spheroid niche to wound site.
4.3.3.1 Immunofluorescence microscopy: MSC differentiation (phosphorylated RUNX-2 and osteopontin staining)
The above mentioned procedure (see Section 4.3.2.1) was followed using fibroblasts and osteoblast cells. The niche model was cultured for either 3 or 14 days with both cell types, to assess migrating MSC staining for early and late osteogenic markers. The media covering the gels was changed twice weekly over the appropriate culture period. MSCs were fixed and stained with phosphorylated RUNX-2 (1:50 dilution), followed by biotinylated secondary (1:50 dilution) and streptavidin-Texas Red (1:50 dilution), then mounted with DAPI on day 3 (see section 3.3.3.3). Whereas, on day 14 the cells were stained for osteopontin (1:50 dilution), followed by biotinylated secondary (1:50 dilution) and streptavidin-Texas Red (1:50 dilution). The cells were then mounted with DAPI (see section 3.3.3.3). Both day 3 and day 14 samples were analysed using a Zeiss Axiovert 200M fluorescent microscope.
4.3.3.2 RNA extraction from the spheroid niche post-exposure to control and wounded osteoblasts
Differentiation analysis of migrated MSCs within niche model: Analysis was conducted on confluent primary osteoblast monolayers, which were either left unscratched or scratched with a P1000 pipette tip. The cells were seeded onto CellBind plates and osteogenic differentiation media (see section 3.3.5) was added for 10 days, prior to the addition of the niche model on top. After 24 hours or 72 hours, the collagen gels containing the spheroids were removed from the wells ready for RNA extraction. Six samples were pooled for each replicate (n=3).
A TRIzol extraction and Qiagen RNeasy micro kit was used as described in Chapter 3, section 3.3.6, to extract the RNA from the MSC spheroids, within the
method described in Chapter 3, section 3.3.7, with a modified primer list (see Table 4-1).
Table 4-1: Fluidigm primers designed for human genes. (*Primers used as housekeeping genes).
Primer Sequence (5’ to 3’)
β-Actin* Forward GTGGGCCGCCCTAGGCACCAG Reverse CACTTTGATGTCACGCACGATTTC RUNX-2 Forward CAGCAGCAGCAACAGCAG
Reverse GGCGATGATCTCCACCAT ACVR1A Forward GCCAAGGGGACTGGTGTAAC
Reverse GAGAATAATGAGGCCAACCTCCA SMAD1 Forward GCTGCTCTCCAATGTTAACCG
Reverse CACTAAGGCATTCGGCATACAC SMAD2 Forward CCACGGTAGAAATGACAAGAAGG
Reverse GATTACAATTGGGGCTCTGCAC SMAD3 Forward GTCTGCGTGAATCCCTACCACReverse GGGATGGAATGGCTGTAGTCG ENOX2* Forward GAGCTGGAGGGAACCTGATTT Reverse CACTGGCACTACCAAACTGCA SMAD4 Forward GGGTCAACTCTCCAATGTCCAC
Reverse GTCACTAAGGCACCTGACCC SMAD5 Forward TGGGTCAAGATAATTCCCAGCCT
Reverse GGCTCTTCATAGGCAACAGGC SMAD6 Forward CTCCCTACTCTCGGCTGTCT
Reverse AGAATTCACCCGGAGCAGTG SMAD7 Forward CCATCACCTTAGCCGACTCT Reverse CCAGGGGCCAGATAATTCGT SMAD9 Forward CTTATCATGCCACAGAAGCCTCT
Reverse GCTCCTCGTAACAAACTGGTCG BMPR1A Forward ACGCCGGACAATAGAATGTTGTC
Reverse GAGCAAAACCAGCCATCGAATG TWY1* Forward ATTGTCATCAAGACGCAGGGC
Reverse GTTGCGAATCCCTTCGCTGTT BMPR1B Forward GGTTCAGACTTCTGCTGATTCAT Reverse CGCAAAAGCATGTTATCAAGG BMP2 Forward CTTCTAGCGTTGCTGCTTCC Reverse AACTCGCTCAGGACCTCGT BMP2 Forward AGACCTGTATCGCAGGCACT Reverse CCACTCGTTTCTGGTAGTTCTTCC BMPR2 Forward AGCCTCTCACACCCACTCC Reverse GCAGAACAACCGTGAGAGG
ACVR1B Forward GACATTGCCCCGAATCAGAGG Reverse GCCCGAGGGCATAAATATCAGC BMP4 Forward CAGCACTGGTCTTGAGTATCCT
Reverse AGCAGAGTTTTCACTGGTCCC CYCR* Forward ACTGCGGGAAGGTCTCTACTT
Reverse GGGTGCCATCGTCAAACTCTA
BMP7 Forward CAGGCCTGTAAGAAGCACGA
Reverse TGGTTGGTGGCGTTCATGTA BMP10 Forward ACCCACCAGAGTACATGTTGG
Reverse GCCCATTAAAACTGACCGGC Nestin Forward GCTCAGGTCCTGGAAGGTC
Reverse AAGCTGAGGGAAGTCTTGGA CD63 Forward CCCTTGGAATTGCTTTTGTT
Reverse TATTCCACTCCCCCAGATGA ALCAM Forward TTCCAGTCCCTCTACTCAGAGC
Reverse GCTAAGAAGGACTCGCAGGA Osterix Forward TGGGCTCCCAACACTATTTC
Reverse GGGAAGACTGAAGCCTGGA UBE2D2* Forward CCATGGCTCTGAAGAGAATCC
Reverse GATAGGGACTGTCATTTGGCC RUNX1T1 Forward ATCACAACAGAGAGGGCCAA
Reverse CTGCAGGTTTCACTCGCTTT SMURF1 Forward ATGCAGTTCGTGGCCAGATA
Reverse CAGGCCCGGAGTCTTCATAC SMURF2 Forward GACAGGATCCTCTCGAGTGC
Reverse AGCTTTCATAGGGTGGAATGTCT INHBA Forward AAGTCGGGGAGAACGGGTAT
Reverse GGTCACTGCCTTCCTTGGAA ACVR2A Forward ACCATGGCTAGAGGATTGGC
Reverse GCCAACCCAAAGTCAGCAAT ACVR2B Forward CTGCAACGAACGCTTCACTC
Reverse CAGGACGATGAGGGAAAGGC RNF20* Forward GGTGTCTCTTCAACGGAGGAA Reverse TAGTGAGGCATCATCAGTGGC TGFB1 Forward CGACTCGCCAGAGTGGTTATC Reverse GTTATCCCTGCTGTCACAGGAG TGFBR1 Forward CGTTCGTGGTTCCGTGAGG Reverse TAATCTGACACCAACCAGAGCTG TRAF6 Forward CGCACTAGAACGAGCAAGTGA
Reverse GCCACACAGCAGTCACTTTCA SNAIL2 Forward TCCTTCCTGGTCAAGAAGCA
Reverse GGTATGACAGGCATGGAGTA Vimentin Forward GGAGAAATTGCAGGAGGAGA
Reverse TGCGTTCAAGGTCAAGACGT IL-8 Forward GTGTGAAGGTGCAGTTTTGCC
Reverse GTGGTCCACTCTCAATCACTC B2M* Forward TTGTCTTTCAGCAAGGACTGG
Reverse ATGCGGCATCTTCAAACCTCC CathepsinB Forward TGTGTATTCGGACTTCCTGC
Reverse TTAAAGAAGCCATTGTCACCC CathepsinD Forward GGTGCTCAAGAACTACATGG
Reverse ATTCTTCACGTAGGTGCTGG CathepsinG Forward AACAGATACACTCCGAGAGG Reverse ACGACTTTCCATAGGAGACG CathepsinL Forward GACTCTGAGGAATCCTATCC Reverse CTTAGGGATGTCCACAAAGC CathepsinS Forward GCGTCATCCTTCTTTCTTCC Reverse CCAGCTGTTTTTCACAAGCC GAPDH* Forward TCAAGGCTGAGAACGGGAA
Reverse TGGGTGGCAGTGATGGCA
Permutmatrix software (Réseau National des plates-formes Bioinformatiques (ReNaBi), France) was used on each cluster gene expression, to convert the numerical values from the collected data into a graduation scale of colours. For example, shades of red were used to represent the degree of up regulated genes, whilst shades of green indicated the degrees of down regulated genes.
4.4 Results
4.4.1 Time-lapse assessment of wound healing assays.
Confluent monolayer culture systems were scratched (to create an artificial wound) and time-lapse experiments were conducted, to assess the ability of the monolayer to repair and close the scratch/wound. Two different cell models were employed; (i) a connective tissue model using fibroblasts (h-TERTS) and (ii) a bone matrix model using primary osteoblasts. The cell models were grown in either the presence or absence of overlaid collagen gels (Figure 4-1). It was necessary to assess whether the presence of collagen gels altered the rate of wound closure.
Figure 4-1 demonstrated both fibroblasts and osteoblasts directionally migrated from the edge of the wound inwards, across the scratch to close the wound. However, in the absence of a collagen gel, fibroblasts closed the wound much quicker, (after only 24 hours) whereas, the osteoblasts took 5 days to close the wound.
However, the scratch (wound) healed slower in the presence of the collagen gel, in both the connective tissue and bone matrix models (Figure 4-1). The connective tissue model, took an extra 5 days for the scratch to close in the presence of collagen gels. Whereas, the bone matrix model took an extra 2 days for the wound to fully heal and close.
Figure 4-1: Light microscopy time lapse images assessing the wound healing process. Two different cell models were used; connective tissue (fibroblasts) and bone matrix (osteoblasts) models, in the presence and absence of an overlaid collagen gel. 10x objective, scale bar = 50 μm.
Subsequently, a viability assessment of the scratched fibroblast monolayer control was carried out. MSCs are known to migrate out of the niche and are guided to the wound, following the release of chemokines and cytokines from the site of injury. Therefore, it was necessary to assess the presence of dead cells at the wound edges, as damaged or dying cells release danger-associated molecular patterns (DAMPs), as part of the pro-inflammatory response by the wound (Tolle and Standiford, 2013).
Figure 4-2 demonstrated there were limited dead cells in the control, non- scratched samples, which were not localised within a specific area but distributed throughout the control samples (highlighted with the white arrows). However, the scratched samples clearly showed a higher concentration of dead cells at the edge of the scratch 24 hours post injury, (highlighted by white arrows in Figure 4-2). Despite this finding, it was noted that the presence of dead cells only comprised a very small percentage, compared to the percentage of living cells at the wound edge.
Figure 4-2: Fibroblast viability assessed over 24 hours, in the presence or absence of a scratch.
Live/dead stain green = living cells, red = dead cells and highlighted with white arrows. (A) 10x objective and (B) 5x objective. Scale bar = 50 μm
Soluble factors including cytokines, released from MSCs following injury, stimulate proliferation and migration of other cell types, to enhance and accelerate wound healing (Hocking and Gibran, 2010). Therefore, further research was conducted to assess whether the MSCs within the spheroid niche model were able to migrate out of the niche, upon injury stimulation.