【作者】 刘世森；

【导师】 刘洪臣；

【作者基本信息】 中国人民解放军军医进修学院 ， 口腔临床医学， 2009， 博士

【摘要】 骨质疏松是以骨量减少、骨组织显微结构退化为特征的一种系统性骨代谢疾病。颌骨作为全身骨骼的一部分,与全身其他骨骼一样会受到骨质疏松的影响,导致颌骨内骨组织的数量和质量下降,使种植体植入后愈合时间延长,BIC、BD、BA下降,对种植牙长期稳定的发挥功能构成了威胁;同时,骨质疏松加快了剩余牙槽嵴吸收速度,降低牙槽嵴高度和宽度,使牙槽骨扩增术面临着更大的失败风险。鉴于此,骨质疏松被认为是种植牙治疗的相对危险症。针对骨质疏松患者,如何使种植体快速达到良好的骨整合状态,如何对萎缩的牙槽骨成功进行扩增,是很多种植科临床医生关注的问题。本研究从外周血中获得可以作为骨组织工程种子细胞的PB-MSCs;改良了壳聚糖凝胶使其能良好的保持种子细胞的活力;利用这两者构建一种可注射组织工程骨,应用到骨质疏松兔种植体周围,结果该可注射组织工程骨在种植体骨外悬空部分周围形成了新生骨组织与种植体紧密接触,在种植体骨内部分近心端形成较强的白色阻射影像。这提示该可注射组织工程骨在体内可以形成新生骨组织,既可以用于牙槽骨扩增术,也可以提高骨质疏松状态下BD、BA,进一步详细的深入研究有待以后进行。实验一骨髓与外周血间充质基质细胞的体外培养目的:研究外周血是否可以与骨髓一样作为骨组织工程种子细胞来源。方法:自腹主动脉抽取1月龄左右新西兰兔外周血,密度离心法分离兔外周血单个核细胞,冲洗收集长骨内骨髓,将两者在体外进行培养。取P₄代细胞,利用RT-PCR法检测表面分子标志物表达情况;进行成脂诱导,油红O染色观察脂滴形成情况;进行成骨诱导,组织学染色观察ALP、Ⅰ型胶原的表达及矿化结节形成情况。结果:骨髓原代培养细胞集落形成旱,细胞形态均一,呈典型的成纤维细胞样细胞,CD29表达阳性,CD34表达阴性。成脂诱导时大部分分化为脂肪细胞。成骨诱导1w时ALP表达阳性,3w时Ⅰ型胶原表达阳性,4w时形成数量较多的矿化结节。外周血原代培养细胞集落形成晚,细胞形态不一,种类多。细胞CD29表达阳性,CD34表达弱阳性。成脂诱导10d时部分细胞分化为脂肪细胞。成骨诱导2w时ALP表达阳性、4w时Ⅰ型胶原表达阳性,5w时有矿化结节形成,同时有管样结构形成。结论:外周血内含有多向分化潜能干细胞,成骨诱导时能分化为成骨细胞并形成矿化结节。外周血可以作为组织工程骨的种子细胞来源。实验二一种细胞活性保持良好的壳聚糖凝胶的制备目的:通过减少盐酸的浓度,升高壳聚糖溶液的PH值,减少交联剂β-GP用量,使壳聚糖凝胶具有生理渗透压,更好的保持细胞活性。材料与方法:用不同浓度盐酸配制2%壳聚糖溶液:A组,0.1M;B组,0.09M;C组,0.08M;D组,0.07M;E组,0.07M;A-D组常温搅拌溶解,E组高温搅拌溶解。持续搅拌6h后高温高压消毒。β-GP溶液分为3种浓度（w/v）:Ⅰ组,11%;Ⅱ组,25%;Ⅲ组,50%。4ml壳聚糖溶液与1mlβ-GP溶液混匀组成壳聚糖凝胶,测量不同组分壳聚糖凝胶PH值并记录其在37℃时凝固所需时间。取E+Ⅰ、D+Ⅱ、D+Ⅲ三组凝胶与外周血间充质基质细胞混合凝固后体外培养3d,利用MTT检测凝胶内细胞活力保持情况。结果:盐酸浓度越低,消毒造成的壳聚糖溶液粘度损失越小,所获得的壳聚糖凝胶PH值越高,由不凝固变为凝固或凝固所需时间越短。E+Ⅰ组凝胶中大多数细胞具有活性,D+Ⅱ组凝胶中少数细胞具有活性,D+Ⅲ凝胶中只有个别细胞具有活性。结论:E+Ⅰ组凝胶含2.2%β-GP,具备生理渗透压,PH值为7.13,凝固时间为20min,能很好的保持细胞的活性。实验三去势法骨质疏松兔模型的建立及下颌牙槽骨的改变目的:去势法建立兔骨质疏松模型并观察下颌牙槽骨的改变。方法:20只雌性新西兰兔平均随机分为两组,手术组行双侧卵巢摘除术,对照组行假手术。检测术前及术后3m、6m血清中OC与TRAP水平、股骨与腰椎BMD.术后6m处死所有实验用兔,下颌骨脱矿后制作5μm切片,组织学染色观察颏孔附近骨形态,光镜下测量下颌中切牙内侧骨层厚度,拍片后使用photoshop软件计算下颌中切牙下方内侧骨面积比例。结果:手术组OC水平术后3m（3.39±0.63μg/L）、6m（3.22±0.98μg/L）高于假手术组（2.46±0.68μg/L;2.1 7±0.57μg/L,P＜0.05）;TRAP术后3m（OD值0.389±0.061）、6m（OD值0.371±0.051）高于假手术组（OD值:0.332±0.039:0.308±0.029,P＜0.05）。术后3m两组的股骨、腰椎BMD无统计学差异.术后6m手术组股骨BMD（0.349±0.056g/cm³）、腰椎BMD（0.257±0.042g/cm³）均低于假手术组（股骨0.481±0.086g/cm3,P＜0.01;腰椎0.360±0.042g/cm3,P＜0.01）。术后6m手术组下颌骨松质骨内骨小梁变细,排列稀疏甚至消失,手术组下颌中切牙内侧骨层厚度（0.52±0.16mm）较假手术组（0.85±0.14mm,P＜0.01）变薄;中切牙内下方骨小梁面积比（51.7±11.6%）明显低于假手术组（82.6±9.2%,P＜0.01）。结论:去势法兔骨质疏松模型成熟可靠。下颌牙槽骨表现出明显的骨质疏松样改变,提示骨质疏松状态下进行种植牙治疗有必要采取一些特殊措施来保证治疗的成功。实验四可注射组织工程骨用于骨质疏松种植修复的初步探索目的:将可注射组织工程骨安全有效的应用于种植体研究,初步观察其在骨质疏松状态下修复种植体周围骨量不足的效果。方法:（1）9只正常新西兰兔分为3组,每组3只,将1ml E+I配制的壳聚糖凝胶按照不同方式应用于种植部位骨内:第一组种植窝制备前注入;第二组种植窝制备后注入,并迅速植入种植体;第三组将壳聚糖凝胶凝固后置入种植窝。观察2h内实验用兔反应。（2）用自体PB-MSCs与E+I配制的壳聚糖凝胶构建可注射组织工程骨。所用种植体为BAM表面羟基磷灰石涂层的圆柱状无螺纹种植体,植入股骨干骺端。12只骨质疏松兔分为3组,每组4只。空白对照组单纯植入种植体;对照组种植体骨内周围及骨外悬空部分应用壳聚糖凝胶;实验组种植体骨内周围及骨外悬空部分应用可注射组织工程骨。术后10w处死所有骨质疏松兔取材,观察种植体悬空部分周围新骨形成情况。X片观察种植体骨内部分近心端的X线阻射情况。结果:（1）所有第一组新西兰兔在壳聚糖凝胶注射后10-30min内死亡。第二组、第三组新西兰兔无死亡。（2）空白对照组与大部分对照组种植体悬空部分周围无骨组织;实验组种植体悬空部分周围有不同程度的新生骨组织形成并与种植体紧密相接。空白对照组种植体近心端无X线阻射影;对照组部分标本种植体近心端有少量模糊的白色阻射影;实验组在种植体近心端有较强条纹状或蜂窝状的白色阻射影,提示有较多骨形成。结论:在生理条件下可以自发凝固的可注射凝胶直接注射入封闭的骨组织内有很高的风险。可注射组织工程骨既能在种植体骨外悬空部分周围形成新生骨组织,也能在种植体骨内部分周围形成X线阻射影像,提示该可注射组织工程骨可以用于牙槽骨扩增术及提高骨质疏松状态下BD、BA,进一步详细的深入研究有待以后进行。更多还原

【Abstract】 Osteoporosis is a systematic bone metabolic disease characterized by decrease of bone mass and degeneration of bone microstructure.Jaw bones,as a part of systematic skeletal tissues,are also affected by osteoporosis.Quantity and quality of bone tissues in jaw bones are both decreased under osteoporotic status which result in a prolonged healing period and lower BIC,BD,BA aider dental implants are implanted.These unfavorable changes threaten the long and stable function of dental implants.Moreover osteoporosis enhances residual ridge resorption and confronts augmentation of alveolar bone with a higher risk of failure.Studies are in progress on how to obtain faster,better osseointegration of dental implants and successful augmentation of alveolar bone.In this paper,several efforts were managed to solve these problems. Mesenchymal stromal cells were obtained from peripheral blood and acted as seed cells.A well-cytoactivity-preserving hydrogel based on chitosan was prepared and acted as scaffold.An injectable tissue engineered bone constructed by these two components was applied to dental implants in an osteoporotic rabbit model.10w later,new bone formed around the extraosseous part of dental implants and contacted tightly with them.The radiographs showed more X-ray radiopaque shadow at the proximal endosseous part of dental implants,which hinted more new bone formed.All of these results indicated the potential osteogenic ability of this injectable tissue engineered bone.It could be applied to augmentation of alveolar bone and improve the lowered BD,BA under osteoporotic status. However,detailed and further studies were necessary to evaluate the overall effects of this kind of bone graft.ExperimentⅠCulture of rabbit bone marrow and peripheral blood derived mesenchymal stromal cells.Objective To investigate whether peripheral blood could act as a source of seed cells for bone tissue engineering like bone marrow.Methods Peripheral blood of lm old New Zealand rabbits was aspirated from abdominal aorta.Mononuclear cells were separated out using density centrifugalization and cultured in vitro.At the same time bone marrow in long bones was washed out and cultured.P₄ cells were used to perform identification of cell markers and differentiation experiments.RT-PCR was used to identify the cell markers.Oil Red o stain was performed to the observe the formation of lipid droplets when adipo-induced.Histological staining was performed to detect the expression of ALP,typeⅠcollagen and deposition of mineralized nodes.Result Cell colonies formed early in primary culture of bone marrow.Cells in the colonies were uniform fibroblast-like cells and positive of CD29,negtive of CD34.When adipo-induced,most of the cells differentiated to lipocytes.When osteo-induced,these cells became positive of ALP at lw,positive of typeⅠcollagen at 3w,and formed numerous bulk calcium nodes at 4w.However,Cell colonies formed relatively late in primary culture of peripheral blood.There were several different morphological cells and thus,forming different morphological cell colonies.These cells were positive of CD29 and weakly positive of CD34. When adipo-induced,part of the cells differentiated to lipocytes.When osteo-induced, they became positive of ALP at 2w,positive of typeⅠcollagen at 4w, and formed calcium nodes at 5w.Although no vascular inducer were added into the culture medium,tubule-like structures were seen.Conclusions Peripheral blood contains multipotent stem cells.When osteo-induced,they can differentiated to osteoblasts and form calcium nodes. Peripheral blood can act as a source of seed cells for bone tissue engineering.ExperimentⅡPreparation of a well eyto-eompatible hydrogel based on chitosan.Objective To prepare a novel injectable hydrogel based on chitosan with physiological osmolality and well-cytoactivity- preserving ability.Materials and methods Chitosan solutions were prepared with hydrochloric acid of different concentrations:group A,0.1M;group B,0.09M, group C,0.08M;group D,0.07M;group E,0.07M.Group A to D were prepared at room temperature and group E at 60℃.All the groups were stirred continuously for 6h and then autoclave sterilized.Aqueousβ-disodium glycerol phosphate was prepared in three concentrations（w/v）:groupⅠ,11%;groupⅡ,25%;groupⅢ,50%.Chitosan based hydrogel were prepared by 4ml chitosan solution and 1 ml aqueousβ-disodium glycerol phosphate.PH of every hydrogel was detected and its gelatification time at 37 was recorded.Three groups of chitosan hydrogel （E+Ⅰ,D+Ⅱ,D+Ⅲ）were mixed with PB-MSCs.They were cultured in vitro for 3d.Live cells were detected by Formazan crystals formed by deoxidized MTT。Result Lower hydrochloric acid concentration resulted in less autoclaving-induced viscosity loss of chitosan solution and higher PH of chitosan based hydrogel which led the hydrogel to be gelable or to gel in a shorter time at 37℃.Most of the cells in E+Ⅰhydrogel were live while part of the cells were live in D+Ⅱhydrogel and few cells were live in D+Ⅲhydrogel.Conclusions E andⅠconstructed hydrogel contained 2.2%β-disodium glycerol phosphate which induced a physiological osmolality.It possessed a PH of 7.13,gelatification time of 20min and well-preserved viscosity.All of these properties it to be a suitable injectable hydrogel for cell-encapsulating. ovariectomy and the changes of mandibular alveolar bone.Objective To establish an osteoporotic rabbit model for the next study just by ovariectomy and observe the changes of mandibular alveolar bone.Methods 20 New Zealand rabbits were equally divided into 2 groups randomly:operation group whose bilateral ovaries were ectomized and shame operation group as a control.OC and TRAP in serum,BMD of femur and lumbar vertebra were detected before,3m and 6m after operation.All the animals were sacrificed 6m after operation.Their demineralized mandibular alveolar bones were performed histological staining in 5μm slices to observe the change of bone near mental foramen.Thickness of medial osteoplaque beside mandibular incisor was measured.Bone percentage of medial side inferior mandibular incisor were measured on computer by photoshop7.01 after slices had been photographed.Result Operation group showed higher OC level than sham operation group 3m（7.39±0.63μg/L versus6.46±0.68μg/L,P＜0.05）,6m（7.22±0.58μg/L versus6.57±0.45μg/L,P＜0.05）after operation.OD value of TRAP was also higher in operation group 3m（0.389±0.061 versus 0.332±0.039,P＜0.01）, 6m（0.371±0.051 versus0.308±0.029,P＜0.01）after operation.BMD of femur and lumbar vertebra showed no statistical difference in these 2 groups 3m after operation.Femur BMD was lower in operation group（0.349±0.056g/cm³）than sham operation group（0.481±0.086g/cm³,P＜0.01）6m after operation and lumbar vertebra had the same case（0.257±0.042g/cm³ versus 0.360±0.042g/cm³, P＜0.01）.Bone trabeculas of cancellous bone around mandibular incisor were thinner,less even vanished in operation group 6m after operation.Thickness of medial osteoplaque beside mandibular incisor in operation group（0.52±0.16mm） was thinner than that in sham operation group（0.85±0.14mm,P＜0.01）.Bone percentage of medial side inferior mandibular incisor in operation group（51.7±11.6%）was lower than that in sham operation group（82.6±9.2%,P＜0.01）.Conclusions Rabbit model of osteoporosis was successfully established. Mandibular alveolar bone showed typical osteoporotic changes.This indicated that special treatments must be considered to ensure the success of dental implants procedures under osteoporotic status.ExperimentⅣPreliminary application of the injectable tissue engineered bone to osteoporotic dental implants.Objective To apply an injectable tissue engineered bone safely,efficiently to dental implants procedures and observe its effect.Methods（1）9 healthy New Zealand rabbits were equally divided into 3 groups,lml chitosan hydrogel was applied to the implant site in different ways. MethodⅠ:chitosan hydrogel was injected into the implant site before preparing the implant hole.MethodⅡ:chitosan hydrogel was injected into the newly prepared implant hole,and the dental implant was placed immediately.MethodⅢ:coagulated chitosan hydrogel was placed into the prepared implant hole.The reaction of rabbits was observed in 2h.（2）Injectable tissue engineered bone was constructed by autologous peripheral blood mesenchymal stromal cells and well-cytoactivity -preserving chitosan hydrogel described in experimentⅡ.Dental implants used in this paper were BAM HA-coated,round,non-threaded dental implants.They were implanted into the lateral metaphysis of femurs.12 osteoporotic rabbits were equally divided into 3 groups.Blank control group: dental implants were implanted only.Control group:coagulated chitosan hydrogel was placed around the endosseous and extraosseous part of dental implants. Experimental group:coagulated injectable tissue engineered bone was placed around the endosseous and extraosseous part of dental implants.10w later,all the osteoporotic rabbits were sacrificed and the femurs were harvested The specimens were observed macroscopicly and radiographicly.Result（1）All the rabbits applied by methodⅠdied in 10～30min after the chitosan hydrogel was injected into the femurs.The rabbits applied by methodⅡor methodⅢseemed to be unaffected and stayed alive.（2）No new bone formed around the extraosseous part of dental implants in blank control and control groups while new bone formed around the extraosseous part of dental implants and contacted tightly with them in experimental group.The radiographs showed black radiolucent shadow at the proximal endosseous part of dental implants in blank control group,while nebulous X-ray radiopaque shadow in control group.In the radiographs of experimental group,more striated or alveolate X-ray radiopaque shadow located proximally to the dental implants,which hinted more new bone formed.Conclusions It was rather hazardous to inject an injectable hydrogel,which could coagulate spontaneously under physical status,into the closed bone circumstance.The injectable tissue engineered bone possessed potential osteogenic ability.It could be applied to augmentation of alveolar bone and improve the lowered BD,BA under osteoporotic status.However,detailed and further studies were to be performed in the future.更多还原