(G-272) Assaying Extracellular Matrix Gene Expression by Vertebral Endplate Chondrocytes in 3D Hydrogel Culture.

Introduction:: Intervertebral disc degeneration (IDD) affects over 50 million American annually. Treatment options are palliative in nature before surgery is required. There are no existing FDA-approved therapies for the prevention or medical treatment of IDD Animal models are powerful tools for the study of IDD; however, mechanistic analyses still require reductionist approaches offered by explant and cells culture systems. Mono-culture and transwell systems have long been tools for drug testing, but have produced little in the development of effective IDD therapies. Human cell-based 3D microphysiological system are hypothesized to provide a stronger test bed for therapeutic testing by building better tissue analogs. While the study and modeling of articular cartilage has long been studied in the context of hydrogels, vertebral endplate cartilage is rarely studied in 3D cultures. The goal of this study is to establish a 3D model of the vertebral endplate inspired by existing cartilage models using human patient-derived vertebral endplate chondrocytes in chemically defined scaffolds and mediums.

Materials and Methods:: Primary human vertebral endplate chondrocytes (VEC) were harvested from the surgical waste following disc fusion surgery of consenting adults according to an IRB-approved protocol using common, previously published methods. Methacrylated hydrogel scaffold components were prepared as previously described.[1,2] VEC constructs were prepared using cells at passage 3 that were encapsulated at 5 x 104 cells in 7 uL constructs (2mm diameter, 1mm high) in methacrylated gelatin only(mGel); 9% gelatin + 1% hyaluronic acid (mGel-HA) or monolayer on tissue cultures plastic (TCP) all formed in silicone moulds with LAP and UV-light for polymerization. All cultures were maintained in basal chondrogenic medium (CM: high glucose DMEM, ITS+, antibiotic/antimycotic, 4 mM proline, 50 µg/mL ascorbic acid, 1 mM sodium pyruvate, and 10−7 M dexamethasone) for 14 days in the presence of one of four growth factor conditions; 10 ng/mL of transforming growth factor-β3 (TGFβ3), 20 ng/ml bone morphogenetic protein-6 (BMP6), TGFβ3 and BMP6 or no additional growth factor. At the 14 day timepoint, cultures were processed for RNA isolation using the RNeasy Universal kit (Qiagen). Reverse transcription was performed using the Maxima system (ThermoFisher) and the subsequent PCR was conducted using the SYBR green™ assay on an ABI Step-One thermocycler (Applied Biosystems) with primer sequences for the anabolic cartilage markers COL2 AND ACAN described in previous publications.[3,4]

Results, Conclusions, and Discussions:: From this study, we observed three important results. First, VEC express highest levels of COL2 and ACAN in 3D culture as compared to 2D TCP controls (Fig 1A). Second, COL2 and ACAN expression by VEC trended higher in than hydrogels of mGel alone, as in compared to mGel-HA. Third, BMP6 stimulation of VEC 3D cultures enhanced COL2 but greatly decreased ACAN expression, and the combination of TGFb3 and BMP6 did not increase VEC matrix gene expression (Fig 1B).

 

The results of this study suggest that the among the conditions tested, VEC culture in 10% gelatin stimulated by TGFβ3 is most effective in inducing VEC expression of COL2 and ACAN.

 

Studies in our lab indicate that patient-derived VEC express COL2 and ACAN at levels is 20-100 fold lower than healthy rat VEC encapsulated within 10% mGel hydrogels (data not shown). Taking cues from optimization of 3D culture conditions of articular chondrocytes, exploring two possibilities to improve primary VEC ECM gene expression: first, the addition of hyaluronic acid (HA) to the scaffold,[5] and second, the addition of BMP6 to the differentiation medium.[6] HA enhances chondrogenesis of mesenchymal stromal cells (MSCs), induced pleuripotent stem cells (iPSCs) and primary chondrocytes via growth factor sequestration and improving their presentation. In this study, addition of HA to the VEC scaffold unexpexectedly reduced ECM production by VEC. Second, BMP6 has been shown to potentiate TGFβ3 intracellular signaling and chondrogenic differentiation in MSCs and iPSCs. Contrary to expectation, combining TGFb3 and BMP6 did not increase VEC matrix gene expression (Fig 1B). The poor matrix production by primary human VEC after spine fusion may be a feature of the aged, diseased tissues obtained during spinal fusion surgery and not reversible in the culture-type explored here. In addition, the failure to enhance ECM gene expression by mechanisms known to positively affect articular cartilage ECM gene expression may suggest that VEC is qualitatively different than articular cartilage as well.[7] Future studies involve obtaining healthy VEC from cadaveric donors and characterizing their phenotype and induced matrix elaboration in 3D culture as performed here for primary patient-derived VEC.

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[1] Lin H, et al. Tissue Eng A. 2014 Sep;20(17-18):2402-11. doi: 10.1089/ten.TEA.2013.0642.

[2] Deng Y, et al. Acta Biomater. 2019 Jan 1;83:167-176. doi: 10.1016/j.actbio.2018.11.022.

[3] Li Z et al. Adv Sci (Weinh). 2022 Jul;9(21):e2105909. doi: 10.1002/advs.202105909.                   

[4] Xiang S et al. Clin Transl Med. 2022 Dec;12(12):e1112. doi: 10.1002/ctm2.1112.

[5] Bian L et al. Biomaterials. 2013 Jan;34(2):413-21. doi: 10.1016/j.biomaterials.2012.09.052.

[6] Diekman BO et al. J Biomed Mater Res A. 2010 Jun 1;93(3):994-1003. doi: 10.1002/jbm.a.32589.

[7] Lakstins K et al. J Orthop Res. 2021 Jun;39(6):1204-1216. doi: 10.1002/jor.24692.