Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Hydrogel-Based Delivery of Hypoxic Preconditioned Stem Cell-Derived Extracellular Vesicles Mitigates Senescence in Nucleus Pulposus Cells: Regulating Redox Homeostasis for Disc Degeneration Treatment (#MP-13b)

MING GUAN 1 2 , CAN LIU 1 , QIANGQIANG ZHENG 1 , YIHE HU 1 , YUE WANG 1
  1. Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
  2. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Introduction

Nucleus pulposus cell senescence plays a significant role in this process, causing cell cycle arrest, resistance to cell death, and the release of inflammatory factors and enzymes that degrade the matrix. This senescence is often driven by oxidative stress, primarily due to excessive production of reactive oxygen species (ROS) or a decline in cellular antioxidant capacity. Recent research has shown promise in using hypoxia-conditioned mesenchymal stem cells (Hyp-MSCs) and their extracellular vesicles (Hyp-EVs) to mitigate oxidative stress and cell senescence.

Methods

(1) Hyp-EVs were isolated and analyzed to understand their therapeutic effects on nucleus pulposus cell senescence induced by oxidative stress. A proteomics analysis identified the presence of the antioxidant protein glutaredoxin 3 (GLRX3) within Hyp-EVs. GLRX3 knockdown in mesenchymal stem cells was achieved through lentivirus transfection, resulting in the isolation of Hyp-EVs with reduced GLRX3 levels for loss-of-function experiments.

(2) Single-cell sequencing data were used to identify subpopulations of senescent cells and explore GLRX3 expression in these cells. Samples from human and rat discs were collected to verify GLRX3 expression in the context of disc degeneration. RNA sequencing and biological experiments were conducted to study the effects of GLRX3 knockdown in nucleus pulposus cells.

(3) Two types of hydrogels were developed for repairing intervertebral disc degeneration: the dopamine-functionalized gelatin-chondroitin sulfate hydrogel (GDC@EVs) and an exosome laden hydrogel based on quaternized chitosan and oxidized starch (QCS-OST@EVs). These hydrogels were assessed for their properties and their ability to regulate redox balance and reduce cell senescence in in vitro experiments. In an in vivo rat model of disc degeneration, GDC@Hyp-EVs and QCS-OST@Hyp-EVs were separately injected to evaluate their effectiveness using various measures.

Results

(1) Hyp-EVs were found to alleviate mitochondrial dysfunction and the senescence of nucleus pulposus cells induced by oxidative stress. Proteomic analysis revealed that Hyp-EVs were enriched with the antioxidant protein GLRX3, and restoring GLRX3 levels in nucleus pulposus cells was associated with protective effects on mitochondrial function and cell senescence. These protective effects were absent when Hyp-EVs with reduced GLRX3 (Hyp-EVs-shGLRX3) were used.

(2) Single-cell transcriptome analysis demonstrated lower GLRX3 expression in senescent nucleus pulposus cells within degenerative intervertebral discs. The downregulation of GLRX3 was further confirmed in human and rat discs with degeneration. Transcriptome analysis of GLRX3 knockdown in nucleus pulposus cells revealed changes in genes related to the cell cycle, defense response, mitochondrial function, and secretion factors.

(3) Both GDC@Hyp-EVs and QCS-OST@Hyp-EVs exhibited favorable properties, including injectability, self-healing abilities, mechanical strength, ROS reduction, and controlled release of Hyp-EVs. In the in vivo experiments, both hydrogel types effectively reduced mitochondrial damage, decreased local senescence, and promoted the deposition of extracellular matrix by regulating the ROS environment.

Discussion

This study highlights the potential of GLRX3-enriched Hyp-EVs in reducing oxidative stress, alleviating nucleus pulposus cell senescence, and repairing disc degeneration. The developed hydrogel systems show promise in mitigating oxidative stress, promoting cell rejuvenation, and restoring extracellular matrix deposition in degenerative discs. These approaches represent promising avenues for the treatment of disc degeneration.

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 Fig. 1 GDC@Hyp-EVs

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 Fig.2 QCS-OST@Hyp-EVs

  1. Guan, M.; Liu, C.; Zheng, Q.; Chu, G.; Wang, H.; Jin, J.; Wu, H.; Chen, J.; Huang, Q.; Deng, Z.; et al. Exosome-laden injectable self-healing hydrogel based on quaternized chitosan and oxidized starch attenuates disc degeneration by suppressing nucleus pulposus senescence. Int J Biol Macromol 2023, 232, 123479. DOI: 10.1016/j.ijbiomac.2023.123479
  2. Liu, C.; Fan, L.; Guan, M.; Zheng, Q.; Jin, J.; Kang, X.; Gao, Z.; Deng, X.; Shen, Y.; Chu, G.; et al. A Redox Homeostasis Modulatory Hydrogel with GLRX3(+) Extracellular Vesicles Attenuates Disc Degeneration by Suppressing Nucleus Pulposus Cell Senescence. ACS Nano 2023, 17 (14), 13441-13460. DOI: 10.1021/acsnano.3c01713