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Patient recruitment

Ethical approval was obtained for the study (reference 06/Q1206/127, Leeds (East)
REC), with all patients providing written informed consent. To address the influence
and role of differing severities of trauma on MSC dynamics, patients were recruited
into two groups based on the severity of injury as defined by their Injury Severity
Score (ISS) 23]. Briefly, patients in the isolated trauma group had ISS 16 and patients in the polytrauma
group had ISS ?16. A third group of patients with no acute trauma or injuries who
were undergoing elective orthopaedic surgery formed the control group. The demographics
of the recruited patients are summarised in Table 1. For MSC proliferation experiments using the patient’s sera, cultured MSCs from four
additional control subjects (two male/two female, median age 31, range 21 to 35),
characterised in our previous study 24], were utilized.

Table 1. Patient demographics (total number =33)

Sample collection

Patients recruited into both trauma groups had collection of IC BM as well as PB.
IC BM aspiration could only be carried out when the patient was undergoing surgery,
with the initial sampling occurring within 24 hours of injury (acting as baseline,
day 0) and the subsequent sampling, dictated by clinical need for a further intervention,
several days later (range 3 to 32 days). In addition, four patients had a third sampling
time point occurring over a year post-trauma (range 397 to 602 days), when they were
admitted for elective (non-acute trauma) operations.

For BM aspiration, consistency was assured in terms of surgeon (same person for all
patient samples), aspirate location (anterior iliac crest, approximately 5 to 6 cm
posterior to the anterior superior iliac spine), tools (Stryker 306–111, 11-gauge,
bevel tipped trocar, Kalamazoo, MI, USA), volume of aspirate (20 ml) and draw method
(single draw to fill full 20 ml syringe 25]). The aspirate was collected into an ethylenediaminetetraacetic acid (EDTA)-containing
tube (BD Vacutainer, Oxford, UK). PB was collected every other day for 14 days and
also time-matched to each BM aspiration episode; samples were taken into three tubes
for different investigations, with 20 ml collected in EDTA for the MSC enumeration
study, 20 ml taken in a clot accelerant tube (BD Vacutainer, Oxford, UK) for serum
isolation, and the final sample (5 ml) sent to the hospital haematology laboratory
for processing of full blood count, including platelet count and CRP.

Sample processing and CFU-F assay

For MSC enumeration in both the IC BM aspirate and PB, samples were initially processed
for mononuclear cell (MNC) isolation 20]. In brief, the sample was diluted 1:1 with phosphate buffered saline (PBS, Invitrogen,
Paisley UK), before being carefully layered onto Lymphoprep (Axis-Shield, Dundee,
UK) and subsequent centrifugation at 650 g for 25 minutes. The MNC fraction was washed
twice in PBS and seeded into 10-cm diameter dishes (Corning, Tewksbury, MA, USA) in
10 ml of MSC culture media (NH Media, Milteny-Biotec, Bisley, UK), at 3 × 10⁶ MNCs/dish, in triplicate, for the CFU-F assay. The remaining MNCs were frozen down
in freezing media and stored in liquid nitrogen for flow cytometry investigations.
Expecting much lower MSC frequency in PB 12], PB MNCs were seeded at a higher density (10 × 10⁶ MNC/dish). CFU-F cultures were maintained for two weeks and CFU-F colonies scored,
as described previously 26]. The technical variation (between the three dishes) in colony counts was approximately
23%. For all patients/data points, the CFU-F data are presented as the number of CFU-F/ml,
calculated according to the formula:

MSC enumeration using flow cytometry

Bone marrow MNCs were defrosted and the MSC frequency was measured based on the CD45-CD271+
phenotype, as previously described 24], with some modifications. Bone marrow MNCs were re-suspended at 1 x 10⁷ cells/ml in FACs buffer (PBS +0.5% bovine serum albumin (BSA) +2 mM EDTA). Antibodies
were added at the manufacturers’ recommended concentrations and the cells were incubated
with the antibodies for 20 minutes. Antibody combinations used were: CD45-PeCy7/CD271-APC/CD140a-PE,
CD45-PeCy7/CD271-APC/CD140b-PE or an isotype controls combination (CD271-APC was from
Miltenyi Biotec, Bergisch Gladbach, Germany, all other antibodies from BD Biosciences,
Oxford, UK). The cells were washed and re-suspended in FACs buffer containing 100 ng/ml
4?,6-diamidino-2-phenylindole (DAPI) before analysing on a BD LSRII flow cytometer.
Dead cells were excluded from the analysis using DAPI before gating on the CD45-CD271+
population and assessing the expression of CD140a and CD140b (PDGF receptors ? and
?, respectively) on these cells.

Cell counts and serum PDGF-AA and PDGF-BB measurements in PB

The total white cell count, platelet counts and CRP measurements were processed by
the Leeds Teaching Hospitals patient diagnostics laboratory. For investigations involving
patients’ sera, PB samples collected in clot accelerant tubes were allowed to stand
for 30 minutes at room temperature (RT) prior to centrifugation for 15 minutes at
2000 g. The serum was aliquoted (1 ml/tube) and frozen at ?80°C. The levels of two
PDGF isoforms, ?AA and –BB, were measured using commercially available enzyme-linked
immunosorbent (ELISA) assay kits (Quantikine® ELISA kits, RD Systems, Abingdon UK).

Both MSC colony counts and PDGF levels were analysed as the raw data (as CFU-F/ml
or PDGF/ml), as well normalized to day 0 (the latter indicative of increase/decrease
compared to the day 0 baseline, that is, 1 decrease, 1 increase). The healthy control
group recruited for PDGF measurements (n =9) comprised five women and four men who
were non post-traumatic healthy volunteers, with median ages of 35 (range 19 to 63 years)
and 35 (range 22 to 63 years), respectively, for the female and male groups.

MSC proliferation in response to patient’s serum

The effect of patient’s serum on MSC proliferation in vitro was assessed in a colorimetric cell proliferation assay based on a tetrazolium salt
XTT (Roche, Welwyn Garden City, UK), as described previously 27]. In brief, the assay was performed using cultured MSCs from four donors, in quadruplicate
for each cell seeding density and each serum sample. Cells were seeded at 125, 250
and 500 MSCs/well in 96-well plates and were allowed to attach for 24 hours in (D)MEM/2%
FCS (both from Invitrogen, Paisley UK); the next day media were replaced with 150 ?l
of either non-haematopoietic (NH) media (positive control wells), (D)MEM/10% FCS not
optimized for MSC growth (negative control wells) or (D)MEM/10% patient’s serum (test
wells). MSCs were allowed to grow and the assay was stopped on day 5 by replacing
the growth media with the XTT labelling mixture; the colour change was read at 450
and 620 nm using Opsys MR Plate reader (Dynex Technologies, Worthing, UK). Optical
densities (ODs) were analysed separately for each seeding density and MSC culture
and normalised to the OD of the positive control (NH media); the values for four MSC
cultures were next averaged for each seeding density and serum proliferative indices
(PIs 1?=?less proliferative than NH, 1?=?more proliferative than NH) were recorded
for each time-point, for each serum.

Statistical analysis

As a Gaussian distribution could not be assumed due to the sample size, non-parametric
tests were carried out. The Mann–Whitney test was used to compare differences between
two independent samples. The Wilcoxon signed-rank test was carried out to compare
differences between paired samples. The Chi-square test was used for comparison of
nominal data. Spearman’s rank correlation coefficient was used to test the relationship
between two variables. A P-value of 0.05 was considered statistically significant and denoted by *. All statistical
analysis was carried out using IBM SPSS statistics 19 and all graphical figures were
made in Prism 6 (GraphPad Software, Inc.).