ROYAN INTERNATIONAL TWIN CONGRESS,2014.

Effect of Umbilical Cord Blood Derived Platelet-lysate on Fibroblast Proliferation and Activation of Smad Signaling Pathway

Wound healing is one of the body's natural mechanisms . But In conditions such as certain illnesses, surgeries, the body is not able to repair the skin simply (1).The healing of Wounds consists of a complex process which require the coordination of different cell types such as keratinocytes, fibroblasts, endothelial and inflammatory cells. Response to injury involves a complex of interactions among epidermal and dermal cells, extracellular matrix, angiogenesis and plasma proteins. This dynamic process is classically divided into three overlapping phases: inflammation, proliferation and remodelling.(2)Platelet with their protein content are another effective cells which facilitate healing.(3)Due to above studies, here we investigated the effect of umbilical cord blood platelet lysate (UCB-PL) on fibroblasts proliferation and motility.

References:

1.Singer AJ, Clark R. Cutaneous wound healing. N Engl J Med. 1999;341(10):738-462.Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. Journal of Leukocyte Biology.

2001;69(4):513-21.3.Ranzato E, Martinotti S, Volante A, Mazzucco L, Burlando B. Platelet lysate modulates MMP‐2 and

MMP‐9 expression, matrix deposition and cell‐to‐matrix adhesion in keratinocytes and fibroblasts. Experimental dermatology. 2011;20(4):308-13.

4. Ranzato E, Mazzucco L, Patrone M, Burlando B. Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKT. Journal of

cellular and molecular medicine. 2009;13(8b):2030-8.5. Parazzi V, Lazzari L, Rebulla P. Platelet gel from cord blood: a novel tool for tissue engineering.

Platelets 2010; 21: 549-54.

UCB-Pl caused to enhance mobility of fibroblasts in presence of 10%of UCB=PL(P<0.05) Fig 3 ,4.

Mahmoodi.M (M.Sc.)1,2, Ebrahimi .M (Ph.D.)1,2,1.Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran2. Department of Regenerative Biomedicine at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

IntroductionIntroduction

Materials & Methods

ResultsResults

ConclusionsConclusions

Our results determined that UCB-PL induces a marked increase of the wound repair capabilities of fibroblasts by stimulating cell proliferation and mobility.

Based on our findings, we conclude that UCB-PL can increase proliferation and migration of fibroblasts through inducing activation of smad pathway.

Fig 3,4: Scratch assay to test mobility of fibroblasts (an in vitro assay for wound healing)

UCB-PL was produced from Platelet Rich Plasma (PRP) at concentration of 2×10 (platelet/ml). Then PRP was frizzed⁹ (-80˚C) and thawed (37˚C) for three cycles. The remaining platelet bodies and debris were eliminated by centrifugation (3000 g ×10 min) and finally supernatant (UCB-PL) was stored at -80˚C until use.

Fibroblasts were obtained from Royan Institute Cell Bank cultured in DMEM supplemented with NEAA, Pen/strep and different concentration of UCB-PL (5%-8%-10%-15%-20%), the mixture of UCB-PL and FBS. Cells treated with 10% FBS and without serum were used as positive and negative control .Cell proliferation and viability was evaluated by trypan blue. Wound scratch assay was used for wound healing evaluation at 6,12 and 24 hrs post scratch.Cellular RNA was extracted from Fibroblasts treated with different concentration of UCB-PL and positive and negative control samples and cDNA synthesis was performed on them. The expression level of Smad2-Smad3 was evaluated by the use of Real time-PCR techniqe.

The level of protein including smad2-Psmad2-smad3-psmad3 were measured by western blot.

ResultsResultsOur results showed that the effect of UCB-PL on fibroblast proliferation is time and dose dependent. As seen in the Figure 1, 10% UCB-PL significantly increased cell proliferation (P<0.05). However cell growth reduced when the concentration of UCB-PL increased to 20% (P<0.05).

Fig 1: the number of cells counted 24. 48 and 72 hours post treatment with different concentration of UCB-PL, FBS and serum free as test, positive and negative control respectively,

Different concentration of pl (%)

0-h 6-h 12-h 24-h

8%

10% 10%

neg neg neg neg

Pl/fbsPl/fbsPl/fbs Pl/fbs

pos pos pos

8% 8%8%

10%

pos

Fig 5,6: The results showed that the mRNA levels of Smad2-Smad3 In the presence of PL, the rates Smad2-Smad3 of were significantly more rapid than in the absence of PL.

Fig 7 :Our data showed that negative control samples caused decrease in levels of smad2-smad3- psmad2-psmad3 In contrast, the level of these proteins were increased in the presence of (UCB-PL)

UCB-PL at dose 1-10%was not cytotoxic , however, at high doses it caused to cell death and decrease in fibroblasts viability (P<0.05) Fig.2.

Fig 2: Cell viability testing 24 hours post treatment with different concentration of UCB-PL, FBS and serum free as test, positive and negative control respectively,

psmad2

Ladder NEG 8% 10% UCB-PL/FBS FBSLadder NEG 8% 10% UCB-PL/FBS FBS60

50

6050

6050

6050

4030

smad3

Ladder 8% 10% UCB-PL/FBS FBS NEG Ladder 8% 10% UCB-PL/FBS FBS NEG

psmad3

smad2

gapdh