Characterisation of human mesenchymal stem cell metabolism during proliferation and differentiation.

PhDMesenchymal stem cells (MSCs) have the ability to differentiate towards cartilage, bone, fat and muscle, and therefore have great therapeutic potential. Human MSCs reside under hypoxia (4-7% oxygen) in vivo and recent investigations have described an increase in population doublings and maintenance of differentiation capacity upon culture under these conditions. The reason for these differences may be related to their cellular metabolism. This thesis examines MSC metabolism during proliferation, chondrogenic and osteogenic differentiation. Furthermore, MSCs were cultured under uninterrupted and controlled hypoxia (5% or 2% oxygen) to observe its effect on proliferation and differentiation. The production of lactate and consumption of oxygen by MSCs indicated a mixed metabolism, with the cells utilising both oxidative phosphorylation and glycolysis under 20% oxygen or normoxic conditions. The majority of cellular ATP production was through glycolysis, whilst the full oxidative capacity of the mitochondria was not fully utilised. During chondrogenic differentiation, oxygen consumption was significantly reduced with time in culture and the cells became highly glycolytic, whilst osteogenic differentiation maintained the mixed metabolism of expanded MSCs. Fewer colonies were formed at 5% oxygen, compared to 2% and 20% oxygen. Hypoxic culture induced fewer cells per colony compared with normoxia. However, MSCs expanded under hypoxia had reduced cellular senescence after five passages, potentially due to the reduced utilisation of oxidative phosphorylation that has been shown to lead to the production of reactive oxygen species (ROS). The oxygen levels during expansion did not affect chondrogenic potential but expansion under hypoxia prevented ostoegenic differentiation. Osteogenesis was also inhibited for MSCs expanded at normoxia and differentiated under hypoxia. These results may be related to the initial colony formation under hypoxia and the changes in cellular metabolism during osteogenic differentiation

Quantitative Analysis of Surface Contouring with Pulsed Bipolar Radiofrequency on Thin Chondromalacic Cartilage

The purpose of this study was to evaluate the quality of surface contouring of chondromalacic cartilage by bipolar radio frequency energy using different treatment patterns in an animal model, as well as examining the impact of the treatment onto chondrocyte viability by two different methods. Our experiments were conducted on 36 fresh osteochondral sections from the tibia plateau of slaughtered 6-month-old pigs, where the thickness of the cartilage is similar to that of human wrist cartilage. An area of 1 cm(2) was first treated with emery paper to simulate the chondromalacic cartilage. Then, the treatment with RFE followed in 6 different patterns. The osteochondral sections were assessed for cellular viability (live/dead assay, caspase (cell apoptosis marker) staining, and quantitative analysed images obtained by fluorescent microscopy). For a quantitative characterization of none or treated cartilage surfaces, various roughness parameters were measured using confocal laser scanning microscopy (Olympus LEXT OLS 4000 3D). To describe the roughness, the Root-Mean-Square parameter (Sq) was calculated. A smoothing effect of the cartilage surface was detectable upon each pattern of RFE treatment. The Sq for native cartilage was Sq=3.8 +/- 1.1 mu m. The best smoothing pattern was seen for two RFE passes and a 2-second pulsed mode (B2p2) with an Sq=27.3 +/- 4.9 mu m. However, with increased smoothing, an augmentation in chondrocyte death up to 95% was detected. Using bipolar RFE treatment in arthroscopy for small joints like the wrist or MCP joints should be used with caution. In the case of chondroplasty, there is a high chance to destroy the joint cartilage

Cell-based treatment options facilitate regeneration of cartilage, ligaments and meniscus in demanding conditions of the knee by a whole joint approach

Purpose This article provides an update on the current therapeutic options for cell-based regenerative treatment of the knee with a critical review of the present literature including a future perspective on the use of regenerative cell-based approaches. Special emphasis has been given on the requirement of a whole joint approach with treatment of comorbidities with aim of knee cartilage restoration, particularly in demanding conditions like early osteoarthritis. Methods This narrative review evaluates recent clinical data and published research articles on cell-based regenerative treatment options for cartilage and other structures around the knee Results Cell-based regenerative therapies for cartilage repair have become standard practice for the treatment of focal, traumatic chondral defects of the knee. Specifically, matrix-assisted autologous chondrocyte transplantation (MACT) shows satisfactory long-term results regarding radiological, histological and clinical outcome for treatment of large cartilage defects. Data show that regenerative treatment of the knee requires a whole joint approach by addressing all comorbidities including axis deviation, instability or meniscus pathologies. Further development of novel biomaterials and the discovery of alternative cell sources may facilitate the process of cell-based regenerative therapies for all knee structures becoming the gold standard in the future. Conclusion Overall, cell-based regenerative cartilage therapy of the knee has shown tremendous development over the last years and has become the standard of care for large and isolated chondral defects. It has shown success in the treatment of traumatic, osteochondral defects but also for degenerative cartilage lesions in the demanding condition of early OA. Future developments and alternative cell sources may help to facilitate cell-based regenerative treatment for all different structures around the knee by a whole joint approach

Cells under pressure – the relationship between hydrostatic pressure and mesenchymal stem cell chondrogenesis

Early osteoarthritis (OA), characterised by cartilage defects, is a degenerative disease that greatly affects the adult population. Cell-based tissue engineering methods are being explored as a solution for the treatment of these chondral defects. Chondrocytes are already in clinical use but other cell types with chondrogenic properties, such as mesenchymal stem cells (MSCs), are being researched. However, present methods for differentiating these cells into stable articular-cartilage chondrocytes that contribute to joint regeneration are not effective, despite extensive investigation. Environmental stimuli, such as mechanical forces, influence chondrogenic response and are beneficial with respect to matrix formation. In vivo, the cartilage is subjected to multiaxial loading involving compressive, tensile, shear and fluid flow and cellular response. Tissue formation mechanobiology is being intensively studied in the cartilage tissue-engineering research field. The study of the effects of hydrostatic pressure on cartilage formation belongs to the large area of mechanobiology. During cartilage loading, interstitial fluid is pressurised and the surrounding matrix delays pressure loss by reducing fluid flow rate from pressurised regions. This fluid pressurisation is known as hydrostatic pressure, where a uniform stress around the cell occurs without cellular deformation. In vitro studies, examining chondrocytes under hydrostatic pressure, have described its anabolic effect and similar studies have evaluated the effect of hydrostatic pressure on MSC chondrogenesis. The present review summarises the results of these studies and discusses the mechanisms through which hydrostatic pressure exerts its effects

The Importance of Physioxia in Mesenchymal Stem Cell Chondrogenesis and the Mechanisms Controlling Its Response

Articular cartilage covers the surface of synovial joints and enables joint movement. However, it is susceptible to progressive degeneration with age that can be accelerated by either previous joint injury or meniscectomy. This degenerative disease is known as osteoarthritis (OA) and it greatly affects the adult population. Cell-based tissue engineering provides a possible solution for treating OA at its earliest stages, particularly focal cartilage lesions. A candidate cell type for treating these focal defects are Mesenchymal Stem Cells (MSCs). However, present methods for differentiating these cells towards the chondrogenic lineage lead to hypertrophic chondrocytes and bone formation in vivo. Environmental stimuli that can stabilise the articular chondrocyte phenotype without compromising tissue formation have been extensively investigated. One factor that has generated intensive investigation in MSC chondrogenesis is low oxygen tension or physioxia (2–5% oxygen). In vivo articular cartilage resides at oxygen tensions between 1–4%, and in vitro results suggest that these conditions are beneficial for MSC expansion and chondrogenesis, particularly in suppressing the cartilage hypertrophy. This review will summarise the current literature regarding the effects of physioxia on MSC chondrogenesis with an emphasis on the pathways that control tissue formation and cartilage hypertrophy

Stem cell differentiation increases membrane-actin adhesion regulating cell blebability, migration and mechanics

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/K. S. is funded by an EPSRC PhD studentship. S.T. is funded by an EU Marie Curie Intra European Fellowship (GENOMICDIFF)

Physioxia Has a Beneficial Effect on Cartilage Matrix Production in Interleukin-1 Beta-Inhibited Mesenchymal Stem Cell Chondrogenesis

Osteoarthritis (OA) is a degenerative condition that involves the production of inflammatory cytokines (e.g., interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)) that stimulate degradative enzymes, matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS) resulting in articular cartilage breakdown. The presence of interleukin-1 beta (IL-1 beta) is one reason for poor clinical outcomes in current cell-based tissue engineering strategies for treating focal early osteoarthritic defects. Mesenchymal stem cells (MSCs) are a potential cell source for articular cartilage regeneration, although IL-1 beta has been shown to inhibit in vitro chondrogenesis. In vivo, articular chondrocytes reside under a low oxygen environment between 2-5% oxygen (physioxia) and have been shown to enhance in vitro MSC chondrogenic matrix content with reduced hypertrophic marker expression under these conditions. The present investigation sought to understand the effect of physioxia on IL-1 beta inhibited MSC chondrogenesis. MSCs expanded under physioxic (2% oxygen) and hyperoxic (20%) conditions, then chondrogenically differentiated as pellets in the presence of TGF-beta 1 and either 0.1 or 0.5 ng/mL IL-1 beta. Results showed that there were donor variations in response to physioxic culture based on intrinsic GAG content under hyperoxia. In physioxia responsive donors, MSC chondrogenesis significantly increased GAG and collagen II content, whilst hypertrophic markers were reduced compared with hyperoxia. In the presence of IL-1 beta, these donors showed a significant increase in cartilage matrix gene expression and GAG content relative to hyperoxic conditions. In contrast, a set of MSC donors were unresponsive to physioxia and showed no significant increase in matrix production independent of IL-1 beta presence. Thus, physioxia has a beneficial effect on MSC cartilage matrix production in responsive donors with or without IL-1 beta application. The mechanisms controlling the MSC chondrogenic response in both physioxia responsive and unresponsive donors are to be elucidated in future investigations

Fibronectin Adherent Cell Populations Derived From Avascular and Vascular Regions of the Meniscus Have Enhanced Clonogenicity and Differentiation Potential Under Physioxia

The meniscus is composed of an avascular inner region and vascular outer region. The vascular region has been shown to contain a progenitor population with multilineage differentiation capacity. Strategies facilitating the isolation and propagation of these progenitors can be used to develop cell-based meniscal therapies. Differential adhesion to fibronectin has been used to isolate progenitor populations from cartilage, while low oxygen or physioxia (2% oxygen) enhances the meniscal phenotype. This study aimed to isolate progenitor populations from the avascular and vascular meniscus using differential fibronectin adherence and examine their clonogenicity and differentiation potential under hyperoxia (20% oxygen) and physioxia (2% oxygen). Human vascular and avascular meniscus cells were seeded onto fibronectin-coated dishes for a short period and monitored for colony formation under either hyperoxia or physioxia. Non-fibronectin adherent meniscus cells were also expanded under both oxygen tension. Individual fibronectin adherent colonies were isolated and further expanded, until approximately ten population doublings (passage 3), whereby they underwent chondrogenic, osteogenic, and adipogenic differentiation. Physioxia enhances clonogenicity of vascular and avascular meniscus cells on plastic or fibronectin-coated plates. Combined differential fibronectin adhesion and physioxia isolated a progenitor population from both meniscus regions with trilineage differentiation potential compared to equivalent hyperoxia progenitors. Physioxia isolated progenitors had a significantly enhanced meniscus matrix content without the presence of collagen X. These results demonstrate that combined physioxia and fibronectin adherence can isolate and propagate a meniscus progenitor population that can potentially be used to treat meniscal tears or defects

Extracellular Vesicles Derived from Osteogenic-Differentiated Human Bone Marrow-Derived Mesenchymal Cells Rescue Osteogenic Ability of Bone Marrow-Derived Mesenchymal Cells Impaired by Hypoxia

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O2) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs’ and norm-naïve EVs’ parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes