In vitro evaluation of sustained release Kartogenin by gelatin methacryloyl microspheres for repairing nucleus pulposus degeneration

doi:10.12307/2024.247

Abstract

Abstract: BACKGROUND: The imbalance of matrix synthesis and degradation is the main cause of nucleus pulposus degeneration. Small molecule drug Kartogenin (KGN) can restore the balance of matrix synthesis and degradation. Sustained release of KGN using an appropriate drug delivery system is essential for the long-term and effective treatment of KGN.
OBJECTIVE: To prepare the injectable hydrogel microspheres by encapsulating KGN with gelatin methacryloyl (GelMA) by microfluidic technology and to investigate the biocompatibility and biological function of nucleus pulposus cells.
METHODS: β-Cyclodextrins (β-CD) and KGN were mixed firstly and then mixed with 10% GelMA at a volume of 1:9. Injectable hydrogel microspheres GelMA@β-CD@KGN were prepared by microfluidic technology. The micromorphology of the microspheres was characterized using a scanning electron microscope. The drug release of hydrogel microspheres immersed in PBS within one month was measured. Nucleus pulposus cells were isolated from SD rats and passage 1 cells were cultured in three groups. In the control group, nucleus pulposus cells were cultured separately. In the other two groups, GelMA@β-CD microspheres and GelMA@β-CD@KGN microspheres were co-cultured with nucleus pulposus cells. Cell proliferation was detected by CCK-8 assay and cell survival was detected by live/dead cell staining. Cells were cultured by two complete media with and without interleukin-1β with two kinds of microspheres. mRNA expressions of matrix synthesis and decomposing proteins in nucleus pulposus cells were detected by RT-PCR.
RESULTS AND CONCLUSION: (1) Under the scanning electron microscope, the GelMA@β-CD@KGN microspheres after lyophilization were regularly spherical, highly dispersed, uniform in size and full in shape. GelMA@β-CD@KGN microspheres sustained drug release in vitro, reaching 62% of the total drug release at 30 days. (2) Live/dead cell staining showed that GelMA@β-CD@KGN could maintain the activity of nucleus pulposus cells. CCK-8 assay showed that GelMA@β-CD@KGN could promote the proliferation of nucleus pulposus cells. (3) In the complete media with and without interleukin-1β, mRNA expression of aggrecan and type II collagen was higher in the GelMA@β-CD@KGN microsphere group than that in the GelMA@β-CD microsphere group (P < 0.05, P < 0.01); mRNA expression of matrix metalloproteinase 13 and platelet reactive protein disintegrin metallopeptidase 5 was lower than that in the GelMA@β-CD microsphere group (P < 0.01). (4) These findings indicate that GelMA@β-CD@KGN microspheres have good biocompatibility and sustained drug release ability. As a drug delivery system, it is a kind of biomaterial with broad application prospects.

Key words: intervertebral disc, nucleus pulposus, microsphere, gelatin methacryloyl, microfluidic, Kartogenin, drug delivery system

CLC Number:

Tian Xin, Liu Tao, Yang Huilin, He Fan. In vitro evaluation of sustained release Kartogenin by gelatin methacryloyl microspheres for repairing nucleus pulposus degeneration[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(5): 724-730.

Figures/Tables 8

References

[1] DEYO RA, WEINSTEIN JN. Low back pain. N Engl J Med. 2001;344(5):363-370.
[2] HARTVIGSEN J, HANCOCK MJ, KONGSTED A, et al. What low back pain is and why we need to pay attention. Lancet. 2018;391(10137):2356-2367.
[3] FRANCISCO V, PINO J, GONZÁLEZ-GAY MÁ, et al. A new immunometabolic perspective of intervertebral disc degeneration. Nat Rev Rheumatol. 2022; 18(1):47-60.
[4] ANTONIOU J, STEFFEN T, NELSON F, et al. The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest. 1996;98(4):996-1003.
[5] BINCH ALA, RATCLIFFE LPD, MILANI AH, et al. Site-Directed Differentiation of Human Adipose-Derived Mesenchymal Stem Cells to Nucleus Pulposus Cells Using an Injectable Hydroxyl-Functional Diblock Copolymer Worm Gel. Biomacromolecules. 2021;22(2):837-845.
[6] JOHNSON K, ZHU S, TREMBLAY MS, et al. A stem cell-based approach to cartilage repair. Science. 2012;336(6082):717-721.
[7] YU C, LI D, WANG C, et al. Injectable kartogenin and apocynin loaded micelle enhances the alleviation of intervertebral disc degeneration by adipose-derived stem cell. Bioact Mater. 2021;6(10):3568-3579.
[8] VAN DEN BULCKE AI, BOGDANOV B, DE ROOZE N, et al. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules. 2000;1(1):31-38.
[9] 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.
[10] XU J, LI J, LIN S, et al. Nanocarrier-mediated codelivery of small molecular drugs and siRNA to enhance chondrogenic differentiation and suppress hypertrophy of human mesenchymal stem cells. Adv Funct Mater. 2016;26: 2463-2472.
[11] MURA P. Advantages of the combined use of cyclodextrins and nanocarriers in drug delivery: A review. Int J Pharm. 2020;579:119181.
[12] XU J, FENG Q, LIN S, et al. Injectable stem cell-laden supramolecular hydrogels enhance in situ osteochondral regeneration via the sustained co-delivery of hydrophilic and hydrophobic chondrogenic molecules. Biomaterials. 2019;210:51-61.
[13] 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.
[14] XU X, LIANG Y, LI X, et al. Exosome-mediated delivery of kartogenin for chondrogenesis of synovial fluid-derived mesenchymal stem cells and cartilage regeneration. Biomaterials. 2021;269:120539.
[15] FRAPIN L, CLOUET J, CHÉDEVILLE C, et al. Controlled release of biological factors for endogenous progenitor cell migration and intervertebral disc extracellular matrix remodelling. Biomaterials. 2020;253:120107.
[16] ZHOU W, DUAN Z, ZHAO J, et al. Glucose and MMP-9 dual-responsive hydrogel with temperature sensitive self-adaptive shape and controlled drug release accelerates diabetic wound healing. Bioact Mater. 2022;17:1-17.
[17] RIBEIRO JS, BORDINI EAF, FERREIRA JA, et al. Injectable MMP-Responsive Nanotube-Modified Gelatin Hydrogel for Dental Infection Ablation. ACS Appl Mater Interfaces. 2020;12(14):16006-16017.
[18] CAI C, ZHANG X, LI Y, et al. Self-Healing Hydrogel Embodied with Macrophage-Regulation and Responsive-Gene-Silencing Properties for Synergistic Prevention of Peritendinous Adhesion. Adv Mater. 2022;34(5): e2106564.
[19] CHANG H, CAI F, ZHANG Y, et al. Silencing Gene-Engineered Injectable Hydrogel Microsphere for Regulation of Extracellular Matrix Metabolism Balance. Small Methods. 2022;6(4):e2101201.
[20] LI L, DU Y, XIONG Y, et al. Injectable negatively charged gelatin microsphere-based gels as hemostatic agents for intracavitary and deep wound bleeding in surgery. J Biomater Appl. 2018;33(5):647-661.
[21] BIAN J, CAI F, CHEN H, et al. Modulation of Local Overactive Inflammation via Injectable Hydrogel Microspheres. Nano Lett. 2021;21(6):2690-2698.
[22] YANG J, LIANG J, ZHU Y, et al. Fullerol-hydrogel microfluidic spheres for in situ redox regulation of stem cell fate and refractory bone healing. Bioact Mater. 2021;6(12):4801-4815.
[23] ZHANG C, GULLBRAND SE, SCHAER TP, et al. Combined Hydrogel and Mesenchymal Stem Cell Therapy for Moderate-Severity Disc Degeneration in Goats. Tissue Eng Part A. 2021;27(1-2):117-128.
[24] SAKAI D, ANDERSSON GB. Stem cell therapy for intervertebral disc regeneration: obstacles and solutions. Nat Rev Rheumatol. 2015;11(4):243-256.
[25] MWALE F, ROUGHLEY P, ANTONIOU J. Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc. Eur Cell Mater. 2004;8:58-63.
[26] HUGHES SP, FREEMONT AJ, HUKINS DW, et al. The pathogenesis of degeneration of the intervertebral disc and emerging therapies in the management of back pain. J Bone Joint Surg Br. 2012;94(10):1298-1304.
[27] LE MAITRE CL, POCKERT A, BUTTLE DJ, et al. Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans. 2007;35(Pt 4):652-655.
[28] LIU T, LI X, WANG T, et al. Kartogenin mediates cartilage regeneration by stimulating the IL-6/Stat3-dependent proliferation of cartilage stem/progenitor cells. Biochem Biophys Res Commun. 2020;532(3):385-392.
[29] BAHARLOU HOUREH A, MASAELI E, NASR-ESFAHANI MH. Chitosan/polycaprolactone multilayer hydrogel: A sustained Kartogenin delivery model for cartilage regeneration. Int J Biol Macromol. 2021;177:589-600.
[30] ZHANG M, HU W, CAI C, et al. Advanced application of stimuli-responsive drug delivery system for inflammatory arthritis treatment. Mater Today Bio. 2022;14:100223.
[31] HUANG Y, JIANG T, CHEN J, et al. Effects of kartogenin on the attenuated nucleus pulposus cell degeneration of intervertebral discs induced by interleukin-1β and tumor necrosis factor-α. Int J Mol Med. 2018;41(2): 749-756.
[32] HOU M, ZHANG Y, ZHOU X, et al. Kartogenin prevents cartilage degradation and alleviates osteoarthritis progression in mice via the miR-146a/NRF2 axis. Cell Death Dis. 2021;12(5):483.
[33] FENG Q, LI D, LI Q, et al. Dynamic Nanocomposite Microgel Assembly with Microporosity, Injectability, Tissue-Adhesion, and Sustained Drug Release Promotes Articular Cartilage Repair and Regeneration. Adv Healthc Mater. 2022;11(8):e2102395.
[34] ZHANG YJ, YIN WL, LIU Y, et al. Dynamic protein hydrogel with supramolecularly enveloped kartogenin promotes cartilage regeneration through mitochondrial activation. Compos B Eng. 2022;246:1359-8368.
[35] MA CJ, LIU X, CHE L, et al. Stem Cell Therapies for Intervertebral Disc Degeneration: Immune Privilege Reinforcement by Fas/FasL Regulating Machinery. Curr Stem Cell Res Ther. 2015;10(4):285-295.
[36] 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.
[37] RUOSLAHTI E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol. 1996;12:697-715.
[38] RUTGES J, CREEMERS LB, DHERT W, et al. Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration. Osteoarthritis Cartilage. 2010;18(3):416-423.