Transforming Growth Factor β Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro

Abstract

The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor β (TGFβ) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFβ1 or TGFβ3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFβ1 nor TGFβ3 were able to restore the notochordal phenotype. Although TGFβ1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation.

Development of 2-D and 3-D culture platforms derived from decellularized nucleus pulposus

“…To assess the effects of culture on the 2-D ECM coatings on the maintenance of the NP cell phenotype following serial passage in monolayer culture, RT-qPCR analysis was used to quantify the expression of NP-associated markers ( KRT8, KRT19, SOX9 ), ECM genes ( COL2A1, COL1A1, ACAN ), and fibroblast-associated markers ( ACTA2, FSP1 ). Similar to previous reports ( Cui et al, 2012 ; Rosenzweig et al, 2017 ; Ashraf et al, 2020 ), our analysis demonstrated a robust decrease in the expression of NP-associated markers in primary cells over serial passage (P0–P3) on TCP ( Figure 4 , grey bars). Compared to primary (P0) NP cells cultured on TCP, primary (P0) NP cells cultured on DNP coatings showed significantly lower expression of the NP-associated markers KRT8 , KRT19 , and SOX9 - a change not induced by culture of P0 NP cells on COL coatings ( Figure 4A ).…”

“…Many in vitro studies involve the expansion of cells in 2-D on rigid tissue culture plastic substrates that are convenient for supporting cell attachment and proliferation, but substantially alter cell-cell and cell-ECM interactions as compared to the native cellular microenvironment, which can markedly affect cell function ( Rubashkin, Ou, and Weaver 2014 ; Rosenzweig et al, 2017 ; Ashraf et al, 2020 ). Notably, monolayer culture of primary bovine NP cells on TCP results in loss of the NP phenotype with serial passaging, as measured by decreased NP-associated marker expression ( Ashraf et al, 2020 ). Recognizing the cell-instructive capacity of the ECM, the primary goal of this study was to develop 2-D and 3-D culture platforms that mimic the complex ECM within the native nucleus pulposus.…”

Controlled Release of TGF-β3 for Effective Local Endogenous Repair in IDD Using Rat Model

“… 39 Ashraf et al reported that TGF-β3 enhanced the ability of NPCs to form tissues, in part by decreasing cell death. 40 Consistent with the results of the present study, TGF-β3 treatment blocked the inhibitory effect of H 2 O 2 on NPC proliferation, likely by suppressing the apoptotic gene BAX and upregulating the proliferative genes MCL1 and BCL2. Further, TGF-β3 alleviated the H 2 O 2 -induced expression of ECM genes (ACAN and Col-II) and partly blocked the stimulatory effect of H 2 O 2 on the expression of catabolic (MMP3 and Adamts5) and inflammatory genes (COX-2 and iNOS).…”

Spheroid-Like Cultures for Expanding Angiopoietin Receptor-1 (aka. Tie2) Positive Cells from the Human Intervertebral Disc

“…Moreover, the monolayer culture of NPCs showed an increased senescence and oxidative stress [18,19]. Previous studies on NPCs achieved better outcomes in cell phenotype and ECM production with 3D culture within a biomaterial scaffold compared to monolayer culture [20][21][22][23].…”

“…Previous research on 2D NPCs culture showed that the gene expression of collagen type 2 (COL2) decreased significantly from passage (P) 1 to P2, and aggrecan (ACAN) decreased significantly from P0 to P1 [17]. Furthermore, 2D NPCs culture revealed that the gene expression change seemed to happen at early P and then tended to stabilize [17][18][19].…”