansan Hospital (30-2022-016)\u003c/li\u003e\n \u003cli\u003eAuthors\u0026apos; information; Department of Orthopaedic, Pusan National University Yangsan Hospital, Yangsan, Kore\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Jae Kyung Lim, Jae Ho Kim, Gyu Tae Park, Min Kyung Cho, Seung Hun Woo\u0026nbsp;\u0026amp;\u0026nbsp;Suk Woong Kang\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Corresponding author : Suk Woong Kang\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMester A, Mester A. The history of photobiomodulation: endre mester (1903\u0026ndash;1984). Photomed Laser Surg. 2017;35:393\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhelan HT, Houle JM, Whelan NT, et al. editors. The NASA light-emitting diode medical program\u0026mdash;progress in space flight and terrestrial applications. AIP Conference Proceedings; 2000: American Institute of physics.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Freitas LF, Hamblin MR. Proposed mechanisms of photobiomodulation or low-level light therapy. IEEE J Sel Top Quantum Electron. 2016;22(3):348\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDompe C, Moncrieff L, Matys J, et al. Photobiomodulation\u0026mdash;underlying mechanism and clinical applications. J Clin Med. 2020;9(6):1724.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatys J, Świder K, Grzech-Leśniak K, Dominiak M, Romeo U. Photobiomodulation by a 635 nm Diode Laser on Peri-Implant Bone: Primary and Secondary Stability and Bone Density Analysis\u0026mdash;A Randomized Clinical Trial. Biomed Res Int. 2019;2019:2785302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArt\u0026eacute;s-Ribas M, Arnabat-Dominguez J, Puigdollers A. Analgesic effect of a low-level laser therapy (830 nm) in early orthodontic treatment. Lasers Med Sci. 2013;28:335\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenc G, Kocadereli İ, Tasar F, Kilinc K, El S, Sarkarati B. Effect of low-level laser therapy (LLLT) on orthodontic tooth movement. Lasers Med Sci. 2013;28:41\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWikramanayake TC, Rodriguez R, Choudhary S, Mauro LM, Nouri K, Schachner LA, Jimenez JJ. Effects of the Lexington LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia areata. Lasers Med Sci. 2012;27:431\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarolet D, Roberge CJ, Auger FA, Boucher A, Germain L. Regulation of Skin Collagen Metabolism In Vitro Using a Pulsed 660nm LED Light Source: Clinical Correlation with a Single-Blinded Study. J Invest Dermatol. 2009;129:2751\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDompe C, Moncrieff L, Matys J, et al. Photobiomodulation\u0026mdash;underlying mechanism and clinical applications. J Clin Med. 2020;9(6):1724.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHosseinpour S, Fekrazad R, Arany PR, Ye Q. Molecular impacts of photobiomodulation on bone regeneration: a systematic review. Prog Biophys Mol Biol. 2019;149:147\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosso MPO, Buchaim DV, Kawano N, Furlanette G, Pomini KT, Buchaim RL. Photobiomodulation therapy (PBMT) in peripheral nerve regeneration: a systematic review. Bioengineering. 2018;5(2):44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu JH, Park J, Kim JW, et al. Exploring the Effects of 630 nm Wavelength of Light-Emitting Diode Irradiation on the Proliferation and Migration Ability of Human Biceps Tendon Fibroblast Cells. Clin Orthop Surg. 2023;12(1):166\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVinck EM, et al. Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation. Lasers Med Sci. 2003;18(2):95\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClayton RA, Court-Brown CM. The epidemiology of musculoskeletal tendinous and ligamentous injuries. Inj. 2008;39(12):1338\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJames R, Kesturu G, Balian G, Chhabra AB. Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options. J Hand Surg. 2008;33(1):102\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMock C, Cherian MN. The global burden of musculoskeletal injuries: challenges and solutions. Clin Orthop Relat Res. 2008;466(10):2306\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVitale MA, Vitale MG, Zivin JG, Braman JP, Bigliani LU, Flatow EL. Rotator cuff repair: an analysis of utility scores and cost-effectiveness. J Shoulder Elb Surg. 2007;16(2):181\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopes Silva RSD, Pessoa DR, Mariano RR, Castro ABS, de Oliveira RA, Ferraresi C. Systematic review of photobiomodulation therapy (PBMT) on the experimental calcaneal tendon injury in rats. Photochem Photobiol. 2020;96(5):981\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosso MPO, Buchaim DV, Kawano N, Furlanette G, Pomini KT, Buchaim RL. Photobiomodulation therapy (PBMT) in peripheral nerve regeneration: a systematic review. Bioengineering. 2018;5(2):44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaffulli N, Barrass V, Ewen S. Light microscopic history of Achilles tendon ruptures. Am J Sports Med. 2000;28(6):857\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEriksen HA, Pajala A, Leppilahti J, Risteli J. Increased content of type III collagen at the rupture site of human Achilles tendon. J Orthop Res. 2002;20(6):1352\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuneja SC, Schwarz EM, O\u0026rsquo;Keefe RJ, Awad HA. Cellular and molecular factors in flexor tendon repair and adhesions: A histological and gene expression analysis. Connect Tissue Res. 2013;54(3):218\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarconi GD, Fonticoli L, Rajan, et al. Epithelial-mesenchymal transition (EMT): The type-2 EMT in wound healing, tissue regeneration and organ fibrosis. Cells. 2021;10(7):1587.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSunwoo JY, Eliasberg CD, Carballo CB, Rodeo SA. The role of the macrophage in tendinopathy and tendon healing. J Orthop Res. 2020;38(8):1666\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Photobiomodulation, Light-emitting diode, Tendon","lastPublishedDoi":"10.21203/rs.3.rs-4578400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4578400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe application of light-emitting diode (LED)-dependent photobiomodulation (PBM) in promoting post-tendon injury healing has been recently reported. Despite establishing a theoretical basis for ligament restoration through PBM, the lack of empirical evidence deems this therapeutic strategy contentious. Therefore, the aim of this study was to investigate the potency of LED-based PBM in facilitating tendon healing in a murine model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMigration kinetics were analyzed at two specific wavelengths: 630 and 880 nm. The Achilles tendon in the hind limbs of Balb/c mice was severed by Achilles tendon transection. Subsequently, the mice were randomized into LED non-irradiation and LED irradiation groups. Mice with intact tendons were employed as healthy controls. The wounds were LED-irradiated for 20 min daily for two days. Histological properties, tendon healing mediators, and inflammatory mediators were screened on day 14.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe roundness of the nuclei and fiber structure, indicating the degree of infiltrated inflammatory cells and severity of fiber fragmentation, respectively, were lower in the LED irradiation group than in the LED non-irradiation group. Immunohistochemical analysis depicted an increase in tenocytes (SCX\u003csup\u003e+\u003c/sup\u003e cells) and a recovery of wounds with reduced fibrosis (lower collagen 3 and TGF-β1) in the LED irradiation group during healing; conversely, the LED non-irradiation group exhibited tissue fibrosis. Overall, the ratio of M2 macrophages to total macrophages in the LED irradiation group was higher than that in the injured group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eLED-based PBM in the Achilles tendon rupture murine model effectuated a rapid restoration of histological and immunochemical outcomes. These findings suggest that LED-based PBM presents remarkable potential as an adjunct therapeutic for tendon healing and warrants further research to standardize various parameters to advance and establish it as a reliable treatment regime.\u003c/p\u003e","manuscriptTitle":"Efficacy of light-emitting diode-mediated photobiomodulation in tendon healing in a murine model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 11:50:03","doi":"10.21203/rs.3.rs-4578400/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d140fbfd-fb4a-4c30-93a3-c8814f35ba9d","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-28T14:23:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-17 11:50:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4578400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4578400","identity":"rs-4578400","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}