Melatonin-loaded gelatin methacryloyl microspheres delay nucleus pulposus degeneration

doi:10.12307/2024.213

Abstract

Abstract: BACKGROUND: Nucleus pulposus degeneration is an important pathological link of intervertebral disc degeneration. Melatonin has a protective effect on cells through anti-inflammatory and antioxidant pathways, but the effect of melatonin on the nucleus pulposus has been less studied. At present, the emergence of various biological scaffolders provides a new idea for the study of drug-material combinations.
OBJECTIVE: To explore whether melatonin can improve the metabolic state of the nucleus pulposus by reducing oxidative stress damage as well as the effect of gelatin methacryloyl (GelMA) microspheres loaded with melatonin on intervertebral disc degeneration in vivo.
METHODS: In vitro, melatonin was combined with GelMA solution, and GelMA hydrogel was prepared into microspheres by microfluidic technology to co-culture with nucleus pulposus cells. The cell proliferation activity was detected by cell counting kit-8 assay, the surface morphology of the microspheres was observed under scanning electron microscopy, and the rate of drug release was detected by ultraviolet spectrophotometer. Then, interleukin-1β was used to induce degeneration of the nucleus pulposus. After treatment, the expression levels of aggrecan, type II collagen α1, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5) in the nucleus pulposus were detected by qRT-PCR. In vivo, nucleus pulposus degeneration was induced by puncture. Subsequently, GelMA and GelMA@MT microspheres were injected. After 6 weeks, the specimens were taken for tissue staining, and the changes in tissue morphology were observed under the microscope for histological analysis and scoring.
RESULTS AND CONCLUSION: (1) When the GelMA and GelMA@MT microspheres were observed under electron scanning microscope, melatonin binding did not change the morphology and external appearance of the microspheres. Drug release experiments showed that the drug release reached about 80% after 40 days. (2) Cell counting kit-8 assay results showed that both GelMA and GelMA@MT microspheres had no obvious cytotoxicity and promoted the proliferation of nucleus pulposus cells. (3) qRT-PCR results revealed that GelMA@MT microspheres increased the expression of aggrecan and type I collagen α1 in the interleukin 1β environment by 42.1% and 27.1%, respectively, and decreased the expression of matrix metalloproteinase 13 and ADAMTS5 by 70.7% and 109.3%, respectively. (4) The level of reactive oxygen species was significantly lower in the interleukin 1β+GelMA@MT group than in the interleukin 1β and interleukin 1β+GelMA groups. (5) Histological staining of the sections showed that melatonin-loaded GelMA microspheres significantly delayed disc degeneration in vivo. (6) These findings indicate that GelMA@MT microspheres made by combining melatonin with GelMA hydrogel have good cytocompatibility in vitro and significantly delay nucleus pulposus degeneration in vitro and in vivo.

Key words: melatonin, nucleus pulposus, intervertebral disc, microsphere, GelMA

CLC Number:

Li Yangfeng, Tian Xin, He Fan, Yang Huilin. Melatonin-loaded gelatin methacryloyl microspheres delay nucleus pulposus degeneration[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(5): 676-681.

Figures/Tables 4

References

[1] BINCH ALA, FITZGERALD JC, GROWNEY EA, et al. Cell-based strategies for IVD repair: clinical progress and translational obstacles. Nat Rev Rheumatol. 2021;17(3):158-175.
[2] LYU FJ, CUI H, PAN H, et al. Painful intervertebral disc degeneration and inflammation: from laboratory evidence to clinical interventions. Bone Res. 2021;9(1):7.
[3] KAMALI A, ZIADLOU R, LANG G, et al. Small molecule-based treatment approaches for intervertebral disc degeneration: Current options and future directions. Theranostics. 2021;11(1):27-47.
[4] KERR GJ, VERAS MA, KIM MK, et al. Decoding the intervertebral disc: Unravelling the complexities of cell phenotypes and pathways associated with degeneration and mechanotransduction. Semin Cell Dev Biol. 2017;62:94-103.
[5] SILAGI ES, SHAPIRO IM, RISBUD MV. Glycosaminoglycan synthesis in the nucleus pulposus: Dysregulation and the pathogenesis of disc degeneration. Matrix Biol. 2018;71-72:368-379.
[6] WANG Y, CHE M, XIN J, et al. The role of IL-1beta and TNF-alpha in intervertebral disc degeneration. Biomed Pharmacother. 2020;131: 110660.
[7] RISBUD MV, SHAPIRO IM. Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol. 2014;10(1): 44-56.
[8] VASEY C, MCBRIDE J, PENTA K. Circadian Rhythm Dysregulation and Restoration: The Role of Melatonin. Nutrients. 2021;13(10):3480.
[9] SATO K, MENG F, FRANCIS H, et al. Melatonin and circadian rhythms in liver diseases: Functional roles and potential therapies. J Pineal Res. 2020;68(3):e12639.
[10] CHENG Z, XIANG Q, WANG J, et al. The potential role of melatonin in retarding intervertebral disc ageing and degeneration: A systematic review. Ageing Res Rev. 2021;70:101394.
[11] NABAVI SM, NABAVI SF, SUREDA A, et al. Anti-inflammatory effects of Melatonin: A mechanistic review. Crit Rev Food Sci Nutr. 2019; 59(sup1):S4-S16.
[12] LI Z, LI X, CHEN C, et al. Melatonin inhibits nucleus pulposus (NP) cell proliferation and extracellular matrix (ECM) remodeling via the melatonin membrane receptors mediated PI3K-Akt pathway. J Pineal Res. 2017;63(3). doi: 10.1111/jpi.12435.
[13] CHEN F, JIANG G, LIU H, et al. Melatonin alleviates intervertebral disc degeneration by disrupting the IL-1beta/NF-kappaB-NLRP3 inflammasome positive feedback loop. Bone Res. 2020;8:10.
[14] HE Q, ZHANG J, LIAO Y, et al. Current advances in microsphere based cell culture and tissue engineering. Biotechnol Adv. 2020;39:107459.
[15] YUE K, TRUJILLO-DE SANTIAGO G, ALVAREZ MM, et al. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials. 2015;73:254-271.
[16] XU P, JIANG F, ZHANG H, et al. Calcium Carbonate/Gelatin Methacrylate Microspheres for 3D Cell Culture in Bone Tissue Engineering. Tissue Eng Part C Methods. 2020;26(8):418-432.
[17] SHAO L, PAN B, HOU R, et al. User-friendly microfluidic manufacturing of hydrogel microspheres with sharp needle. Biofabrication. 2022; 14(2). doi: 10.1088/1758-5090/ac57a5.
[18] XU H, SUN M, WANG C, et al. Growth differentiation factor-5-gelatin methacryloyl injectable microspheres laden with adipose-derived stem cells for repair of disc degeneration. Biofabrication. 2020;13(1): 015010.
[19] CHEN P, XIA C, MEI S, et al. Intra-articular delivery of sinomenium encapsulated by chitosan microspheres and photo-crosslinked GelMA hydrogel ameliorates osteoarthritis by effectively regulating autophagy. Biomaterials. 2016;81:1-13.
[20] FONTANA G, SEE E, PANDIT A. Current trends in biologics delivery to restore intervertebral disc anabolism. Adv Drug Deliv Rev. 2015;84: 146-158.
[21] ZHAO X, MA H, HAN H, et al. Precision medicine strategies for spinal degenerative diseases: Injectable biomaterials with in situ repair and regeneration. Mater Today Bio. 2022;16:100336.
[22] CLOUET J, FUSELLIER M, CAMUS A, et al. Intervertebral disc regeneration: From cell therapy to the development of novel bioinspired endogenous repair strategies. Adv Drug Deliv Rev. 2019;146:306-324.
[23] ROH EJ, DARAI A, KYUNG JW, et al. Genetic Therapy for Intervertebral Disc Degeneration. Int J Mol Sci. 2021;22(4):1579.
[24] YANG W, YU XH, WANG C, et al. Interleukin-1beta in intervertebral disk degeneration. Clin Chim Acta. 2015;450:262-272.
[25] FENG C, YANG M, LAN M, et al. ROS: Crucial Intermediators in the Pathogenesis of Intervertebral Disc Degeneration. Oxid Med Cell Longev. 2017;2017:5601593.
[26] HE R, CUI M, LIN H, et al. Melatonin resists oxidative stress-induced apoptosis in nucleus pulposus cells. Life Sci. 2018;199:122-130.
[27] LI B, LI H, YANG H, et al. Preparation and antibacterial properties of an AgBr@SiO(2)/GelMA composite hydrogel. Biomed Mater. 2022;17(2). doi: 10.1088/1748-605X/ac49f7.
[28] CHEN J, HUANG D, WANG L, et al. 3D bioprinted multiscale composite scaffolds based on gelatin methacryloyl (GelMA)/chitosan microspheres as a modular bioink for enhancing 3D neurite outgrowth and elongation. J Colloid Interface Sci. 2020;574:162-173.
[29] GUPTA V, KHAN Y, BERKLAND CJ, et al. Microsphere-Based Scaffolds in Regenerative Engineering. Annu Rev Biomed Eng. 2017;19:135-161.
[30] DONG Z, MENG X, YANG W, et al. Progress of gelatin-based microspheres (GMSs) as delivery vehicles of drug and cell. Mater Sci Eng C Mater Biol Appl. 2021;122:111949.
[31] JIANG G, LI S, YU K, et al. A 3D-printed PRP-GelMA hydrogel promotes osteochondral regeneration through M2 macrophage polarization in a rabbit model. Acta Biomater. 2021;128:150-162.
[32] XIAO L, LIN J, CHEN R, et al. Sustained Release of Melatonin from GelMA Liposomes Reduced Osteoblast Apoptosis and Improved Implant Osseointegration in Osteoporosis. Oxid Med Cell Longev. 2020; 2020:6797154.