| Large resections around bone tumors, complications after bone fracture, orinflammatory bone diseases often result in major bone loss or bone defect. Bone defecthealing is one of the challenges of orthopaedic surgery, for which bone grafts are widelyused. However, bone grafts have several drawbacks and are far from ideal, including thehigh donor-site morbidity and a high complication rate. So the new alternatives areneeded to satisfy clinical treatment. In search of alternative therapies, growth factorsgain a significant importance. At present, the growth factors approvalled by FDA for theclinical bone repair treatments are mainly BMPs family protein, in which rhBMP-2isthe mosr active one. But there are some drawbacks for rhBMP-2treatment: high cost,ectopic bone, excessive bone, and stimulate broken bone cell differentiation leading tofast absorption after the new bone formation. We aims at the problems of rhBMP-2treatment in the clinical application, and try to search an alternative factor for rhBMP-2,or one factor can have synergistic effect with rhBMP-2on bone repair. In recent year,C-terminal24-a.a. peptide of mechano growth factor, MGF24E gained an increasingattention because of its regenerative effects and the tissue-protective actions. MGF is theinitial splice-variant of IGF-1when tissue or cells suffer from damage.According to theprevious research results of our team on MGF24E, the studies are undertaken toinvestigate the roles and mechanism of mechano growth factor on bone injury repair, inorder to solve the problem of rhBMP-2on the clinical treatment. The main works andconclusions are included as follows:1. The1mm or so bone fractures in radius of SD rats were prepared, and MGFprotein expression in jury was analised by Western blot with rabbit anti-MGF/MGF24Epolyclonal antibody. Results showed that MGF protein expression one daypostoperatively, rise to higher value, to peak3days postoperatively, and then slightlyfall down but still maintain a high expression in a relatively long period.2.5mm segmental fracture in the radius of rabbit was treated by exogenousMGF24E injection, and the repair effects were assessed as radiographical andhistological observation and quantitative analysis.①The clinical-healing time in high-dose treatment group was shorter2weeksthan that in the control group and low-dose treatment group. The regenerating bones inhigh-dose treatment group are tube-shape bone with reopening of the medullary cavity and the repairment was significantly better than the control group and low-dosetreatment group.②The high-dose treatment significantly improves angiogenesis of regeneratingbone around defective areas, new blood vessels of high-dose group are more2foldsthan that of the control group.3. The roles of MGF24E on angiogenesis and the underlying mechanisms wereinvestigated. The cell proliferation, migration, and tubulogenesis of the human vascularendothelial EA.hy926cells co-treated with2%serum and MGF24E were determined toassess angiogenesis in comparison with100ng/ml of VEGF165-positive control orvehicle control (phosphate-buffered saline).①The pro-proliferation capacity of10ng/mL MGF24E was same to that of100ng/mL VEGF165. MGF24E promoted cell proliferation by inducing the cell cycleS-phase entry and transition to DNA replication and via an MAPK/ERK-dependentsignaling pathway.②The capacities of MGF24E on promoting migration and tubulogenesis wasstronger than those of the control, but weaker than VEGF165.MGF24E-induced highermigratory activity and tubulogenesis partially involved ERK-signaling pathway.③The suppression of vascular endothelial growth factor and angiopoietin-Iexpressions by2%serum starvation was reversed by the addition of10ng/ml ofMGF24E in2%serum medium, and the MGF24E-upregulated VEGF and Ang-I werehigher than those of the cells without serum starvation, which partially involvedERK-signaling pathway.4. The effects of MGF24E on the osteogentic behaviors of primary osteoblastsand the underlying mechanisms were investigated. The cell proliferation, migration,ALP activity, mineralization and calcium secretion expression of primary osteoblastsco-treated with7%serum and dual growth factors (containing25ng/mL MGF24E+50ng/mL rhBMP-2) were determined to assess physiological behaviors of primaryosteoblasts in comparison with100ng/mL VEGF165+50ng/mL rhBMP-2-positive controlor vehicle control (phosphate-buffered saline) or25ng/mL MGF24E or50ng/mLrhBMP-2, and meanwhile the molecular mechanism of osteogenesis in vitro wereinvestigated.①The pro-proliferation capacity of MGF24E was stronger than that of rhBMP-2or vehicle control. The activities of dual treatment (MGF24E+rhBMP-2) on osteoblastsproliferation, migration, osteodifferentiation and mineralization were higher than those of MGF24E treatment or rhBMP-2treatment. Although pro-migration activity ofpositive control (VEGF165+rhBMP-2) was higher than that of rhBMP-2treatment, theactivities of positive control on proliferation, osteodifferentiation and mineralizationwere lower than those of rhBMP-2treatment.②The synergistic effect of dual treatment mainly involved the upregulations ofRunx2and OPN mRNA expressions5. The composite scaffold containing different growth factors was constructedwith the carrier of absorbable gelatin sponge by the manner of negative pressurephysical adsorption at4℃,and the morphology structure, porosity of scaffold, releasecharacteristics of growth factors in vitro and cells compatibility of scaffold wereevaluated.①The results of scanning electron microscopy (SEM) showed that growth factordistributed in scaffold appearing as a ball-shape, pore size and porosity can satisfy thegrowth of osteoblast and new capillary;②The results of release characteristics in vitro showes that sudden release ofdifferent growth factores was observed in the initial period; MGF24E release was fastestand rhBMP-2release was the slowest. the release characteristics of MGF24E can meetthe demand of injury repair, and the release characteristics of rhBMP-2can meet theway of high concentration at one-time in clinical treatment;③The results of cell activity of different scaffolds showed, different growthfactors led to different cytocompatibilities, and cytocompatibilities of dual treatmentgroup and MGF24E treatment group were better than those of rhBMP-2group andvehicle control.6. The15mm segmental critical bone defect models in radius of rabbits wereestablished. The radiographical investigation, histological investigation andbiomechanics measurement were taken to evaluate the bone-defect repair capability ofdual treatment (containing200μg/mL MGF24E+80μg/mL rhBMP-2), in comparisonwith80μg/mL rhBMP-2-positive control or vehicle control (phosphate-buffered saline)or200μg/mL MGF24E, and meanwhile the repair mechanism of bone-defect in vitrowere investigated according to the expressions of OPN and CD31.①The radiographical results showed, that healing time of cortex-bridging in dualtreatment groups was12weeks, and clinical-healing time of callus-bridging in dualtreatment groups was4weeks,which was earlier than that of rhBMP-2treatment groups;after samples of rhBMP-2treatment group appeared excessive bone, regenerating bone appeared fast absorption (radius-ulna fusion phenomenon), but dual treatmentcompletely reversed the abnormal bone-healing phenomena induced by rhBMP-2,suggesting a synergistic effect; Although during12weeks of repair period, only halfsamples of MGF24E treatment group appeared cortex-bridging, but were still far morethan that of the negative control group; the radiographical scores of different treatmentsmeted the linear function;②The histological results showed, the differences of histocompatibilities ofdifferent growth factors treatment scaffolds were observed in the initial periodpostoperatively, and histocompatibilities of dual treatment scaffold and MGF24Etreatment scaffold were better than that of rhBMP-2treatment scaffold and the vehiclescaffold; regenerating bone mass and histological scores showed that bone-healingcapability of dual treatment group was the best, better than rhBMP-2treatment groupand MGF24E treatment group, suggesting the synergistic effect; The histological scoresmeted the logarithm function, according with the S curve law of biological communitiesgrowth.③The biomechanical test results showed that, even if the regenerating bone ofdifferent treatment groups appeared bone mass bridging, mechanical properties ofregenerating bone were still lower than native radius; the stiffness and bending intensityof regenerating bone samples in MGF24E treatment group were about a third of thoseof native radius; the stiffness of rhBMP-2treatment group was nearly the half of that ofnative radius, and the bending intensity of rhBMP-2treatment group is about1/5of thatof native radius, the stiffness and bending intensity of dual treatment group were64%and54%of those of native radius respectively, suggesting the synergistic effect;④The results of osteogenesis mechanism showed that, bone-defect repair modeof different growth treatments was endochondral ossification; the treatments containingMGF24E can induce early bone matrix synthesis and blood supply recovery, thus thenew bone formation of samples in dual treatment group was better than that of samplesin rhBMP-2treatment group, but the capability of MGF24E on promoting bone matrixsynthesis and blood supply recovery displayed in the initial period postoperatively, asthe gradual blood supply recovery of samples in rhBMP-2treatment group,thedifferences of dual treatment group and rhBMP-2treatment group in new boneformation was smaller.To sum up, MGF24E not only can significantly promote angiogenesis, but also canpromote bone formation in vivo, so dual treatment of MGF24E and rhBMP-2can promote critical bone-defect healing in advance, and its repair quality was better thanregenerating bone in rhBMP-2or MGF24E treatment groups, suggesting the synergisticeffect; The key was that but dual treatment completely reversed the abnormalbone-healing phenomena induced by rhBMP-2, contributing to some further studies asan therapy of critical bone defect healing and showing an significance for clinicaltreatment. |
PDF Full Text Request