CELL-DERIVED EXTRACELLULAR MATRIX TAILORING FOR INTERVERTEBRAL DISC REGENERATION

• DOI: 10.1302/1358-992x.2024.2.115
• Publication Date: 2024-01-02T16:19:59Z
• Authors: C. Milheiro; R.M. Gonçalves; M. Amendola; M. Barbosa; J. Caldeira
• Keywords: Decellularization; Intervertebral Disc

Intervertebral disc (IVD) degeneration is characterized by tissue loss of function and major structural changes, which can lead to Low Back Pain. Current treatments fail tackle the disc's hindered degenerative alterations. Our goal promote IVD regeneration, through recapitulation a fetal-like microenvironment based on tailored cell-derived matrix (CDM), rich in two fetal exclusive collagens – COLXII COLXIV. We hypothesize that this engineered CDM will have pro-regenerative potential allow functional restoration. Immortalized mesenchymal stromal/stem cells (iMSCs) were transduced with CRISPR/dCas9-VP64 lentiviral system, targeting COL12A1 or COL14A1 . Gene protein expression evaluated qRT-PCR, Western Blot immunofluorescence. Simultaneously, optimization decellularization protocol was performed. Process efficacy validated DNA content quantified using Quant-iT™ PicoGreen™ Kit. Successful iMSCs engineering confirmed an average increase mRNA relative for both (2.12-fold 11.7-fold COL14A1) Protein level assessment western blot immunofluorescence increased Decellularized production optimized. Cell removal demonstrated nuclear actin staining reduction, when compared non-decellularized control. showed marked decrease NH4OH- SDS-based protocols. Matrix preservation after fibronectin only NH4OH-based decellularization. Preliminary proteomic characterization data as shown some impact namely Sonic Hedgehog (Shh) pathway repression pain DREAM (Downstream regulator element antagonist modulator). In conclusion, we able establish CRISPR activating system COL14A1, allowing use biofactories fetal-tailored CDM. The customized ECM recapitulations innovative strategy restoration, its versatility opens new avenues regeneration field.