Formula A: Percentage Wound Closure: % C = percentage wound closure, A0 = Area of wound at day 0, Ai = Area of wound at 7 days.
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1 Formulae for Wound Closure and Stereology (15, 16, 20) Formula A: Percentage Wound Closure: % C = percentage wound closure, A0 = Area of wound at day 0, Ai = Area of wound at 7 days. A0 Ai 100 %C = x A0 1 Formula B. Surface Density I Sv = 2 x LT Formula C. Length Density (2 x IL) Lv TS Formula D. Radial Diffusion Distance 1 R diff = π x Lv Formula E. Blood Vessel Diameter Sv d = Lv π Formula F. Inflammation Inflammatory Cell Fraction = PP PT
2 Supplementary Table 1. Pearson s correlation values between wound healing parameters. WC = Percentage wound closure, I = inflammatory cell infiltrate, Sd = Surface density of blood vessels, Ld = Length density of blood vessels, RDD = Radial diffusion distance. BVD = Blood vessel diameter. TL BV = Total Length of Blood Vessels. *signifies P < For wounds treated with 1,000,000 and 100,000 MSCs seeded on a collagen scaffold, the total length of blood vessels positively correlated with inflammatory cell fraction. In the treatment group of 1,000,000 MSCs seeded on a collagen scaffold, inflammatory cell fraction correlates with length density of blood vessels. These correlations were not observed in other groups. 1,000,000 MSCs + Collagen 100,000 MSCs + Collagen WC Ld Sd RDD BVD TL BV WC Ld Sd RDD BVD TL BV Ld Sd * 0.92* RDD * 0.88* * 0.92* BVD TLBV * * 0.78* I * * * 0.68* 50,000 MSCs + Collagen Collagen Ld Sd * 0.88* RDD * 0.80* * 0.95* BVD TLBV * 0.74* 0.75* 0.77* I Untreated Wound Ld 0.14 Sd * RDD * 0.75* BVD * TLBV * 0.73* 0.67* I
3 Supplementary Figure 1. Blood Glucose Readings of rabbits post alloxan treatment. The serum glucose of animal remained > 20 mmol/l for 35 days post alloxan up until time of surgery and treatment application.
4 Supplementary Figure 2A. Differentiation assays of rabbit MSCs. These images demonstrate differentiation into adipocytes (oil red O) and osteocytes (Alizerin Red, Alkaline Phosphatase and Von Kossa). Control cells are in the column on the left and treated cells in the right column. (Supplementary figures 2a and 2b) depict results of differentiation assays and confirm the ability of nondiabetic rabbit bone marrow derived MSCs to differentiate into osteogenic, chondrogenic and adipogenic lineages
5 Supplementary Figure 2B. Rabbit MSCs demonstrate differentiation into chondrogenic lineage as evidenced by increased amount of glycosaminoglycan (GAG)/DNA in MSCs at 14 and 21 days. Supplementary Figure 3. Timecourse of Metabolic Activity of MSCs seeded on a collagen scaffold. Metabolic activity is assessed using Alamar Blue (resazurin) (Invitrogen). Rabbit MSCs retained metabolic activity after seeding for 24 hours. The assay was performed at 16 hours, 96 hours, 144 hours and 336 hours. The metabolic activity of rabbit MSCs seeded on a collagen scaffold is maintained for two weeks in vitro as compared to media control. This is evident for cells seeded at doses of 50,000 and 1,000,000 cells per scaffold.
6 Supplementary Figure 4. Five micrometer histological sections of collagen scaffold seeded with 1,000,000 rabbit MSCs. A. Masson s trichome stained section (2X magnification) Scale bar = 1 mm. B. Masson s trichome stained section 10X magnification. Scale bar = 200 micrometers. MSCs are located predominantly on the superior surface of the collagen scaffold. MSCs are stained purple and collagen stained green. Supplementary Figure 5. Fluorescently labeled MSCs in diabetic wound 1 week after treatment. (40X Magnification) A. PKH26 labelled MSCs (red). B. DAPI stained cells in wound. (blue) C. Overlay of Labeled MSCs and DAPI stained cells. D. Overlay brightfield image and labeled MSCs (red) and DAPI stained cells (blue). This provides evidence that the cells were located in the wound site at one week post treatment. The cells dualstained with DAPI, a nuclear stain confirming dualstained cells are transplanted MSCs. This observation demonstrates that MSCs were present in the wound for at least 7 days and that the collagen scaffold was successful in mediating cell delivery to the wound.
Corresponding Author: Timothy O Brien
Page 1 of 36 Diabetes Topical Administration of Allogeneic Mesenchymal Stem Cells Seeded in a Collagen Scaffold Augments Wound Healing and Increases Angiogenesis in the Diabetic Rabbit Ulcer Aonghus O
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