| ObjectiveFor severe injury of ligaments, natural healing fails to take place and replacementtissues or grafts are required. Autografts tissues have been widely used in clinic. Butdonor site morbidity, prolonged surgery time, scar proliferation and sacrifice ofautogenous tissues remain to be the limiting factors of autografts. Tissue-engineeredligaments (TELs) have become the research focus of injuried ligaments replacement,which have the following advantages:1) scaffolds and seed cells ware fabricated in vitro,which could relieve patients’ suffering.2) TELs can be constructed in different shapeand size, which can well deal with the massive defect of ligament.3) TELs caneffectively lower the decline of immune response and mechanical capacity.TEL is mainly composed of seed cells and the decellularized ligament scaffolds, thechoice of seeded cells is a key step. Research shows that bFGF can promote BMSCdifferentiation into tendon cells. BMSCs from adult human bone marrow that can beobtained by needle biopsy, can be well selected and expanded in vitro. Animal studiesrevealed that bFGF has a significant impact on the healing of tendons and ligaments. Butthe shortcomes of local application of the growth factors are only short-time effect due torapid metabolism of the growth factors, expensive, and intense local immune reaction. Toreinforce the function of seeded-cells is one of the foci on tissue engineering. Applicationof growth factors through gene transfection into seeded-cells is considered superior tomaking use of recombinant protein directly. This study tries to construct recombinantadenovirus vectors Ad.bFGF-eGFP coding for basic fibroblast growth factor (bFGF)gene and enhanced green fluorescent protein (eGFP) report gene; investigate expression,secretion of bFGF after transfection, and the effect of bFGF on proliferation,differentiation, and production of specific extracellular matrix proteins of BMSCs; and tomake sure the best method to construct the TEL.Methods1．Construction of recombinant adenovirus vectors Ad.bFGF-eGFP coding for bFGFgene and eGFP report gene, identification and amplification (already completed);2．Isolation, culture of h-BMSCs, and investigate expression, secretion of bFGFafter transfection, and the effect of bFGF on proliferation, differentiation, and production of specific extracellular matrix proteins of BMSCs;(1)Isolation, culture of human BMSCs: after approval of ethics committee andvolunteers informed consent, bone marrow was aspirated from iliac crest of volunteers.BMSCs were obtained from isolation of the aspirate with Percoll step gradient, culturedand expanded in DMEM medium. The bionomics of BMSCs was characterized.(2)Identification: General morphological characteristics, immuno-fluorescenttechniques and multi-directional derivation of BMSCs were observed to identifyh-BMSCs3. Transfect the Ad.bFGF-eGFP into h-BMSCs in vitro, and then the experimentswere divided into3groups：treatment group, control group and carrier-without group.(1)Transfection efficiency of adenovirus vector to h-BMSCs with different MOIvalue was identified by flow-cytometry analysis of the expression of eGFP, and thenselect optimum MOI. After Ad.bFGF-eGFP transfection, bFGF protein expression inh-BMSCs was measured by Western-blot, immunohistochemical staining. Quantityanalysis of ELISA was used to measure bFGF secretion in culture medium.(2)The effect of bFGF on proliferation, multi-differentiation differentiation andproduction of specific extracellular matrix proteins of BMSCs after transfection intoh-BMSCs: observe the state of morphologic changes and proliferation of h-BMSCsduring h-BMSCs culture. MTT assay was performed to analysis of cell proliferation. Theeffect of bFGF on mRNA and protein expression of collagen Ⅰ, collagen Ⅲ,fibronectin, α-SMA, and vimentin was investigated by RT-PCR and Western-blot.Results1．In original culture h-BMSCs attached and spread with circular,triangular,polygonal or spindle-shaped morphology. After one week cultrure single colony wasformed. h-BMSCs isolated from10ml bone marrow were expended to2×107cells aftercultured in vitro for three weeks.2．h-BMSCs were positive for CD29and CD90but negative for CD34and CD45.In the process of cultivation, with appropriate medium, the cells appeared multiplexdifferentiation potential and with the capacities in the osteogenic, chondrogenic andadipogenic differentiation.3．h-BMSCs were high effectively transfected by Ad in vitro. Over90%ofh-BMSCs were transfected at MOI50.Human bFGF protein was detected by ELISA、RT-PCR and Western-blot in h-BMSCs and culture medium2d after Ad.bFGF-eGFP transfer. Expression reached peak after7d, then decreased gradually.4. The Ad.bFGF-eGFP gene transfer group demonstrated a more homogeneousfibroblast-like, spindle-shaped morphology, and higher cell densities than the other twogroups after7, and14d. Western-blot and RT-PCR revealed that expression of collagentype Ⅰ、Ⅲ, α-SMA, vimentin and fibronectin was higher significantly than the othertwo groups at the levels of gene and protein.Conclusions1．BMSCs were easily achieved, obtained with less injury, which are the ideal cellsource for TEL, and had the potential to extend to2×107cells in vitro after3weeksculture.2. h-BMSCs can effectively transfected by Ad.bFGF-eGFP, and secrete bFGF. AndbFGF can enhanced the ability of cell proliferation, differentiation to fibroblasts andexpression of specific extracellular matrix proteins of ligaments. |
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