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Mesenchymal stem cells (MSCs) are increasingly being used in tissue engineering and cell-based therapies in all fields ranging from orthopedic to cardiovascular medicine. Despite years of research and numerous clinical trials, MSC therapies are still very much in development and not considered mainstream treatments. The majority of approaches rely on an in vitro cell expansion phase in monolayer to produce large cell numbers prior to implantation. It is clear from the literature that this in vitro expansion phase causes dramatic changes in MSC phenotype which has very significant implications for the development of effective therapies. Previous reviews have sought to better characterize these cells in their native and in vitro environments, described known stem cell interactions within the bone marrow, and discussed the use of innovative culture systems aiming to model the bone marrow stem cell niche. The purpose of this review is to provide an update on our knowledge of MSCs in their native environment, focusing on bone marrow-derived MSCs. We provide a detailed description of the differences between naive cells and those that have been cultured in vitro and examine the effect of isolation and culture parameters on these phenotypic changes. We explore the concept of "one step" MSC therapy and discuss the potential cellular and clinical benefits. Finally, we describe recent work attempting to model the MSC bone marrow niche, with focus on both basic research and clinical applications and consider the challenges associated with these new generation culture systems.

This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-β1 (TGF-β1) concentrations (0, 1 or 10 ng/ml).The role of the TGF-β signalling pathway in this process was also studied. Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 × 106 cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression, DNA content and glycosaminoglycan amount. Addition of TGF-β1 in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-β1 concentration of the culture medium, mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds, with a much stronger effect on gene expression at lower TGF-β1 concentrations. With TGF-β1 absent in the culture medium, mechanical load stimulated gene transcripts and protein synthesis of TGF-β1 and TGF-β3. TGF-β type I receptor inhibitor LY364947 blocked the up-regulation on TGF-β1 and TGF-β3 production stimulated by mechanical load, and also blocked the chondrogenesis of hMSCs. Taken together, these findings suggest that mechanical load promotes chondrogenesis of hMSCs through TGF-β pathway by up-regulating TGF-β gene expression and protein synthesis.

Immortalized cell lines are used more frequently in basic and applied biology research than primary bone-derived cells because of their ease of access and repeatability of results in experiments. It is clear that these cell models do not fully resemble the behavior of primary osteoblast cells. Although the differences will affect the results of biomaterials testing, they are not clearly defined. Here, we focused on comparing proliferation and maturation potential of three osteoblast cell lines, SaOs2, MG-63, and MC3T3-E1 with primary human osteoblast (HOb) cells to assess their suitability as in vitro models for biomaterials testing. We report similarities in cell proliferation and mineralization between primary cells and MC3T3-E1. Both, SaOs2 and MG-63 cells demonstrated a higher proliferation rate than HOb cells. In addition, SaOs2, but not MG-63, cells demonstrated similar ALP activity, mineralization potential and gene regulation to HOb's. Our results demonstrate that despite SaOs-2, MG63, and MC3T3 cells being popular choices for emulating osteoblast behavior, none can be considered appropriate replacements for HOb's. Nevertheless, these cell lines all demonstrated some distinct similarities with HOb's, thus when applied in the correct context are a valuable in vitro pilot model of osteoblast functionality, but should not be used to replace primary cell studies.

Background Mechanical stimuli are of crucial importance for the development and maintenance of articular cartilage. For conditioning of cartilaginous tissues, various bioreactor systems have been developed that have mainly aimed to produce cartilaginous grafts for tissue engineering applications. Emphasis has been on in vitro preconditioning, whereas the same devices could be used to attempt to predict the response of the cells in vivo or as a prescreening method before animal studies. As a result of the complexity of the load and motion patterns within an articulating joint, no bioreactor can completely recreate the in vivo situation. Questions/purposes This article aims to classify the various loading bioreactors into logical categories, highlight the response of mesenchymal stem cells and chondrocytes to the various stimuli applied, and determine which data could be used within a clinical setting. Methods We performed a Medline search using specific search terms, then selectively reviewed relevant research relating to physical stimulation of chondrogenic cells in vitro, focusing on cellular responses to the specific load applied.

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The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

Articular cartilage is a load-bearing tissue playing a crucial mechanical role in diarthrodial joints, facilitating joint articulation, and minimizing wear. The significance of biomechanical stimuli in the development of cartilage and maintenance of chondrocyte phenotype in adult tissues has been well documented. Furthermore, dysregulated loading is associated with cartilage pathology highlighting the importance of mechanical cues in cartilage homeostasis. The repair of damaged articular cartilage resulting from trauma or degenerative joint disease poses a major challenge due to a low intrinsic capacity of cartilage for self-renewal, attributable to its avascular nature. Bone marrow-derived mesenchymal stem cells (MSCs) are considered a promising cell type for cartilage replacement strategies due to their chondrogenic differentiation potential. Chondrogenesis of MSCs is influenced not only by biological factors but also by the environment itself, and various efforts to date have focused on harnessing biomechanics to enhance chondrogenic differentiation of MSCs. Furthermore, recapitulating mechanical cues associated with cartilage development and homeostasis in vivo, may facilitate the development of a cellular phenotype resembling native articular cartilage. The goal of this review is to summarize current literature examining the effect of mechanical cues on cartilage homeostasis, disease, and MSC chondrogenesis. The role of biological factors produced by MSCs in response to mechanical loading will also be examined. An in-depth understanding of the impact of mechanical stimulation on the chondrogenic differentiation of MSCs in terms of endogenous bioactive factor production and signaling pathways involved, may identify therapeutic targets and facilitate the development of more robust strategies for cartilage replacement using MSCs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:52-63, 2018.