Differentiation Of Somatic Cells In The Peripheral Blood Of Smooth Muscle In Vascular Tissue Engineering Applications

Vascular diseases are common and critical human diseases for which substitution of the damaged vessels are common treatments.However,the substitution treatments are meeting a lot of limitations.With the development of stem cell research and the advancement of material science,it is possible to construct tissue engineering blood vessels(TEBV) to serve as blood vessel substitutes.To construct TEBV,the key processes are to obtain,culture and plant seeding cells in scaffolds.Blood vessels are mainly composed of endothelial cells(EC) and smooth muscle cells(SMC),both of them are important seeding cells in TEBV.SMC help to maintain the contraction function of blood vessels and have the capacity to secrete ECM and angiogenic factors,such as elastin and vascular endothelial growth factor(VEGF).SMC used in TEBV largely come from mature blood vessels.However,the limited proliferation ability of mature SMC,traumatic obtaining processes and even deficiency of healthy blood vessels impede the application of SMC in TEBV.Thus,we try to isolate smooth muscle progenitor cells(SPC) among mononuclear cells from rat peripheral blood and let them differentiate into smooth muscle cells,then implant them onto synthetic scaffolds and collagen gel to assess their potential in the construction of TEBV.Mononuclear cells were isolated from fresh rat peripheral blood by density gradient centrifugation,induced and cultured in EGM-2-MV kit with PDGF-bb.And then the differentiated SMC were characterized by morphological observation, immuno-fluorescent staining and Western blotting.RT-PCR was employed to examine mRNA expression level of smooth muscle markers and function molecules. After that,the SMC from SPC were seeded onto the PHBHHx scaffold(with or without silk fibroin modification) at 2×10~5 cells/cm~2 and cultured under static conditions for three weeks.The growth and proliferation of seeded cells on scaffold was tested by MTT assay and scanning electron microscopy(SEM).We also implanted SPC in typeⅠCollagen gel with endothelial progenitor cells(EPC) to see whether SPC could improve the vasculogenic activity of EPC.The results showed that a part of adherent mononuclear cells appeared "polarity" after three days culture;One week later,increasing number of cells changed toward a spindle-shaped morphology.Three weeks later,most outgrowth cells displayed specific "hill and valley" morphology and showed impressive growth potential.Immuno-fluorescent staining and Western blotting demonstrated the differentiated smooth muscle cells expressed SMα-actin,Calponin and SM MHC,the expression level of these proteins being parallel to mature smooth muscle cells. RT-PCR showed the SMC highly expressed SMα-actin,Calponin,SM MHC, SM22α,elastin,MGP and VEGF at mRNA level.The outcome proteins of these mRNAs play vascular contraction function and consist of main components of extra-cellular matrix in blood vessel.After seeding in silk fibroin-modified PHBHHx scaffold for three days,the SMC differentiated from SPC adhered to the surface of the scaffold with filopodia. Three weeks later,SPC spread between scaffold pores,grew into deeper section and formed confluent lamellar structure.Dynamic MTT evaluation showed that the modification of silk fibroin greatly improved SPC activity and proliferation. Moreover,in typeⅠCollagen gel,EPC formed micro-vascular network only one week after co-cultured with SPC.Our results showed that the SPC in rat peripheral blood can differentiate into smooth muscle cells with contractile and synthetic function.The differentiated SMC grew well in PHBHHx scaffold and accelerated EPC to form micro-vascular network in collagen gel.Since SPCs in peripheral blood are easy to obtain and hold an extensive proliferation capacity in vitro they will become a promising seeding cell resource for TEBV.