| Oral implant as a new subject has been favored by people in recent years.Oral implants are called human the third concomitant teeth, and become themost ideal short of teeth repair way. But many concomitant systemic diseases,such as cardiovascular disease, diabetes, metabolic bone disease, hemorrhagicdiseases, autoimmune disease, etc., which often lead to the final failure ofimplanting, so it has greatly restricted the implanting technology promotion.With people living standard riseing and diet structure changing, the case rate isincreasing year by year. Based on the situation, our subject team have done theprevious animals experiments, and confirmed that the main reason of failure isthe implant surrouding does not get good osseous union, and we think the keyof osseointegration is the formation and potential differentiation of osteoblast.So it is very necessary to research biological characteristics of alveolar bone osteoblast in type2diabetes patients from cell biology angle, and then releasethe system with ideal microspheres local constantly release of growth factors ofpeptide interference,and then we use microspheres with ideal controlled releasesystem which sustained release polypeptide growth factors to carry outinterference.Finally,we analysis induction differentiation role forosteoblast.We provide the theoretical and experimental basis for the nextclinical research.on how to improve the patients with type2diabetes successrate of dental implant.Study Content and Objective:In clinical,we gather to accept the oral implant type2diabetes andhealthy patient alveolar bone remaining bone debris and culture in vitrorespectively, analyzes the biological characteristics.Through a comparativestudy, confirmed type2diabetes patients alveolar bone cells of the biologicalactivities is more poor than normal persons’ones and provide the carrier forfollowing study.Using good biocompatibility, degradation, and no side effect ofbiological polymer PLGA and adopting use water/oil/water (W/O/W) doubleemulsion parcel polypeptide growth factor rhIGF-1and prepared for aslow-release ability microspheres.their size and shape are observated underscanning electron microscope (SEM), and carry out release dynamicsexperimental in vitro, draw the curve, analysis consistent release ability ofmicrospheres.In the same cell count, the two kind microspheres that PLGA packagedrhIGF-1and placebo join two groups type2diabetes alveolar osteoblast culturedish respectively with titanium piece and use rhIGF-1persistent release functionto analysis induction differentiation role to osteoblasts. For the next clinical application research,they laid experimental basis, and also solve the toughquestions that type2diabetes patients implant high failure rate to undertakeeffective exploration.Method and Results:Part1:Methods:Under aseptic conditions, collect three groups received oral implanttype2diabetes patients and normal patients alveolar bone remaining bone debris.These chips were cultured in vitro using the tissue explants adherent method(TEAM). The biological properties of these cells were characterized using thefollowing methods: alkaline phosphatase (ALP) chemical staining for cellviability, Alizarin red staining for osteogenic characteristics, MTT-test for cellproliferation, enzyme dynamics for ALP contents, radio-immunoassay for bonegla protein (BGP) concentration and ELISA assays for the concentration of type Icollagen (COL-I) in the supernatant.Results:(1) The two forms of cells have no significant difference in morphology underthe same culture conditions. However, the alveolar bone osteoblasts receivedfrom type2diabetic patients have a slower growth, lower cell activity andcalcium nodules formation than the normal ones. The concentration of ALP,BGP and COL-1in supernatant of alveolar bone osteoblasts received from type2diabetic patients is lower than the normal ones (P﹤.05).(2) Our results also show that osteoblasts from type2diabetic patients wereslow to grow in low carbohydrate DMEM, while grew faster in the high sugarmedium. The osteoblasts from healthy patient have no obvious differences inlow or high sugar medium.Part2: Methods: Use rhIGF-1and distilled water to form W1phase, Span80and PLGAas O phase.Both phase combine to form W1/O initial emulsion, finally theybind W2phase (tween80and PVA composition) and form W1/O/W2doubleemulsion. After solvent evaporation,form required microspheres.Using scanningelectron microscopy (SEM) observe their size, shape and texture is whethereven or not. Through the cumulative release rate to draw release kinetics curvein order to observe the persistent release efficiency.Results:(1) The surface morphous of the experiment microspheres form is smooth andsize is even. Its particle diameter is1.4076±0.5238μm. The envelope rate isabout82.3±1.8%.(2) From drawing release dynamics curve we can see microspheres requiredwith have good ability to release.At the beginning they release fast.The first20days cumulative release rate is about84%, and then gradually slow down.andabout40days release is over.Part3:Methods: In the cultivationhole put into titanium piece, join cell suspensionliquid, after culturing some time, take out of the titanium piece, carry out cellnonspecific fluorescent dye to confirm the stick ability of two kinds of cellsand titanium piece. In the same cell count, six holes culture plate each holeplaced eight titanium slices. After cells are full to make the cell count, analysisthe combination ability of two kinds of cells and titanium slices in petri dishes.Applying the same way(each hole placed six titanium slices), PLGA packagedrhIGF-1and placebo join culture dishes respectively with titanium slices of type2diabetes alveolar osteoblast. After cells crawling fully and count, confirm thatrhIGF-1induction of role to cells of titanium. Results:(1) Nonspecific fluorescence staining showed the two cells evenly stick thesurface of titanium and observed clearly the nucleus and cell membrane.(2) Through the cell count, compute the attached number of two cells andtitanium slices.Type2diabetes alveolar bone osteoblasts are less healthypatientsones, both have statistically significant (P﹤0.05).(3) Type2diabetes alveolar bone osteoblasts joined two kind microspheres,through the cell count, the number of diabetes patients osteoblasts joinedrhIGF-1group are more than ones joined the placebo group.They arestatistically significant (P﹤0.05). confirmed that the PLGA which packagedrhIGF-1microspheres have the role of induction differentiation for type2diabetes alveolar osteoblasts.Conclusion:(1) Our results suggest that alveolar bone osteoblasts from type2diabeticpatients can be successfully cultured in vitro and they have specific morphologycompared to normal osteoblasts. Moreover, we confirmed that they are differentin their growth rate, activity and mineral secretion. The differences betweendiabetic alveolar bone osteoblasts have strong memory function for pathologicalveolar bone lesions.(2) The prepared microspheres whose size, the encapsulation efficiency andrelease the capacity are more ideal, and they have good biocompatibility andpromote the osteoblast differentiation as controlled release system.(3) Confirmed that PLGA is a kind of ideal carrier materiall without any badreaction with cells, can promote cells appreciation and can improve type2diabetes early synosteosis after implant as the next step effective means in theclinical. |
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