In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair

Abstract

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

A Physiology-Inspired Multifactorial Toolbox in Soft-to-Hard Musculoskeletal Interface Tissue Engineering

“…Among different biochemical factors, oxygen is a crucial molecule and we will discuss the influence of oxygen tension on cellular behavior. Additionally, small molecules, like ascorbic acid, can be employed to accelerate ECM deposition, particularly for cell sheet engineering [56][57][58][59], or to promote cellular proliferation in cell expansion protocols (e.g., glucose and essential amino acids).…”

Mechanical and molecular parameters that influence the tendon differentiation potential of C3H10T1/2 cells in 2D- and 3D-culture systems

“…The tendon phenotype is not maintained in 2D-cultures of tendon cells over passages (Hsieh et al, 2018;Shukunami et al, 2018;Yao et al, 2006). 3D-culture systems in which tendon cells are embedded in hydrogels are recognized to provide an environment closer to that experienced by tendon cells in vivo (Kapacee et al, 2010;Kuo et al, 2010;Marturano et al, 2016;Yeung et al, 2015).…”

“…of six biological samples. The mechanical environment provided to tendon cells homogeneously embedded within hydrogel in 3D-culture systems is recognized to act on tendon gene expression (Hsieh et al, 2018;Marturano et al, 2016). Most of the analyses of the effects of 2D and 3D environments have been performed with tendon stem/ progenitor cells; however, the optimum culture conditions that drive tendon cell differentiation from MSCs have not been yet identified.…”

Mechanisms of Action of Multipotent Mesenchymal Stromal Cells in Tendon Disease

“…In vitro and ex vivo models relevant to MSC mechanisms of action in tendon regeneration comprise two major groups, with some overlap (Figure 1). The first includes the wide range of models for tenogenic differentiation [10,. Among these, approaches in three-dimensional dynamic cultures appear most representative for MSC mechanisms in vivo [57,58,64,70,74,77,79,83,84,86,87].…”

“…Once at a diseased state, the intrinsic regenerative capacity of tendons is poor. Although endogenous mesenchymal stem-like cells with high tenogenic potential reside within tendons [10][11][12], these are susceptible to damage and suffer age-related changes [13,14]. In pathological states, they could even contribute to fatty degeneration, fibrosis, and heterotopic ossifications [15,16].…”