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
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
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
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. Porcine articular cartilage and cartilaginous fishes yield high purity type II collagen. Type II collagen isolated from cartilaginous fishes exhibited similar crosslinking density and thermal stability. Among various porcine cartilaginous tissues, articular cartilage was the most resistant to enzymatic degradation and female trachea exhibited the highest cross-linking density. The biological, biochemical and thermal properties of type II collagen are dependent on the tissue and gender from which the collagen was extracted.Introduction
Conclusion
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
Cell-based therapies require removal of cells from their optimal 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
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.
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. Tendon and ligament injuries constitute an unmet clinical need with approximately 100,000 new cases annually in US alone. Tissue grafts are considered the gold standard in clinical practice. However, allografts and xenografts can lead to potential disease transmission, whilst the limited supply of autografts in severe injuries and degenerative conditions restricts their use. To this end, scaffold and scaffold-free therapies are under development to address the tissue grafts shortage. Herein, we describe biophysical, biochemical and biological methods to maintain tendon derived cell phenotype and/or differentiation of other cell types towards tenogenic lineage; development of tendon-equivalent facsimiles; and ultimately functional neotendon formation.Summary
Introduction