Effects Of⁶⁰COγ-rays On Osteoblasts On Different Titanium Surfaces And On P38Mapk Pathway

The vast majority of maxillofacial cancer patients have soft and hard tissuedefects and prosthetic rehabilitation is one of the most important restoration way.Implant-retained prostheses can greatly improve the treatment effects. However,most of these patients are likely to be subjected to preoperative or postoperativeradiotherapy. It is concerned by most doctors that placing an implant inirradiated sites would have more risks than in non-irradiated sites due to thereduced viability of the compromised bone. With the development of rapidprototyping technology, implants can be inserted during ablative surgery andthen radiotherapy or chemotherapy be carried out, i.e. pre-radiotherapy implantinstallation. In order to elucidate the effects of irradiation on osteoblasts ontitanium surfaces and the molecular mechanisms about the changeddifferentiation ability of osteoblasts by irradiation, the effects of irradiation onproliferation, differentiation and osteogenesis-related gene expressions ofMC3T3-E1cells on micro-arc oxidation modified titanium surfaces (MAO),polished titanium surfaces (PT) and tissue culture polystyrene plates (TC) wereinvestigated. Furthermore, the influences of irradiation on p38MAPK pathway were examined.The major results are as follows:1. Screening of radiation dose and the surface properties of MAOFirst, cells were irradiated on TC surface24h after initial seeding.Gamma-radiation at2,4,6,8or10Gy was administered as a single dose usinga60Co source. Control samples were subjected to a sham radiation procedure.Using MTT method, we convinced that4Gy of irradiation resulted in asignificant decrease in cellular proliferation on TC surface from d3. Second,polished titanium samples were treated by MAO in an aqueous electrolyticsolution contained Ca and P. Compared with PT surface, MAO surface showedenhanced surface roughness and wettability.2. The effects of irradiation on proliferation of MC3T3-E1cells on twotitanium surfacesCells were irradiated on MAO, PT and TC surfaces24h after initialseeding. Gamma-radiation at2,4,6,8or10Gy was administered as a singledose. Control samples were subjected to a sham radiation procedure. Cellproliferation was measured by MTT method at day7post-irradiation. Comparedwith non-irradiated group, cells exposed to4Gy began demonstratingsignificantly lower proliferation rates on three surfaces.The effects of three surfaces on proliferation rates of osteoblasts at thesame radiation dose were also evaluated. The results showed that no significantdifferences were found except the8Gy group, in which the osteoblastsproliferation on MAO was higher than those on TC (p＜0.05).3. The effects of irradiation on differentiation ability of MC3T3-E1cellson two titanium surfacesCells were irradiated on three surfaces24h after initial seeding. Gamma-radiation at4,8Gy was administered as a single dose. Control sampleswere subjected to a sham radiation procedure. Endpoints included alkalinephosphatase activity (ALP), collagen secretion, matrix mineralization and theexpression of osteogenesis-related genes. The results showed that8Gy ofirradiation decreased collagen secretion of osteoblasts and no statisticallysignificant changes in ALP activity. On PT and TC surfaces, significantlydiminished mineralization in cells irradiated by8Gy were found. RT-PCRshowed that8Gy dose inhibited the expression of osteogenesis-related genes(OSX, COL-Iα1and OCN) on three surfaces on post-irradiation day16.The effects of three surfaces on differentiation ability of osteoblasts at thesame radiation dose were also evaluated. At three radiation doses, MAO surfaceinduced more collagen secretion of osteoblasts than PT or TC surface. However,the ALP activities of the cells on MAO were lower than PT or TC. RT-PCRshowed that at three radiation doses, for ALP gene, the expression trend was TC＞PT＞MAO.4. The effects of irradiation on p38MAPK pathway of MC3T3-E1cellsCells were irradiated on TC surface24h after initial seeding. Gamma-radiationat8,16Gy was administered as a single dose. Control samples were subjectedto a sham radiation procedure. MTT assay showed that16Gy dose inhibitedcellular proliferation totally. Furthermore,16Gy of irradiation decreased theALP activity and mineralization of MC3T3-E1cells significantly.Cells were irradiated by16Gy on TC surface24h after initial seeding. Controlsamples were subjected to a sham radiation procedure. On day3,6,9of culture,the expressions of BMP2and BMP4genes were recorded using RT-PCR. At1h,8h after radiation and on day3,9of culture, the level of total p38and itsactivation (phosphorylation) of p38were determined by Western blot analysis. RT-PCR showed that no significant differences were noted in the mRNA levelsof BMP2and BMP4after irradiation at16Gy. Western blot showed that thelevel of total p38was not influenced by radiation at different time points.Compared with the control group, the phosphorylation of p38increaseddramatically at1h post-irradiation at16Gy. However, in irradiated cells thephosphorylation of p38declined from day3and this down-regulation was mostsignificant at day9, indicating that16Gy of irradiation might suppressosteoblasts differentiation ability through changing the phosphorylation of p38.Conclusions:1. In this study, the effects of radiation on biologic activities of MC3T3-E1cells were similar on three different surfaces (MAO，PT，TC). Our studyprovided theoretical basis for the choice of pre-radiotherapy implantinstallation.2. MAO surface induced more collagen secretion of osteoblasts than PTsurface at the same radiation dose, indicating that MAO modified implantsshould be chosen in defect area.3.16Gy of radiation might suppress the differentiation ability of MC3T3-E1cells through changing the phosphorylation of p38. Our study may providethe necessary foundation for attenuating the impaired ability of irradiatedosteoblasts and improving the success rates of implantation in irradiatedareas.