| ObjectiveThe repair and healing of skin wound is achieved by proliferation and expansion of healthy cells from the surrounding tissue. However, this self-reconstruction is not sufficient for the recovery of larger area damage such as severe burns, large scar, skin ulcer and other traumatic defect, in which case the skin substitute such as skin graft is needed. Clinically the skin graft can be either autologous, which results in new skin lesion, or allogeneic which might be immune intolerant. There is much bigger chance of infection and scar formation in deeper skin lesions that always take longer to heal without proper surface cover. It is of both theoretical and therapeutical importance to immediately cover the skin lesion.Stem cell technology could provide an answer. Stem cells can be expanded in large numbers and retain their ability to differentiate into mature functional cells. There are two categories; adult and embryonic stem cells. The research in embryonic stem cells is restricted by its limited source and ethical controversy, so research on adult stem cells attracts more attentions. It is demonstrated that bone marrow mesenchymal stem cells (MSCs) have multiple potential to differentiate into muscle, bone, cartilage,adipose, blood vessel endothelial, liver and neuron cells. MSCs can be used as cell source in tissue engineering, cell implant and gene therapy with the possibility to be distributed to different tissue and organs by normal circulation. They hold great promise in future therapeutic application in that they are easy to obtain, separate, expand and transfect, the procedure of bone marrow extraction is less harmful and the replantation induces no immune rejection.The differentiation of MSCs is closely related to the microenvironment they grow in. it is probably because certain factors in the microenvironment are necessary for MSCs to differentiate into target cell lineages. In this study, porcine bone marrow MSCs were harvested and cultured in vitro, labelled and replanted into skin; or induced by different culture condition with different induction factor in vitro; and then observe the possible differentiation into blood vessel endothelial and epidermal cells in the skin microenvironment.Methods1. Isolation and identification of MSCs1.1 .Isolation, culture and passage: bone marrow was extracted through iliac puncture from adult healthy minipigs. Pellet cells were seeded into culture media after density gradient centrifugation. The culture media was replaced 24 hours later to allow removal of non-adherent cells. Media were changed every 3 to 4 days. Cells were passaged with ratio of 1:3 when almost confluent. Passage 3 cells were used for furtherexperiment.1.2.Cell cycle examination of MSCs: active growing cells were detached by 0.25% trypsin and fixed with 4C ethanol for 30 minutes in suspension, washed 3 times with ice cold PBS, treated with RNAase Img/ml for 30 minutes in 37C, centrifuged down, resuspended in 10 mg/ml propidium iodide for 15 minutes in dark 4C, then passed through flow cytometer.1.3.Osteogenesis induction and characterisation: passaged cells were induced with osteogenetic reagent, stained for alkaline phoshatase and Von-Kossa.2. In vitro induction of MSCs into endothelial cells and epithelial cells2.1. Differentiation into endothelial cells: passage 3 MSCs were cultured in the following 2 different conditions: a. F-12 media with 10% foetal bovine serum (FBS); b. F-12 media with 10% FBS, 10 ng/ml VEGF, 1 ng/ml bFGFand 2 ng/ml IGF.2.2. Differentiation into epithelial cells: passage 3 MSCs were cultured in the following 5 different conditions: a. F-12 media with 10% FBS; b. F-12 media with 8 ng/ml EGF; c. F-12 media with 10% FBS and 8 ng/ml EGF; d. F-12 media with 10% FBS, 8 ng/ml EGF and 30% conditional media; e. F-12 media with 10% FBS and 30% conditional media.Cells were stained for factor VIII (FVIII) and cytokeratin (CKp) and analysed with immnohistochemistry and flow cytometer on the 3rd and 7th day post induction.3.
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