Mesenchymal stromal cell derived CCL2 is required for accelerated wound healing

Mesenchymal stromal cells (MSC) have immunomodulatory effects impacting macrophages, promoting polarisation towards a reparative phenotype. CCL2 is a potent cytokine involved in the recruitment of macrophages. We hypothesised that MSC derived CCL2 may be involved in the MSC therapeutic effect by facilitating macrophage repolarisation. To further delineate this mechanism, MSC isolated from CCL2 deficient mice (MSC-KO) were applied to excisional wounds in wild-type (WT) mice. CCL2 deficiency in MSC completely abrogated the therapeutic response compared to MSC-WT. MSC-KO were unable to repolarise macrophages to the same extent as WT and this was accompanied by a reduced angiogenesis and re-epithelialisation of the wounds at day 10. This study demonstrates that MSC derived CCL2 is required for MSC induced accelerated wound healing. The role of CCL2 in the interaction between MSC and Macrophages has not been previously demonstrated in accelerated wound healing. CCL2 has a potent effect on the ability to reduce the inflammatory response through local recruitment of macrophages. This research highlights CCL2 as a possible target for augmentation of MSC therapy to enhance therapeutic potential.


Flow Cytometry Analysis of Cell Surface Antigens
Expression of associated MSC markers CD44, CD90 and Sca-1 along with CD45 were evaluated to characterise cell population. Cultured cells at P5 were washed once with PBS and detached from the tissue culture flask by trypsinisation followed by neutralisation with equal volume of serum supplemented medium. Cells were then recovered by centrifugation at 500 g for 5 minutes, followed by re-suspension in PBS aliquoted out into several tubes contain 1x10 5 cells for primary antibody staining. Cells were centrifuged and re-suspended in 100 μl of diluted antibody (see Supplementary Table 1) in FACS buffer and incubated for 1 h at 4°C. Cells were then washed in PBS centrifuged and re-suspended in 100 μl of secondary antibody solution for 30 min in the dark at room temperature (rt). Cells were then re-suspended in 100 μl FACS buffer and analysed on flow cytometer. Control isotypes were used to determine positive cell populations.

Lineage Differentiation assays
MSC phenotype was validated by subjecting BM-MSC isolated from both WT and CCL2 KO mice to in vitro differentiation assays to confirm their potential to differentiate into adipocytes, chondrocytes and osteocytes. Tri-differentiation potential was verified using well established previously published protocols 39, 40 .
Briefly, for adipogenic differentiation, cells in culture were subjected to 3 cycles of adipogenic induction media for 3 d followed by adipogenic maintenance media for 1 d. Adipogenic induction media contained dexamethasone 1 mM, insulin 1 mg/ml (Sigma Aldrich, Ireland), indomethacin 100 mM (Sigma Aldrich) and 500 mM MIX (Sigma Aldrich), the adipogenic maintenance media contained insulin 1 mg/ml. Adipogenic differentiation was confirmed with oil red O (Sigma Aldrich, Ireland) staining.
For osteogenic differentiation, cells were cultured in osteogenic differentiation media containing β glycerol phosphate (Sigma Aldrich) 1 M and ascorbic acid (Sigma Aldrich) 2-P For chondrogenic differentiation, pelleted cells were cultured for 21 d in chondrogenic differentiation media containing 10 ng/ml TGF-β3 (Sigma Aldrich). Chondrogenic differentiation was confirmed by mounting the pellets in OCT, sectioning them at 10 μm, and staining with toluidine blue (Sigma Aldrich).

ELISA
To determine concentrations of IL-10, TNFα and CCL2, ELISAs were performed on culture supernatants using kits provided by R&D systems (MN, USA). Following manufacturer protocols 96 well clear plastic plates were coated overnight at room temperature with capture antibody (0.2 μg/ml) (R&D systems). 100 μl of diluted culture supernatants was added to the plate for 2 h at room temperature. The plate was washed and detection antibody (50 ng/ml) (R&D systems) applied for 2 h. After washes, streptavidin conjugated horseradish peroxidase (R&D systems) was incubated for 20 min on the plate followed by substrate colour solution (hydrogen peroxide and tetramethylbenzidine) (R&D systems). The reaction was then stopped after 20 min by addition of 100 μl of 2N sulphuric acid (Sigma Aldrich). The optical density was measured using microplate reader (Molecular Devices, Spectra Max, UK) and sample concentration was calculated from a generated standard curve (Soft Max pro v5.4.3, UK). Each standards and sample was measured in duplicate and experiments had multiple replicates.

Re-epithelialisation
Wounds were excised, and trimmed to 1 cm 2 . This was bisected and one half was stored in 4% PFA o/n. To determine re-epithelialisation, wound sections were stained with H&E. The wound section was imaged at 4x magnification. Unepithelialised length (or wound gap) was measured using ImageJ software by blinded observers

Granulation tissue area
The area of granulation tissue was assessed from previously stained H&E sections. The area was calculated using image J software to trace the region of granulation tissue from images taken at 4x magnification. An image of a microscope ruler slide taken at the same magnification was used to convert pixels to distance in image J and subsequently granulation area was calculated.

Immunofluorescent staining
Wounds were excised, and trimmed to 1 cm 2 containing both wound and healed wound margins. This was bisected and one half was stored in 4% PFA for 1 h at 4°C, then dehydrated in graded sucrose solution and then embed in OCT. Frozen sections were cut at 10 μm thickness in cryostat and stored at -80°C until required. Prior to staining, slides were removed and left to defrost for 30 min at rt.
Slides were rehydrated in PBS (10 min) and then blocked for 1 h in 10% normal goat serum in PBS-Tween (0.5%) solution. Sections were then incubated o/n at 4°C with primary antibody diluted in antibody buffer (1% BSA PBST). The next day, sections were washed x3 in PBST and slides were incubated with secondary antibody solution in antibody buffer for 2 h at rt, followed by 3x washes in PBST. Nuclei were stained with DAPI (1 μg/ml) for 10 min followed by 3x washes in PBS prior to mounting in immunomount and cover slipping.

Vessel Density quantification
Vessel density was calculated by measuring the quantity CD31 positive vessel like structures within the wound bed from images taken at 40x. Random images were taken within the wound bed through 5 non-consecutive tissue sections. The average quantity of vessels per high power field for each wound (vessel density = vessels per HPF) was then calculated.
Average vessel density for each wound was then averaged among the replicate animals.