Low intensity near-infrared light promotes bone regeneration via circadian clock protein cryptochrome 1

Abstract

Bone regeneration remains a great clinical challenge. Low intensity near-infrared (NIR) light showed strong potential to promote tissue regeneration, offering a promising strategy for bone defect regeneration. However, the effect and underlying mechanism of NIR on bone regeneration remain unclear. We demonstrated that bone regeneration in the rat skull defect model was significantly accelerated with low-intensity NIR stimulation. In vitro studies showed that NIR stimulation could promote the osteoblast differentiation in bone mesenchymal stem cells (BMSCs) and MC3T3-E1 cells, which was associated with increased ubiquitination of the core circadian clock protein Cryptochrome 1 (CRY1) in the nucleus. We found that the reduction of CRY1 induced by NIR light activated the bone morphogenetic protein (BMP) signaling pathways, promoting SMAD1/5/9 phosphorylation and increasing the expression levels of Runx2 and Osterix. NIR light treatment may act through sodium voltage-gated channel Scn4a, which may be a potential responder of NIR light to accelerate bone regeneration. Together, these findings suggest that low-intensity NIR light may promote in situ bone regeneration in a CRY1-dependent manner, providing a novel, efficient and non-invasive strategy to promote bone regeneration for clinical bone defects.

References

[1] De Santis, R., Guarino, V. & Ambrosio, L. In Bone Repair Biomaterials (eds. Planell, J.A.et al.) 252–270 (Woodhead Publishing, 2009).

[2] Campana V, . Bone substitutes in orthopaedic surgery: from basic science to clinical practice. J. Mater. Sci. Mater. Med., 2014, 25: 2445-2461.

[3] Sun H, . CD271 antibody-functionalized microspheres capable of selective recruitment of reparative endogenous stem cells for in situ bone regeneration. Biomaterials, 2022, 280: 121243.

[4] Holt, B. D., Wright, Z. M., Arnold, A. M. & Sydlik, S. A. Graphene oxide as a scaffold for bone regeneration. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. https://doi.org/10.1002/wnan.1437 (2017).

[5] Walmsley GG, . Nanotechnology in bone tissue engineering. Nanomed. Nanotechnol. Biol. Med., 2015, 11: 1253-1263.

[6] Sanders DW, . Critical-sized defect in the tibia: is it critical? Results from the SPRINT trial. J. Orthop. trauma, 2014, 28: 632-635.

[7] Van Heest A, Swiontkowski M. Bone-graft substitutes. Lancet (Lond., Engl.), 1999, 353: Si28-Si29.

[8] Gaharwar AK, Singh I, Khademhosseini A. Engineered biomaterials for in situ tissue regeneration. Nat. Rev. Mater., 2020, 5: 686-705.

[9] Yu X, . Mechanically reinforced injectable bioactive nanocomposite hydrogels for in-situ bone regeneration. Chem. Eng. J., 2022, 433: 132799.

[10] Cipitria A, . In-situ tissue regeneration through SDF-1α driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect. Acta Biomater., 2017, 60: 50-63.

[11] Park IS, Chung PS, Ahn JC. Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hind limb ischemia mice. Biomaterials, 2014, 35: 9280-9289.

[12] Nagata MJH, . Bone marrow aspirate combined with low-level laser therapy: a new therapeutic approach to enhance bone healing. J. Photochem. Photobiol. B Biol., 2013, 121: 6-14.

[13] Choi K, . Low-level laser therapy promotes the osteogenic potential of adipose-derived mesenchymal stem cells seeded on an acellular dermal matrix. J. Biomed. Mater. Res. B Appl. Biomater., 2013, 101: 919-928.

[14] Yang CC, Wang J, Chen SC, Hsieh YL. Synergistic effects of low-level laser and mesenchymal stem cells on functional recovery in rats with crushed sciatic nerves. J. Tissue Eng. Regen. Med., 2016, 10: 120-131.

[15] Dos Santos KW, Hugo FN, da Cunha Rodrigues E, Stein AT, Hilgert JB. Effect of oral exercises and photobiomodulation therapy in the rehabilitation of patients with mandible fractures: randomized double-blind clinical trial. Lasers Med. Sci., 2022, 37: 1727-1735.

[16] Park JH, . Effect of photobiomodulation therapy on radiodermatitis in a mouse model: an experimental animal study. Lasers Med. Sci., 2021, 36: 843-853.

[17] Nelidova D, . Restoring light sensitivity using tunable near-infrared sensors. Science, 2020, 368: 1108-1113.

[18] Al-Shammari, A. M., Syhood, Y. & Al-Khafaji, A. S., Use of low-power He-Ne laser therapy to accelerate regeneration processes of injured sciatic nerve in rabbit. Egypt J. Neurol. Psychiatr. Neurosurg. https://doi.org/10.1186/s41983-018-0047-6 (2019).

[19] Wang C-Z, . Low-level laser irradiation improves functional recovery and nerve regeneration in sciatic nerve crush rat injury model. PLoS One, 2014, 9: e103348.

[20] Salehpour F, . Penetration profiles of visible and near-infrared lasers and light-emitting diode light through the head tissues in animal and human species: a review of literature. Photobiomodul. Photomed. Laser Surg., 2019, 37: 581-595.

[21] Metin R, Tatli U, Evlice B. Effects of low-level laser therapy on soft and hard tissue healing after endodontic surgery. Lasers Med. Sci., 2018, 33: 1699-1706.

[22] Ip D, Fu NY. Can combined use of low-level lasers and hyaluronic acid injections prolong the longevity of degenerative knee joints?. Clin. Interv. Aging, 2015, 10: 1255-1258.

[23] Melo Mde O, . Effects of neuromuscular electrical stimulation and low-level laser therapy on the muscle architecture and functional capacity in elderly patients with knee osteoarthritis: a randomized controlled trial. Clin. Rehabil., 2015, 29: 570-580.

[24] Bai J, . Low level laser therapy promotes bone regeneration by coupling angiogenesis and osteogenesis. Stem Cell Res. Ther., 2021, 12: 432.

[25] Hashmi JT, . Role of low-level laser therapy in neurorehabilitation. PM R, 2010, 2: S292-S305.

[26] Xie Y, . New insights into the circadian rhythm and its related diseases. Front. Physiol., 2019, 10: 682.

[27] Haltaufderhyde K, Ozdeslik RN, Wicks NL, Najera JA, Oancea E. Opsin expression in human epidermal skin. Photochem. Photobiol., 2015, 91: 117-123.

[28] Campbell SS, Murphy PJ. Extraocular circadian phototransduction in humans. Science, 1998, 279: 396-399.

[29] Dibner C, Schibler U, Albrecht U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu. Rev. Physiol., 2010, 72: 517-549.

[30] Xu C, . Circadian clock regulates bone resorption in mice. J. Bone Miner. Res. Res., 2016, 31: 1344-1355.

[31] Lieben L. Bone: the circadian clock controls bone remodelling. Nat. Rev. Rheumatol., 2016, 12: 132.

[32] He Y, Chen Y, Zhao Q, Tan Z. Roles of brain and muscle ARNT-like 1 and Wnt antagonist Dkk1 during osteogenesis of bone marrow stromal cells. Cell Prolif., 2013, 46: 644-653.

[33] Guntur AR, . An essential role for the circadian-regulated gene nocturnin in osteogenesis: the importance of local timekeeping in skeletal homeostasis. Ann. N. Y. Acad. Sci., 2011, 1237: 58-63.

[34] McElderry JD, . Tracking circadian rhythms of bone mineral deposition in murine calvarial organ cultures. J. Bone Miner. Res., 2013, 28: 1846-1854.

[35] Yu S, . Circadian BMAL1 regulates mandibular condyle development by hedgehog pathway. Cell Prolif., 2020, 53: e12727.

[36] Zhou X, . BMAL1 deficiency promotes skeletal mandibular hypoplasia via OPG downregulation. Cell Prolif., 2018, 51: e12470.

[37] Zhao J, . BMAL1 deficiency contributes to mandibular dysplasia by upregulating MMP3. Stem Cell Rep., 2018, 10: 180-195.

[38] Bass J, Lazar MA. Circadian time signatures of fitness and disease. Science, 2016, 354: 994-999.

[39] Busino L, . SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science, 2007, 316: 900-904.

[40] Hirano A, Braas D, Fu YH, Ptáček LJ. FAD regulates CRYPTOCHROME protein stability and circadian clock in mice. Cell Rep., 2017, 19: 255-266.

[41] Hirota T, . Identification of small molecule activators of cryptochrome. Science, 2012, 337: 1094-1097.

[42] Ukon Y, . Prostaglandin EP4 selective agonist AKDS001 Enhances New Bone Formation by Minimodeling in a Rat Heterotopic Xenograft Model of Human Bone. Front. Bioeng. Biotechnol., 2022, 10: 845716.

[43] Mao W, . Phloretin ameliorates diabetes-induced endothelial injury through AMPK-dependent anti-EndMT pathway. Pharmacol. Res., 2022, 179: 106205.

[44] Wu, Z. et al. Regulating macrophage polarization in high glucose microenvironment using lithium-modified bioglass-hydrogel for diabetic bone regeneration. Adv. Healthcare Mater. 11, e2200298 (2022).

[45] Yu LN, . Direct modulation of hepatocyte hepcidin signaling by iron. World J. Hepatol., 2021, 13: 1378-1393.

[46] Kim, Y. J., Park, W. R., Choi, B., Choi, H. S. & Kim, D. K. Epigallocatechin-3-gallate suppresses BMP-6-mediated SMAD1/5/8 transactivation of hepcidin gene by inducing SMILE in hepatocytes. Antioxidants (Basel) 10, 1590 (2021).

[47] Katakawa Y, Funaba M, Murakami M. Smad8/9 is regulated through the BMP pathway. J. Cell. Biochem., 2016, 117: 1788-1796.

[48] Nemoto E, . Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis. Biochemical Biophys. Res. Commun., 2012, 422: 627-632.

[49] Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in human bone marrow-derived mesenchymal stem cells: Consequences for cell therapies. Mechanisms Age. Dev., 2008, 129: 163-173.

[50] Mohamad SA, Milward MR, Hadis MA, Kuehne SA, Cooper PR. Photobiomodulation of mineralisation in mesenchymal stem cells. Photochem. Photobiol. Sci., 2021, 20: 699-714.

[51] Okuno T. Thermal effect of visible light and infra-red radiation (i.r.-A, i.r.-B and i.r.-C) on the eye: a study of infra-red cataract based on a model. Ann. Occup. Hyg., 1994, 38: 351-359.

[52] Cho S, . Effects of infrared radiation and heat on human skin aging in vivo. J. Investigative Dermatol. Symp . Proc., 2009, 14: 15-19.

[53] Glavaš H, Vukobratović M, Keser T. Infrared thermography as control of handheld IPL device for home-use. J. Cosmet. Laser Ther., 2018, 20: 269-277.

[54] Huang YY, Sharma SK, Carroll J, Hamblin MR. Biphasic dose response in low level light therapy—an update. Dose Response, 2011, 9: 602-618.

[55] Tan L, . Rapid biofilm eradication on bone implants using red phosphorus and near-infrared light. Adv. Mater., 2018, 30: e1801808.

[56] Chen Q, . Photothermal therapy promotes tumor infiltration and antitumor activity of CAR T cells. Adv. Mater., 2019, 31: 1900192.

[57] Ma L, . A novel photothermally controlled multifunctional scaffold for clinical treatment of osteosarcoma and tissue regeneration. Mater. Today, 2020, 36: 48-62.

[58] Chow, R. W. & Vermot, J. The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology. F1000Res. https://doi.org/10.12688/f1000research.10617.1 (2017).

[59] Nagel G, . Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl Acad. Sci. USA, 2003, 100: 13940-13945.

[60] Grimm C, Remé CE, Rol PO, Williams TP. Blue light’s effects on rhodopsin: photoreversal of bleaching in living rat eyes. Investigative Ophthalmol. Vis. Sci., 2000, 41: 3984-3990.

[61] Tsukamoto H, Furutani Y. Optogenetic modulation of ion channels by photoreceptive proteins. Adv. Exp. Med. Biol., 2021, 1293: 73-88.

[62] Paoletti P, Ellis-Davies GCR, Mourot A. Optical control of neuronal ion channels and receptors. Nat. Rev. Neurosci., 2019, 20: 514-532.

[63] Chiou YY, . Mammalian period represses and de-represses transcription by displacing CLOCK-BMAL1 from promoters in a Cryptochrome-dependent manner. Proc. Natl Acad. Sci. USA, 2016, 113: E6072-e6079.

[64] St John PC, Hirota T, Kay SA, Doyle FJ 3rd. Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock. Proc. Natl Acad. Sci. USA, 2014, 111: 2040-2045.

[65] Yumimoto K, Muneoka T, Tsuboi T, Nakayama KI. Substrate binding promotes formation of the Skp1-Cul1-Fbxl3 (SCFFbxl3) protein complex*. J. Biol. Chem., 2013, 288: 32766-32776.

[66] Kim YY, . Hepatic GSK3β-dependent CRY1 degradation contributes to diabetic hyperglycemia. Diabetes, 2022, 71: 1373-1387.

[67] Czarna A, . Structures of drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function. Cell, 2013, 153: 1394-1405.

[68] Prabhakar, S.S. in Medical Secrets 5th edn (ed. Harward, M.P.) Ch. 9 (Mosby, Saint Louis, 2012).

[69] Yasuda S, . Stress- and ubiquitylation-dependent phase separation of the proteasome. Nature, 2020, 578: 296-300.

[70] Jaitovich A, Bertorello AM. Intracellular sodium sensing: SIK1 network, hormone action and high blood pressure. Biochimica et. Biophysica Acta, 2010, 1802: 1140-1149.

[71] Chaves, I. et al. Functional evolution of the photolyase/cryptochrome protein family: importance of the C terminus of mammalian CRY1 for circadian core oscillator performance. Mol Cell Biol 26, 1743–1753 (2006).

[72] Levraut J, . Initial effect of sodium bicarbonate on intracellular pH depends on the extracellular nonbicarbonate buffering capacity. Crit. Care Med., 2001, 29: 1033-1039.

[73] Parico GCG, Partch CL. The tail of cryptochromes: an intrinsically disordered cog within the mammalian circadian clock. Cell Commun. Signal., 2020, 18

[74] van der Horst GT, . Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature, 1999, 398: 627-630.

[75] Vitaterna MH, . Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc. Natl Acad. Sci. USA, 1999, 96: 12114-12119.

[76] Okano S, Akashi M, Hayasaka K, Nakajima O. Unusual circadian locomotor activity and pathophysiology in mutant CRY1 transgenic mice. Neurosci. Lett., 2009, 451: 246-251.

[77] Hatori M, Panda S. CRY links the circadian clock and CREB-mediated gluconeogenesis. Cell Res., 2010, 20: 1285-1288.

[78] Yang L, . Overexpression of CRY1 protects against the development of atherosclerosis via the TLR/NF-κB pathway. Int. Immunopharmacol., 2015, 28: 525-530.

[79] Zhou L, . Cryptochrome 1 regulates osteoblast differentiation via the AKT kinase and extracellular signal-regulated kinase signaling pathways. Cell. Reprogram., 2019, 21: 141-151.

[80] Fu L, Patel MS, Bradley A, Wagner EF, Karsenty G. The molecular clock mediates leptin-regulated bone formation. Cell, 2005, 122: 803-815.

[81] Chen L, . A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity. Nat. Genet., 2019, 51: 777-785.

[82] Antebi YE, . Combinatorial signal perception in the BMP pathway. Cell, 2017, 170: 1184-1196.e1124.

[83] Xue X, . Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nat. Mater., 2018, 17: 633-641.

[84] Narasimamurthy R, . Circadian clock protein cryptochrome regulates the expression of proinflammatory cytokines. Proc. Natl Acad. Sci. USA, 2012, 109: 12662-12667.

[85] Zhou W, . Endogenous parathyroid hormone promotes fracture healing by increasing expression of BMPR2 through cAMP/PKA/CREB pathway in mice. Cell. Physiol. Biochem., 2017, 42: 551-563.

[86] Wang C, . Defect-related luminescent hydroxyapatite-enhanced osteogenic differentiation of bone mesenchymal stem cells via an ATP-induced cAMP/PKA pathway. ACS Appl. Mater. interfaces, 2016, 8: 11262-11271.

[87] Allada R, Bass J. Circadian mechanisms in medicine. N. Engl. J. Med., 2021, 384: 550-561.

[88] Huang Z, . Icariin promotes osteogenic differentiation of BMSCs by upregulating BMAL1 expression via BMP signaling. Mol. Med. Rep., 2020, 21: 1590-1596.

[89] Zhu M, . BMAL1 suppresses ROS-induced endothelial-to-mesenchymal transition and atherosclerosis plaque progression via BMP signaling. Am. J. Transl. Res., 2018, 10: 3150-3161.

[90] Nam D, . The adipocyte clock controls brown adipogenesis through the TGF-β and BMP signaling pathways. J. Cell Sci., 2015, 128: 1835-1847.

[91] Tsukamoto-Yamauchi N, Terasaka T, Iwasaki Y, Otsuka F. Interaction of pituitary hormones and expression of clock genes modulated by bone morphogenetic protein-4 and melatonin. Biochemical Biophysical Res. Commun., 2015, 459: 172-177.

[92] Diamantino AG, . Effect of non-coherent infrared light (LED, λ945 ± 20 nm) on bone repair in diabetic rats-morphometric and spectral analyses. Lasers Med. Sci., 2017, 32: 1041-1049.

[93] Gunji, H. et al. Effect of high-frequency near-infrared diode laser irradiation on periodontal tissues during experimental tooth movement in rats. Lasers Surg. Med. https://doi.org/10.1002/lsm.22797 (2018).

[94] Agas, D. et al. Photobiomodulation by near-infrared 980-nm wavelengths regulates pre-osteoblast proliferation and viability through the PI3K/Akt/Bcl-2 pathway. Int. J. Mol. Sci. 22, 7586 (2021).

[95] Liu, Y. et al. Built-in electric fields dramatically induce enhancement of osseointegration. Adv. Funct. Mater. 27, 1703771 (2017).

Funding

This work was funded by the National Key Research and Development Program of China (2021YFC2400404, to LC), the Key Program of National Natural Science of China (82030070, to LC), the National Science Foundation for Excellent Young Scholars of China (31725011, to LC), and the Youth Clinical Research Fund of Chinese Stomatological Association (CSA-O2020-10, to QT).