Current cell-based tissue engineering strategies have limited clinical applicability due to the need for large cell numbers and prolonged culture periods that lead to phenotypic drift.
Phenotypic drift of stem cells and insufficient production of extracellular matrix (ECM) are frequently observed in tissue-engineered cartilage substitutes, posing major weaknesses of clinically relevant therapies targeting cartilage repair. Microenvironment plays an important role for stem cell maintenance and differentiation and therefore an optimal chondrogenic differentiation protocol is highly desirable. Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates biological processes by several orders of magnitude. MMC was recently shown to significantly increase ECM deposition and to promote chondrogenic differentiation of stem cells. We hypothesise that the addition of sulphated high-molecular weight polysaccharides (carrageenan) to the media positively affects stem cell maintenance and chondrogenic differentiation. Herein, we venture to assess the impact of MMC on the maintenance of stem cell phenotype and multipotency, and ECM deposition in xeno-free human bone marrow mesenchymal stem cell (BMSCs) cultures. We investigate different xeno- and serum-free stem cell media with MMC for expansion of BMSCs, assessing multipotency maintenance (FACS analysis), cell viability, metabolic activity, proliferative capacity and matrix deposition (SDS-PAGE, ICC) at day 4 and day 10. Experiments will be conducted at 2 different passages (p3, p7). Medium without MMC will be used as control. Based on these results, cells expanded with the best protocol will be subsequently investigated for chondrogenic differentiation comparing different xeno-/serum-free and serum containing differentiation media. Chondrogenic differentiation will be assessed via Alcian blue and Safranin O stainings, gene expression for chondrogenic marker genes and quantification of GAG content. Finally, these findings will pave the way for developing more effective strategies for cartilage tissue engineering.
Cell-based tissue engineering strategies for tendon repair have limited clinical applicability due to delayed extracellular matrix (ECM) deposition and subsequent prolonged culture periods, which lead to tenogenic phenotypic drift. Deposition of ECM in vitro can be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the intra- and extra-cellular milieu of multicellular organisms, which has been described to accelerate ECM deposition in human tenocytes. A variety of cell sources have been studied for tendon repair including tenocytes, dermal fibroblasts (DFs) and mesenchymal stem cells (MSCs) and various biophysical, biochemical and biological tools have been used to mimic tendon microenvironment. Therefore, we propose to assess the combined effect of MMC and mechanical loading on different cell sources to determine their suitability for the in vitro fabrication of tendon-like tissue. The uniaxial strain induced differential cell orientation based on the differentiation state of the cells: tenocytes and DFs, both permanently differentiated cells exhibited alignment perpendicular to the direction of the load, similarly to what is seen in native tendon environment. Immunocytochemistry showed that, when MMC is used, the DFs and MSCs showed increased deposition of collagen type I, one of the main components in tendon ECM. It is also seen that the ECM deposited follows the alignment of the cell cytoskeleton. However, for tenocytes, deposition of collagen type I is only seen when MMC is used in combination with mechanical loading, indicating that mechanical loading led to increased synthesis of collagen I, suggesting maintenance of the tenogenic phenotype. Other collagen types relevant to native tendon composition were also analysed, including types III, V and VI, and their deposition was also shown to be modulated by the use of MMC and mechanical loading. This appears to recreate the events of tendon tissue formation during development, where these collagen types are involved in regulation of collagen I fibrillogenesis and fibril diameter. Preliminary data also indicates that, under mechanical loading and MMC, expression of tenogenic genes is upregulated whilst chondrogenic and osteogenic markers are downregulated. This indicates the suitability of the combination of MMC and mechanical stimulation for modulating tenogenic phenotype of various cell sources and fabricating tendon-like tissue.
Cell-based tissue engineering strategies for tendon repair have limited clinical applicability due to delayed extracellular matrix (ECM) deposition and subsequent prolonged culture periods, which lead to tenogenic phenotypic drift. Deposition of ECM Human dermal fibroblasts, tenocytes and bone marrow mesenchymal stem cells were cultured for 3 days with 100 µg/ml of carrageenan (MMC) under static and dynamic culture conditions. Cyclic uniaxial strain was applied using a MechanoCulture FX (CellScale) at 1 Hz and 10% strain for 12 hours a day. Cell morphology and alignment were evaluated by fluorescein isothiocyanate (FITC) labelled phalloidin and 4’,6-diamidino-2-phenylindole (DAPI) staining. Extracellular matrix composition was evaluated by immunocytochemistry. Cell phenotype maintenance/differentiation (tenogenic, chondrogenic and osteogenic lineages) were assessed by gene and protein analysis. After 12 hours of exposure to the uniaxial load, permanently differentiated cells are strictly aligned in the direction perpendicular to the load while the MSCs do not show preferential alignment. ECM deposition (e.g. collagens type I, III, V, VI) is increased in the presence of MMC and this effect is maintained under mechanical loading. ECM deposited under mechanical loading is also aligned in the direction perpendicular to the load. Tenogenic, osteogenic and chondrogenic markers are being tested to assess cell phenotype. Mechanical loading and macromolecular crowding can induce cell and ECM alignment and increased ECM deposition without affecting cell metabolic activity or viability. Cell and ECM alignment alongside ECM composition and tenogenic marker expression suggest this approach might be suitable to maintain or differentiate towards tenogenic lineage.
