Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude. Herein, we ventured to identify the optimal crowder and to assess the influence of MMC in umbilical cord mesenchymal stem cell. 7 types of carrageenan (κ&λ, κ-LV1, κ-LV2, λ-MV, λ-HV, ι-MV, ι-HV) acted as crowder and biophysical properties were assessed respectively. Human umbilical cord mesenchymal stem cells were seeded at 15,000 cells/cm² in 24 well plates and allowed to attach for 24 h. Subsequently, the medium was changed to medium with 7 types of carrageenan (10, 50, 100, 500 μg/ml) and 100 μM L-ascorbic acid phosphate (Sigma Aldrich). Medium without carrageenan was used as control. Cell morphology and SDS-PAGE analysis were conducted after 3, 5 and 7 days. Biophysical assessment showed 7 types of carrageenan have increased particle size with concentration, good polydispersity and negative charges. SDS-PAGE and densitometric analyses revealed significant increase (p < 0.001) in collagen deposition in the presence of 10 μg/ml carrageenan λ and ι at all the time points. SDS-PAGE and densitometric analysis also showed that the highest collagen deposition was observed in culture at 50 μg/ml carrageenan λ. No significant difference was observed in cell morphology between the groups. Collectively, these data primarily illustrate the beneficial effect of carrageenan λ in human umbilical cord mesenchymal stem cell culture.

Macromolecular crowding (MMC) accelerates matrix deposition through excluded volume effect (EVE). Herein, we ventured to identify the optimal decellularisation protocol of MMC enhanced fibroblast cultures as a new cell formed platform model. Human dermal fibroblasts (hDF), human lung fibroblasts (hLF), and human mammary fibroblasts (hMF)seeded at 50,000 cells/cm² were cultured for 10 days without and with MMC (100 μg/mL carrageenan) and 100 μM L-ascorbic acid phosphate. Subsequently, the cultured cell layers were decellularised using various decellularisation protocols [i.e., ammonium hydroxide (NH₄OH), sodium deoxycholate (DOC), SDS-EDTA mixed buffer, and nonident P40 (NP40)]. SDS-PAGE, hydroxyproline assay, sGAG assay, SEM, histological staining (i.e., picrosirius red stain and H&E), immunocytochemistry (i.e., collagen I, III and fibronectin), PicoGreen® assay. SDS-PAGE with complementary density and hydroxyproline analysis for assessing collagen deposition, and sGAG assay for total sGAG content assessment demonstrated significantly increased (p< 0.001) in the presence of MMC. SEM, histological and immunocytochemistry displayed enhanced ECM deposition, integrity, and maintenance of the matrix composition in the presence of MMC. PicoGreen® assay revealed efficient decellularisation with significant removal of DNA (p <0.001) in all matrices. MMC can be used effectively to accelerate ECM deposition by fibroblast from various tissue sources, to facilitate production of cell-derived matrix-rich constructs feasible as robust platform models.

Mesenchymal stromal cells (MSCs) have been one of the most widely studied cell types in preclinical and clinical trials, due to their self-renewing, multipotent capacity, immunomodulatory properties and relative ease of isolation from multiple tissues. Despite limitations and safety concerns, fetal bovine serum (FBS) is still predominantly used for MSC expansion in clinical protocols. In addition, the undefined nature of serum composition and lot-to-lot variability have been linked to reduced reproducibility and efficiency of MSC bioprocessing. Moreover, use of animal serum in human cell culture increases the risk of contamination with adventitious pathogenic microorganisms, such as viruses, prions and bacteria. Hence, a defined serum-free formulation can provide increased safety, better control over physiological responsiveness, consistent performance and reproducible results. Here we present preliminary data on a prototype serum-free medium optimized for in vitro tenogenic differentiation of human bone marrow-derived MSCs. This serum-free formulation is capable of generating tenocyte-like cells in vitro expressing tenogenic markers such as Scx, Tnmd, TnC, Collagen I and Collagen III, whilst repressing expression of specific markers of other mesenchymal lineages.

Introduction

Collagen is the predominant component of extracellular matrix in various connective tissues and makes up to 25% to 35% of the whole protein content in animal bodies. Type II collagen was first introduced from chicken sternal cartilage and presents supportive function in cartilaginous tissue. Since type II collagen is the major component of cartilage in joint, this study is aiming to determine an optimal type II collagen material for the development of medical devices for articular cartilage regeneration. In order to make more effective use of underutilized food waste, type II collagens from mammalian tissue sources (porcine tracheal cartilage; auricular cartilage; articular cartilage) and marine tissue sources (cuckoo ray, blonde ray, thorn back ray, lesser spotted dogfish) were isolated through acid-pepsin digestion under 4°C and characterized by various biological, biochemical and biophysical analysis. Pepsin cleaves the telopeptide region of the collagen molecule and pepsin treated collagen extraction ensures higher collagen yield. Telopeptide-free collagen reveals cytocompatibility, biodegradability and lower toxicity. The number and size of collagen chains were revealed by SDS-polyacrylamide gel electrophoresis. Intermolecular crosslinking density was quantified by Ninhydrin assay. Thermal stability was tested by differential scanning calorimetry (DSC) and enzymatic degradation was assessed by collagenase assay. Human chondrocytes were seeded on to collagen sponges at a density of 30,000 cells per sponge. Cell morphology (DAPI/ Rhodamine Phalloidin), viability(LIVE/DEAD®), proliferation(PicoGreen®) and metabolic activity (alamarBlue®) were analysed. Quantitative morphometric analysis was carried out using ImageJ software.

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.

The main limitation of autologous chondrocyte implantation techniques is the necessity for in vitro cell expansion, which is associated with phenotypic drift and loss of extracellular matrix synthesis. Although media supplements (e.g. TGF-β) are extensively used to mitigate the tendency of de-differentiation, the lack of extracellular matrix is still one of the major obstacles to obtaining engineered cartilage substitutes with long-term clinical efficacy. Macromolecular crowding (MMC) is a biophysical phenomenon that increases tissue-specific extracellular matrix deposition. This study aimed to test whether MMC can be used to enhance hyaline-like ECM deposition in human chondrocyte culture: this hypothesis was tested in cells at P2 and at P7. Cells at P2 were cultured using a standard medium (DMEM/F12) in monolayer or alginate beads, whilst cells at P7 were cultured and re-differentiated using the system Clonetics™ of Lonza in the presence of 5 % HS or 5 % FBS, in monolayer and alginate beads. Macromolecular crowding medium was added 14 days after the start of re-differentiation. Collagen deposition was evaluated after 2, 5 and 10 days using SDS-PAGE and immunocytochemistry. MMC enhanced matrix deposition in all the conditions tested. However, although cells at P7 were cultured using a commercially available system, their deposited matrix was richer in collagen type I, whilst collagen type II was barely detectable. This was even more evident for cells in monolayer in HS and indicates that cells acquired a fibroblastic phenotype. To conclude, we showed that MMC increased matrix deposition in chondrocyte culture and that, unfortunately, commercially available systems are not always able to maintain chondrogenic phenotype. Since ECM produced is often undetectable and collagen expression and synthesis are not always correlated with its secretion, we propose to use MMC to assess chondrocyte phenotype maintenance and effectiveness of re-differentiation media.

Cell-based therapies require removal of cells from their optimal in vivotissue context and propagation in vitroto attain suitable number. However, bereft of their optimal tissue niche, cells lose their phenotype and with it their function and therapeutic potential. Biophysical signals, such as surface topography and substrate stiffness, and biochemical signals, such as collagen I, have been shown to maintain permanently differentiated cell phenotype and to precisely regulate stem cell lineage commitment (1, 2). Herein, we developed and characterised substrates of variable rigidity and constant nanotopographical features to offer control over cellular functions during ex vivoexpansion.

PDMS substrates with varying ratios of monomer to curing agent (0:1, 1:1, 5:1) were fabricated based on established protocols. Grooved substrates were created using a silinated wafer with groove dimensions of 2µm × 2µm × 2µm; planar control groups were created using flat glass. The aforementioned PDMS solutions were poured onto the wafer/glass, cured at 200 ºC and treated with oxygen plasma. Substrates were then investigated with/without collagen I coating. (0.1, 0.5, and 1 mg/ml). Atomic force microscopy (AFM) and optical profilometry were used to assess the topographical features of the substrates. Dynamic mechanical analysis (DMA) was used to determine the mechanical properties of the substrates. The simultaneous effect of surface topography / substrate rigidity on cell phenotype and function was assessed using human permanently differentiated cells (dermal fibroblasts, tenocytes) and stem cells (human bone marrow stem cells) and various morphometric and gene / protein assays.

PDMS substrates of varying stiffness (1000 kPa, 130 kPa, 50 kPa) can be made by varying the Sylgard ratio, while maintaining topographical features. Human adult dermal fibroblasts, tenocytes, and tenocytes attach, align, elongate and deposit aligned extracellular matrix on the grooved PDMS substrate surface of all 3 stiffnesses.

Preliminary in vitrodata indicate that surface topography and substrate stiffness play crucial role in maintaining cell phenotype and the prevention of phenotypic drift in vitro.

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 vitrocan be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the intra- and extra-cellular milieu of multicellular organisms², which has been described to accelerate ECM deposition in human tenocytes¹. A variety of cell sources have been studied for tendon repair including tenocytes, dermal fibroblasts and mesenchymal stem cells (MSCs)³and various biophysical, biochemical and biological tools have been used to mimic tendon microenvironment and induce phenotype maintenance in long term cultures or differentiation⁴. Therefore, we propose to assess the combined effect of macromolecular crowding and mechanical loading on different cell sources to determine their suitability for the in vitro fabrication of tendon-like tissue.

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.

Summary

Tissue grafts fail to recapitulate native tendon function, imposing the need for development of functional regeneration strategies. Herein, we describe advancements in tendon repair and regeneration using functionalised natural and synthetic devices and scaffold-free cell-based therapies.