Martin J. Stoddart

Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic.

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. Stem Cells 2014;32:1713–1723

Physical Stimulation of Chondrogenic Cells In Vitro: A Review

BackgroundMechanical 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/purposesThis 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.MethodsWe 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.ResultsThere is much data pertaining to increases in chondrogenic gene expression as a result of controlled loading protocols. Uniaxial loading leads to selective upregulation of genes normally associated with a chondrogenic phenotype, whereas multiaxial loading results in a broader pattern of chondrogenic gene upregulation. The potential for the body to be used as an in vivo bioreactor is being increasingly explored.ConclusionsBioreactors are important tools for understanding the potential response of chondrogenic cells within the joint environment. However, to replicate the natural in vivo situation, more complex motion patterns are required to induce more physiological chondrogenic gene upregulation.

Cells and biomaterials in cartilage tissue engineering

Cartilage defects are notoriously difficult to repair and owing to the long-term prognosis of osteoarthritis, and a rapidly aging population, a need for new therapies is pressing. Cell-based therapies for cartilage regeneration were introduced into patients in the early 1990s. Since that time the technology has developed from a simple cell suspension to more complex 3D structures. Cells, both chondrocytes and stem cells, have been incorporated into scaffold material with the aim to better recreate the natural environment of the cell, while providing more structural support to withstand the large forces applied on the de novo tissue. This review aims to provide an overview of potential cell sources and different scaffold materials, which are in development for cartilage tissue engineering.

Chondrogenesis of Human Bone Marrow Mesenchymal Stem Cells in Fibrin–Polyurethane Composites

This study investigated whether a three-dimensional (3D) fibrin gel-polyurethane scaffold composite can provide an environment for chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs) that is as supportive as pellet culture, which is an established model for evaluating chondrogenesis. Pellet culture was carried out in serum-free medium in the absence or presence of transforming growth factor beta 1 (TGF-beta1) and dexamethasone. hMSCs were seeded into a fibrin gel-biodegradable polyurethane scaffold at cell densities of 2 x 10(6), 5 x 10(6), and 10 x 10(6) cells per scaffold and cultured in serum-free medium supplemented with TGF-beta1 and dexamethasone. With comparable proteoglycan synthesis and type I and type X collagen gene expression levels, scaffolds seeded with 5 x 10(6) cells expressed higher type II collagen and aggrecan gene transcripts than pellets on day 14. The deposition of proteoglycan and type II collagen was detected on the top layer of scaffolds seeded with 10 x 10(6) cells and was more evenly distributed in the scaffolds seeded with 5 x 10(6) cells. The scaffold composite culture system shows chondrogenesis of hMSCs comparable with that of pellet culture. Initial cell seeding density influences the ability and process of hMSC chondrogenesis. This study founded a basic system for cartilage neo-tissue formation in vitro.

Epsilon-Aminocaproic Acid Is a Useful Fibrin Degradation Inhibitor for Cartilage Tissue Engineering

Fibrin is a hydrogel carrier widely used in cartilage tissue engineering. It is rapidly degraded by plasmin, which is produced by the cells. epsilon-Aminocaproic acid (EACA) can be used to inhibit this enzyme and thus save the fibrin carrier. In this study we investigated the effect of EACA on the transforming growth factor beta-1-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). To assess this, we used the standard pellet culture system, and EACA treatment was compared to an untreated chondrogenic control. To investigate differentiation, real-time RT-PCR was used on chondrocytic marker genes: aggrecan, collagen types II and X, and the SRY-related HMG-box gene 9 (SOX9). Also, specific glycosaminoglycan production was measured. Safranin-O/fast green staining was used to localize proteoglycans and collagens within the pellet. All results concur that EACA did not affect the chondrogenic differentiation process at 5 muM concentration, which is adequate to inhibit fibrin degradation. Therefore, it is a useful plasmin inhibitor for cartilage tissue engineering with hMSCs.

Statin-induced calcification in human mesenchymal stem cells is cell death related

Statins are widely used in clinics to lower cholesterol levels. Recently, they have been shown to positively affect bone formation and bone mass in a rat model. The aim of this study was to investigate the effect of pravastatin, simvastatin and lovastatin on the osteoblastic differentiation of human mesenchymal stem cells (MSCs) in vitro. Cell number, alkaline phosphatase (ALP) activity, matrix mineralization and gene expression pattern were determined. Pravastatin did not affect cell differentiation. Simvastatin and lovastatin enhanced bone morphogenetic protein 2 (BMP‐2) mRNA levels. In contrast, ALP activity and mRNA levels were suppressed by statins, as well as the DNA content and cell activity (MTT). An increase in apoptotic events was observed at high concentrations of statins, along with high Ca‐45 incorporation. Lower concentrations of statins did not increase apoptotic staining, but also failed to induce calcification. When statin‐induced calcification did occur, the morphology of the deposits was very different from the conventional nodule formation; the calcium was laid down along the membranes of the rounded cells suggesting it was as a result of cell death. Our results indicate that statins are not able to differentiate human MSCs into osteoblasts and that high concentrations of statins (>1 μM) have a cytotoxic effect.

Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis.

Mechanical and chemical stimulation have been shown to enhance in vitro chondrogenesis. The aim of this study was to analyze and compare combined physicobiochemical effects. Bovine articular chondrocytes were retrovirally transduced to express bone morphogenetic protein-2 (BMP-2) or left as naïve controls. Cells were seeded in three-dimensional polyurethane scaffolds and further cultured under static conditions or exposed to dynamic compression and shear in a joint-specific bioreactor. Four groups: control (A), load (B), BMP-2-infected (C), and BMP-2-infected plus load (D) were analyzed for DNA and glycosaminoglycan (GAG) content; collagen I, II, and X; aggrecan, (cartilage oligomeric protein (COMP), superficial zone protein, matrix metalloproteinase (MMP)-3; MMP-13 mRNA; histology; and immunohistochemistry at 7, 21, and 35 days post-seeding. Synergistic effects (D) were higher than the sum of the individual treatments (B and C) for GAG/DNA, collagen II, and COMP. Histology revealed a functional organization in D including an intense safranin O staining in C and D superior to that in A and B. Immunostaining for collagen II and aggrecan was detected in C and D and was strongest in D. The results show that both stimuli augment in vitro chondrogenesis better than in controls. Biochemical manipulation proved to be predominantly more effective than load, and synergistic effects were demonstrated.

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3‐D constructs for cartilage tissue engineering

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production.

In vitro gene transfer to chondrocytes and synovial fibroblasts by adenoviral vectors.

The major requirement of a successful gene transfer is the efficient delivery of an exogenous therapeutic gene to the appropriate cell type with subsequent high or regulated levels of expression. In this context, viral systems are more efficient than nonviral systems, giving higher levels of gene expression for longer periods. For the application of osteoarthritis (OA), gene products triggering anti-inflammatory or chondroprotective effects are of obvious therapeutic utility. Thus, their cognate genes are candidates for use in the gene therapy of OA. In this chapter, we describe the preparation, the use, and the effect of the transduction of chondrocytes or synovial fibroblasts with an adenoviral vector encoding the cDNA for glutamine: fructose-6-phosphate amidotransferase (GFAT). This is intended to serve as an example of a technology that can be used to evaluate the biological effects of overexpression of other cDNAs.

Cells and secretome--towards endogenous cell re-activation for cartilage repair.

Regenerative medicine approaches to cartilage tissue repair have mainly been concerned with the implantation of a scaffold material containing monolayer expanded cells into the defect, with the aim to differentiate the cells into chondrocytes. While this may be a valid approach, the secretome of the implanted cells and its effects on the endogenous resident cells, is gaining in interest. This review aims to summarize the knowledge on the secretome of mesenchymal stem cells, including knowledge from other tissues, in order to indicate how these mechanisms may be of value in repairing articular cartilage defects. Potential therapies and their effects on the repair of articular cartilage defects will be discussed, with a focus on the transition from classical cell therapy to the implantation of cell free matrices releasing specific cytokines.