3D Clumps/Extracellular Matrix Complexes of Periodontal Ligament Stem Cells Ameliorate the Attenuating Effects of LPS on Proliferation and Osteogenic Potential.

Spoorthi Ravi Banavar,Fabian Amalraj Davamani,Mikihito Kajiya,Eng Lai Tan,Hidemi Kurihara,Umer Daood,Suan Phaik Khoo,Shaju Jacob Pulikkotil,Ian C Paterson,Swati Yeshwant Rawal

doi:10.3390/jpm11060528

Abstract

Background: The effects of lipopolysaccharide (LPS) on cell proliferation and osteogenic potential (OP) of MSCs have been frequently studied. Objective: to compare the effects of LPS on periodontal-ligament-derived mesenchymal stem cells (PDLSCs) in monolayer and 3D culture. Methods: The PDLSCs were colorimetrically assessed for proliferation and osteogenic potential (OP) after LPS treatment. The 3D cells were manually prepared by scratching and allowing them to clump up. The clumps (C-MSCs) were treated with LPS and assessed for Adenosine triphosphate (ATP) and OP. Raman spectroscopy was used to analyze calcium salts, DNA, and proline/hydroxyproline. Multiplexed ELISA was performed to assess LPS induced local inflammation. Results: The proliferation of PDLSCs decreased with LPS. On Day 28, LPS-treated cells showed a reduction in their OP. C-MSCs with LPS did not show a decrease in ATP production. Principal bands identified in Raman analysis were the P–O bond at 960 cm−1 of the mineral component, 785 cm−1, and 855 cm−1 showing qualitative changes in OP, proliferation, and proline/hydroxyproline content, respectively. ELISA confirmed increased levels of IL-6 and IL-8 but with the absence of TNF-α and IL-1β secretion. Conclusions: These observations demonstrate that C-MSCs are more resistant to the effects of LPS than cells in monolayer cell culture. Though LPS stimulation of C-MSCs creates an early pro-inflammatory milieu by secreting IL-6 and IL-8, PDLSCs possess inactivated TNF promoter and an ineffective caspase-1 activating process.

Highlights

Multipotent, self-renewing mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of cell types
The cryopreserved periodontal-ligament-derived mesenchymal stem cells (PDLSCs) were retrieved from the cryo-tank and expanded in a T75 flask with complete culture media (CCM) consisting of 88% Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, New York, NY, USA), 10% fetal bovine serum (Gibco, New York, USA), 1% GlutaMAXTM L-glutamine (Gibco, New York, NY, USA), 1% penicillin-streptomycin (Gibco, New York, NY, USA), ten ng/mL
In the current in vitro study, we evaluated the effects of LPS on proliferation, osteogenic abilities, and cytokine profiles of the 2D and 3D PDLSC model

Summary

Introduction

Multipotent, self-renewing mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of cell types. Since the early 1900s, two-dimensional (2D) cell cultures have been the preferred method for in vitro studies. Multicellular tumor spheroids were first described in the 1970s by culturing tumor cells in a nonadherent environment, resulting in scaffold-free, self-assembled, three-dimensional cellular aggregates [6]. Significant advances in three-dimensional (3D) cell cultures, including organ-on-a-chip technologies, have allowed researchers to review complex aspects of human physiology, pathology, and drug responses in vitro [7]. The effects of lipopolysaccharide (LPS) on cell proliferation and osteogenic potential (OP) of MSCs have been frequently studied. Objective: to compare the effects of LPS on periodontal-ligament-derived mesenchymal stem cells (PDLSCs) in monolayer and 3D culture

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