Elucidating Nanoclay-Stem Cell Interactions for Enhancing Bone Regeneration

Abstract

Bone fractures and degenerative diseases are a major socioeconomic problem that is on the rise with an aging population. Therefore, there is a pressing, yet unmet, clinical need for treatment approaches to repair damaged/degenerated bone tissues. A promising approach to address this problem is to develop new bioactive materials that can direct and enhance the osteogenic differentiation of human bone marrow stromal cells (hBMSCs). Recently, clay
nanoparticles (herein Laponite) have received growing interest in this context due to their exciting potential to enhance cellular functions including adhesion, proliferation and differentiation, most notably for osteogenesis. However, the mechanism(s) of interaction between nanoclay and hBMSCs, which is the key for the successful harnessing of nanoclay for bone regeneration, remain poorly understood. In this thesis the ability of clay nanoparticles to promote hBMSCs osteogenesis (differentiation into the osteogenic lineage) was investigated followed by an investigation of various modes through which nanoclay may influence hBMSCs osteogenic function, in particular, the of Laponite endocytosis and subsequent release of Laponite degradation products (Si(OH)4, Mg2+ and Li+).

The osteogenic effects of clay nanoparticles on hBMSCs were determined at varying doses and culture time by determination of alkaline phosphatase (ALP) activity, calcium phosphate mineralization and osteogenic gene expression. The role of nanoclay endocytosis and subsequent release of degradation products, in particular lithium, was tested through the use of the endocytosis inhibitor chlorpromazine hydrochloride (CPZ) and the use of lithium modified Laponite formulations developed with BYK-ALTANA (Widnes, Cheshire, UK). Finally, the cellular uptake kinetics, intracellular transport pathway(s), and fate of internalized clay nanoparticles were tracked using inductively coupled plasma mass spectrometry (ICPMS), confocal and electron microscopy.

Clay nanoparticles were biocompatible up to a concentration of 100 μg/mL and promoted osteogenic differentiation of hBMSCs in a dose- and time-dependent manner. Inhibition of nanoclay endocytosis significantly attenuated (reduced) the ability of nanoclay to enhance ALP activity of hBMSCs, while nanoclay degradation products, applied at concentrations equivalent to that present in Laponite, failed to induce any significant increase in ALP
activity. Furthermore, no significant differences were observed in ALP activity and calcium deposition between standard Laponite and lithium modified Laponite structures and both null and high lithium nanoclays showed similar effects on osteogenic gene expression of hBMSCs indicating that nanoclay osteogenic bioactivity is not principally an effect of nanoclay degradation products.

Clay nanoparticles strongly interacted with hBMSCs distributing intracellularly, extracellularly and on the cell membrane as clusters/aggregates. Clay nanoparticles were readily internalized by hBMSCs through clathrin-mediated endocytosis and their uptake kinetics followed a linear increase with incubation time. Following endocytosis, clay nanoparticles were observed entrapped within endosomal and lysosomal compartments, but clay nanoparticles were not free in other cell organelles. Internalized nanoclay underwent degradation with endosomal-lysosomal maturation and exocytosis. Nanoclay uptake influenced cell physiological functions as demonstrated by nanoclay-induced hBMSCs autophagy which stands as potential mechanism for Laponite osteogenic bioactivity.

This thesis provides a clear understanding of how nanoclay interact with hBMSCs for enhanced osteogenic differentiation and sets a basis for the successful control and manipulation of nanoclay for biomaterial design and regenerative medicine.

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Identifiers

ORCID for Jonathan Dawson: orcid.org/0000-0002-6712-0598

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