title: Characterising Load-Induced Changes in 3D Cultured Mesenchymal Stem Cells Through Collagen Isoform Composition and Arrangement
Creators: name: Janvier, Adam
Publisher: name: University of Liverpool Repository
Year: 2021

description: Tissue engineering has been highlighted as a potential regenerative medicine therapy for the regeneration of musculoskeletal tissues, many of which have poor healing capacity. Currently there is no ‘gold standard’ approach to tissue engineering with many researchers investigating the effects of different stimuli on different cells in different culture environments. One of these stimuli is mechanical stimulation, a variety of which naturally occur in the body. Mechanical stimulation is often used in tissue engineering to recapitulate the structure, extracellular matrix composition (ECM) and biomechanics of tissues such as tendon, bone and cartilage. The extracellular matrix of these tissues is primarily composed of one fibril forming collagen, for tendon and bone this is collagen type I whilst for cartilage it is collagen II. However an array of additional collagen isoforms play important roles in ECM architecture and maturation. The aim of this thesis was to investigate if collagen synthesis can be used to assess human mesenchymal stem cells (hMSCs) differentiation in response to different mechanical stimulation. Typically tissue engineering studies use the most populous ECM components to highlight the success of the engineered tissues, whilst this makes sense it neglects the minor ECM components. For musculoskeletal tissues fibril forming collagens are routinely the dominant component of the ECM, however without the minor collagens these structure would not function appropriately. The composition of collagens varies across all musculoskeletal tissues, therefore by investigating the complete collagen composition the differentiation of the cells can be identified and the quality of the tissue being engineered can be established. Tensile stimulation, hydrostatic pressure and microgravity were applied to hMSCs seeded within fibrin hydrogels, chosen as it acts as a blank slate material for collagen investigation. These mechanical stimuli were selected as they have all routinely been used to show enhanced or inhibited MSC differentiation, offering a well-established set of mechanical stimulations to investigate their role in the differentiation of hMSCs and how the subsequent collagen production can be used to identify it. Molecular (qPCR and western blot), imaging (histology, TEM and fluorescence) and structural (mechanical testing and μCT) analytical techniques have been used to assess what collagens have been produced and how this relates to the structural development of the engineered tissue. The cell embedded hydrogels had varying responses to the different percentages of cyclic tensile stimulation (0%, 3%, 5% and 10%). These specific strains were selected to assess how hMSCs would respond to the static culture (0%), low physiological dynamic strain (1-4%), high physiological dynamic strain (5-6%) and degenerative dynamic strain (&gt;6%). The 0% and 10% strain groups indicated some osteogenic differentiation through Alizarin red staining and ALP analysis from the culture media. Suggesting that physiologically relevant dynamic strain inhibited osteogenic differentiation. 3% cyclic strain saw a two-fold increase in maximum stress and a slight decrease in fibril diameter compared to the control. The 5% strain group saw increases in tendon collagens COL3A1 and COL11A1 as well as tenogenic markers SCXA and TNMD though expression of the negative tendon marker COL2A1 was also increased. At the protein level collagen II was downregulated whilst collagen III was upregulated compared to the control. The fibril diameter and fibre alignment was found to be highest in the 10% strain group, typically a marker of increased mechanical properties, however with 10% strain only the rate of stress relaxation was increased compared to other groups with a decrease in maximum stress compared to the 3% strain group. The microtissues used for hydrostatic pressure were cultured in one of three culture medias, basic, chondrogenic or osteogenic and with one of four hydrostatic pressure condition, control, 100 kPa, 200 kPa or 300 kPa. The effects of hydrostatic pressure was largely overridden by the differentiation media supplements with the basic media group showing the biggest changes in response to different levels of hydrostatic pressure. The chondrogenic media group displayed the highest level of COL1A1, COL2A1 and COL10A1 suggesting that the hMSCs within this media group were undergoing hypertrophy. At the protein level no microtissues saw significance within a media group suggesting that hydrostatic pressure was not influencing the collagen synthesis of the hMSCs as much as the media types. The μCT analysis showed within the media groups the density of the mineralised particles was largely unchanged for the basic media, chondrogenic media and control and 100 kPa osteogenic media samples with the osteogenic 200 and 300 kPa being near two fold greater than all other conditions, suggesting that with appropriate media the higher loading regimens generated a more developed mineralised structure than the lower hydrostatic pressure. It appeared that the microgravity microtissues were all pre-disposed to spontaneously differentiate towards the osteogenic lineage as seen through the collagen gene expression. Further ALP concentration in the media increased in all culture condition across the three week culture period. PCA analysis showed evidence that the static culture was acting separately from the dynamic and microgravity culture suggesting that the increased nutrient diffusion within the RCCS 4H bioreactor was having a significant effect on the culture. Analysis of the ratio of COL14A1 to COL12A1 was used to demonstrate which culture was the most mature, COL14A1 indicating immaturity and COL12A1 maturity. The microgravity group had the least developed ECM due to the highest ratio, whilst the static group had the most developed ECM due to the lowest ratio. This was further supported through the PCA analysis highlighting COL12A1 as one of the largest contributing variables to the statics groups separation from the other two. This indicated that increased nutrient diffusion was inhibiting the maturation of the MSCs compared to static culture and microgravity was amplifying this effect.