| Anterior cruciate ligament (ACL) is a ligament inside of knee joint capsule, playsimportant roles to maintain knee normal motion and stability. The most patients who havean ACL injured need the ACL graft reconstruction surgery because of insufficient capacityof healing by itself. Meanwhile, the gift material currently used is limited in source and afew of choices which need to be faced and solved. In recent years, with the rapiddevelopment of tissue engineering technology, the ligament tissue engineering has becomethe research hotspot in this field, which may potentially answer the above problems. Inthis experiment, two selected growth factors were combined to use to inducemesenchymal stem cells (MSC) into the ligament cell and promote it secreted collagen. Then, the induced MSC was combined to the acellular dermal matrix (ADM) and finallytissue engineering tendon was reconstructed. In the experiment, we aimed to test thefeasibility of single or combined use of transforming growth factorβ1(TGF-β1) and basicfibroblast growth factor (bFGF) to induce the seed cells in ligament tissue engineeringtechnology repectivly.The experimental rational and objective:Mesenchymal stem cells were harvested form baby rabbit and cultured in vitroculture and then marked with BrdU fluorescent tags. After that, the third generation cellswere induced with transforming growth factor β1and basic fibroblast growth factors, eachand both respectively, to compare which one was the best stimulator to make MSCdifferentiated into ligament cells. The induced MSC was adhered to the acellular dermalmatrix (ADM) and assessed by growth, proliferation and secretion of collagen. The goalof our experiment was to test possibility of combination using growth factor β1and basicfibroblast growth factors as a seed cells stimulator in ligament tissue engineeringtechnology.Experimental method:1. MSC’s cultivation and inductionMesenchymal stem cells were harvested form baby rabbit and cultured in vitroculture. The third generation rabbit’s MSC was selected and the MSC was marked withBrdU fluorescent tags. After that, the cells were induced with25μg/L basic fibroblastgrowth factors and10μg/L transforming growth factor β1. Four groups were settled:namely blank control group, transforming growth factor β1group, basic fibroblast growthfactor group and transforming growth factor β1+basic fibroblast growth factor group(combination group). The effects of both growth factors on the growth and morphology ofbone marrow mesnchymal stem cells were investigated with using microscope, CCK-8test, MTT test to assess the effects of growth factors on MSC biological activity.2. Induced MSC’s collagen staining and quantitative analysis As the same, the third generation rabbit’s MSC was used and marked with BrdUfluorescent tags. The cells were induced with25μg/L basic fibroblast growth factors and10μg/L transforming growth factor β1. Each one of four groups as designated above werestained by Picro-Sirius red and observed under inverted microscope, analyzed by theImage Pro Plus quantitative detection and collagen secrete were compared between thefour groups respectively.3. BrdU immunohistochemical fluorescence staining of MSC in ADMThe third generation rabbit’s MSC was chosen and marked with BrdU fluorescenttags. The BrdU marker positive rate was calculated. Then the experiment was carried outin divided two groups: the blank control group and the transforming growth factor β1+basic fibroblast growth factor group (combination group). Cell’s growth and proliferationwere observed with and without adhering to ADM scaffold by BrdU fluorescent tags at thesame time.The result:From experiment1and experiment2, the outcomes suggested that the transforminggrowth factor β1+basic fibroblast growth factor group had the best effectiveness on thecell proliferation and collagen secretion, respectively. In the TGF-β1+bFGF group, bothMSC proliferation and collagen secretion are the highest than any other blank group orsingle factor groups. Additionally, the cells wer arranged cross stratification structure. Inthe experiment3, the induced MSC in ADM was also well in growth and proliferation.In the TGF-β1+bFGF group, MSC cells grown throughout the scaffold material anddistributed more even compared with blank control group. From the software Imagepro-Plus measurement and statistics, fluorescence ratio per unit area was37.11±7.8%inblank control group, but76.81±3.9%in TGF-β1+bFGF group. Results show that oflatter growth factor combination group was better than that of blank control group.The conclusions:These findings suggest that transforming growth factor β1combined with basicfibroblast growth factor can promote bone marrow mesenchymal stem cells todifferentiate into ligament cell-like cells, which can adhere to acellular dermal matrix in a good growth. The experiment shows that transforming growth factor β1combined withbasic fibroblast growth factor exhibits significant effectiveness to construction oftissue-engineered anterior cruciate ligament. |
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