兔下颌骨来源骨髓间充质干细胞生物学行为及其体内成骨的研究

目的:探讨兔下颌骨来源的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)体外分离、培养及其生物学行为,评价下颌骨BMMSCs体内成骨的效果。方法:取兔下颌骨中的松质骨,贴壁培养,取第P2或P3代检测:1)倒置显微镜下观察细胞生长状态;2)MTT法测定细胞增殖并绘制细胞生长曲线;3)向成骨细胞和脂肪细胞诱导分化;4)将P3代BMMSCs与羟基磷灰石/磷酸三钙(Hydroxyapatite/tricalcium phosphate,HA/TCP)复合,自体回植;单纯HA/TCP植入做对照。术后8周和20周,通过组织学观察评价体内成骨的效果。结果:1)细胞经传代后形态一致;2)细胞生长曲线显示下颌骨BMMSCs经历潜伏期、对数生长期和平台期;3)成骨诱导、成脂诱导后形成钙结节及脂滴,茜素红、油红O染色呈阳性;4)体内成骨显示术后8周BMMSCs-HA/TCP组空隙内新骨形成;术后20周大量新骨形成,HA/TCP大部已降解。单纯HA/TCP植入组术后8周未见新骨形成;20周大部HA/TCP降解,仍未见新骨形成。结论:兔下颌骨分离出的BMMSCs,具有强大的自我复制、增殖能力及多向诱导分化的潜能,并能体内成骨。     

牙周膜干细胞用于犬牙槽嵴组织工程化增高的实验研究

目的：探讨牙周膜干细胞复合生物支架材料增高重建牙槽嵴效果,为缺牙后牙槽嵴重度萎缩的种植义齿修复奠定基础.方法：分离培养牙周膜干细胞（PDLSCs）,将其分别与骨组织工程支架材料羟基磷灰石-磷酸三钙（HA/TCP）及附着龈组织工程材料脱细胞真皮基质（ADM）复合诱导后,植入犬双尖牙缺失致牙槽嵴缺损部位,PDLSCs-HA/TCP植入骨缺损区,PDLSCs-ADM植入粘骨膜缺损区.植入32周,大体测量植入前后增高牙槽嵴高度;X-线片显示牙槽嵴高度与密度变化;组织学观察边界区新生牙槽骨结构,未植入做对照.结果：细胞-材料复合体植入后牙槽嵴高度显著增高,由移植前4.15＆#177;0.20,增加到移植后9.30＆#177;0.15（P＜0.05）.X-线显示邻牙标记部位冠方有类骨密度硬组织形成,牙槽嵴明显增高.组织学显示：与对照组比较,细胞材料移植组可见缺损区有新生骨样组织形成,可见骨陷窝和骨细胞样结构.结论：PDLSCs分别与骨及粘骨膜组织工程支架材料HA/TCP、ADM复合后体内移植,可显著增高牙槽嵴高度,是牙槽嵴增高重建、实现无牙合种植修复的有效途径.     

自体骨髓间充质干细胞移植对小型猪下颌骨放射性骨坏死的预防作用

目的：观察和分析自体骨髓间充质干细胞（ bone marrow mesenchymal stem cells,BMMSCs）静脉输注移植是否对小型猪下颌骨放射性骨坏死（ osteoradionecrosis,ORN）有预防作用。方法照射前1个月分离培养扩增BMMSCs,在照射后不同时点实验组进行自体BMMSCs静脉输注移植,对照组静脉输注同体积不含细胞的细胞培养液。照射2个月后拔除左侧下颌第一恒磨牙,通过肉眼、CT以及组织病理学观察小型猪动物模型下颌骨是否发生ORN。结果照射2个月拔牙创伤后,两组动物均出现了组织水肿、皮肤溃烂、骨质破坏等。5个月后实验组动物皮肤愈合,随后CT显示破坏骨质修复,组织病理学显示为接近正常的骨组织,而对照组动物下颌骨仍呈典型ORN表现。结论自体BMMSCs移植对下颌骨ORN有一定的预防作用,其预防机制尚有待进一步研究。     

骨形成蛋白与胶原基纳米骨修复牙周骨缺损的实验研究

目的 探讨重组人骨形成蛋白-2(rhBMP-2)和胶原基纳米骨复合材料(nHAC)修复牙周骨缺损的效果.方法 制备小型猪牙周骨缺损模型,设立rhBMP-2-nHAC复合材料植入组、空白对照组和单纯植入胶原基纳米骨组,术后8周观察各组间修复效果及组织病理学变化,通过骨组织形态计量学测定分析评价牙周骨组织的再生情况.结果 术后8周可见复合材料植入组大量新生牙周组织生长,四环素单标记表面、四环素双标记表面、骨矿化沉积率、骨体积、类骨质表面、成骨细胞表面等骨组织形态计量学结果 与空白对照组和单纯胶原基纳米骨组比较,差别均有统计学意义(P ＜ 0.05).结论 rhBMP-2和nHAC可明显促进牙周骨缺损修复.     

人颌骨骨膜成骨细胞体外成骨的实验研究

目的:研究人下颌骨骨膜来源的种子细胞组织工程化成骨的可行性.方法:从人下颌骨骨膜内分离出成骨细胞,纯化后进行体外培养扩增,将其接种至异体部分脱钙松质骨支架内,复合物植入裸鼠皮下培养2个月,观察成骨细胞在支架内的粘附、生长和成骨情况.结果:从人下颌骨骨膜成功分离培养出成骨细胞,细胞在异体部分脱钙松质骨内粘附、生长,植入裸鼠皮下培养2个月后,能在支架内形成新生骨组织.结论:人下颌骨骨膜成骨细胞能在体外支架内组织工程化形成新生骨组织.     

Beagle犬骨髓基质细胞复合A-PCPC裸鼠皮下成骨的实验研究

目的:观察支架材料活性磷酸钙骨水泥(active porous calcium phosphate cement,A-PCPC)及其与Beagle犬骨髓基质细胞(bone marrow stromal cells,BMSCs)复合体在裸鼠体内的成骨性能.方法:抽取Beagle犬骨髓,体外分离培养、成骨诱导、扩增BMSCs并通过细胞化学方法检测其成骨表型.体外构建BMSCs/A-PCPC复合体,并行扫描电镜超微结构观察.将复合体植于裸鼠背部皮下,同期植入单纯A-PCPC作为对照组.术后2、4、8周取材,HE染色,进行组织学及组织形态计量学分析.结果:ALP染色阳性,Von Kossa染色可见钙结节形成.扫描电镜可见细胞贴附于材料表面及孔隙表面生长.HE染色显示,2周时,复合体组可见少量编织骨,单纯材料组成骨较少.4周时,复合体组可见编织骨向小梁骨转化,单纯材料组可见大量骨样组织.8周时,复合体组新骨趋于成熟,单纯材料组骨成熟度较差.结论:A-PCPC支架接种BMSCs后显示良好的成骨活性,能够促进未成熟骨矿化.可以作为骨组织工程支架材料.     

骨膜下成骨修复小型猪下颌骨节段性缺损的初步研究

目的 探讨单纯骨膜下成骨修复小型猪下颌骨缺损的可能性。方法选择18个月龄雌性小型猪10只，拔除其右侧下颌骨的前磨牙及第一磨牙，两个月后随机将动物分为保留骨膜与不保留骨膜两群．其中保留骨膜群6只，不保留骨膜群4只。保留骨膜群根据骨块切除长度不同分40、50、60mm组，不保留骨膜群根据骨块切除长度不同分10mm组与20mm组，每组各2只动物。骨块切除后均行坚固内固定，术后4、8、12周分别拍X线片。结果保留骨膜群40、50mm组及不保留骨膜群10mm组术后4、8、12周X线片均显示下颌骨缺损处被钙化新生骨所充填．完成了骨连接。保留骨膜群60mm组及不保留骨膜群20mm组术后4、8、12周X线片显示下颌骨缺损未完成骨连接。结论 骨膜原位成骨能修复小型猪下颌骨50mm及其以下节段性骨缺损，为临床骨缺损的修复另辟新径。     

低细胞密度状态下的人骨髓间质干细胞成骨能力研究

目的　研究低密度人骨髓间质干细胞(Humanbonemarrow -derivedmesenchymalstemcells,hMSCs)体内成骨能力及影响因素。方法　从人髂骨骨髓分离培养hMSCs。以不同细胞密度与块状和颗粒型磷酸钙陶瓷(HA/TCP)载体混合,移植于裸小鼠体内,分别观察2和3个月,组织学评价研究hMSCs异体体内成骨的最小细胞密度。按3.3×1 0 6个细胞/cm3 载体的比例混合细胞与载体(HA/TCP和鼠尾胶原包被的HA/TCP) ,体外共培养一周后移植裸鼠皮下,3个月后,组织学评价细胞载体体系体外培养和胶原包被载体对hMSCs体内成骨能力的影响。结果　只有以3.3×1 0 6个细胞/cm3 载体在体内移植3个月后可观察到在块状和颗粒HA/TCP表面有骨组织生成。用鼠尾胶原包被的颗粒HA/TCP和细胞与载体体外共培养并无明显促进骨组织生成作用。结论　保证hMSCs异体成骨的最小细胞密度为3.3×1 0 6个细胞/cm3 载体。在低密度hMSCs状态下,鼠尾胶原和细胞与载体体外共培养并不能提高hMSCs的成骨能力。     

兔髁突来源骨髓间充质干细胞体内对骨缺损修复的实验研究

目的:研究兔下颌骨髁突来源骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)的体外分离、培养、鉴定及其体内成骨情况。方法:从兔下颌骨髁突中分离干细胞进行培养,对BMMSCs向成骨、成骨骼肌细胞的分化进行鉴定。实验组将BMMSCs复合于载黄芪多糖(APS)支架材料上,植入骨缺损区;对照组1植入单纯珊瑚羟基磷灰石(CHA)材料;对照组2植入APS/CHA复合材料;空白组不植入任何材料。应用组织学和扫描电镜观察,并比较植入材料与骨组织交界处的成骨修复情况。结果:细胞经过传代后形态一致,呈梭形;ALP染色呈阳性,成骨诱导后形成钙结节;成骨骼肌诱导后desmin免疫细胞化学染色阳性。植入体内修复骨缺损12周,实验组见大量新生骨及活跃的骨细胞;对照组1仅在材料表面见少量新骨形成;对照组2材料内有部分新骨形成;空白组基本未见骨修复。结论:从兔下颌骨髁突中分离、培养出的BMMSCs,具有体内、体外成骨的能力。     

小型猪牙周膜细胞体外培养及与三维支架材料的生物相容性研究

目的建立小型猪牙周膜细胞的体外培养方法,研究其与三维支架材料的生物相容性,为牙齿再生和牙周组织工程研究提供依据.方法无菌条件下,拔除12月龄小型猪上颌尖牙,剥离牙根外牙周组织,用3g/LⅠ型胶原酶和4g/L Dispase 37℃消化1 h,用70μm滤网收集细胞,1000 r/min离心10 min,培养液重新悬浮成单细胞悬液,α-MEM(α-modification of Eagle's medium)培养基培养.每天用倒置显微镜观察细胞生长情况,流式细胞仪检测抗Stro-1抗体的表达情况.取第3代牙周膜细胞接种于HA/TCP上,扫描电镜观察培养3天和5天后细胞在三维支架材料上的生长情况.结果原代培养的小型猪牙周膜细胞生长良好,呈多角形或梭性.流式细胞仪结果显示,分离培养的牙周膜细胞中有7.6%抗Stro-1抗体表达阳性.扫描电镜观察结果表明,牙周膜细胞在HA/TCP三维支架上生长良好.结论小型猪牙周膜细胞可被成功的分离培养,在三维支架材料HA/TCP上表现出良好的生长状态,有望为牙齿再生和牙周组织工程研究提供有用的细胞来源.     

兔骨髓基质干细胞自体嚼肌内移植异位成骨实验研究

目的：观察骨髓基质干细胞成骨向分化诱导后植入嚼肌内异位成骨的过程。方法：从兔髂骨分离骨髓基质干细胞,经体外诱导分化,与磷酸三钙／羟基磷灰石（tricalcium phosphate／hydroxyapatite,TCP／HA）支架复合,植入兔自体嚼肌内,形态学、免疫细胞化学、组织学观察其成骨特征。结果：体外培养的兔骨髓基质干细胞性状稳定,经矿化诱导培养的骨髓基质干细胞表现为成骨细胞分化,表达Ⅰ型胶原蛋白,与支架材料复合植入自体嚼肌内可见成熟骨组织形成。结论：利用骨髓基质干细胞为种子细胞植入嚼肌内开展功能性组织工程化颌骨移植方法可行。     

Allogeneic Stem Cells From Deciduous Teeth in Treatment for Periodontitis in Miniature Swine

Background: Regeneration of lost periodontium in periodontitis is a challenge in that alveolar bone, cementum, and periodontal ligament need to be restored to their original architecture. Stem cells from exfoliated deciduous teeth (SHEDs) appear to be an attractive candidate for periodontium tissue regeneration. Previously, the authors successfully regenerated periodontal defects using autologous and allogeneic periodontal ligament stem cells (PDLSCs). The purpose of the present study is to investigate the ability of allogeneic SHEDs to regenerate lost periodontium in a swine periodontitis model. Methods: Animal models of periodontitis were established in miniature pigs, and allogeneic stem cells were isolated from miniature pig deciduous teeth (SPDs). The animal models were treated with SPDs plus hydroxyapatite/tricalcium phosphate (HA/TCP). Allogeneic PDLSCs plus HA/TCP or HA/TCP alone were set as positive control or control, respectively. Clinical assessments, computed tomography (CT) scanning, and histologic examination were used to evaluate the outcome of tissue regeneration. Results: Clinical indices including probing depth, gingival recession, and attachment loss showed significant restoration in the SPD and PDLSC treatment groups, compared to the HA/TCP group 12 weeks post‐transplantation. Meanwhile, CT scans showed that 75% of the samples had successful hard‐tissue regeneration in both PDLSC and SPD groups, compared to the HA/TCP group, where the success rate was only 25%. In addition, histologic examination demonstrated that SPD and PDLSC treatment brought about remarkable regeneration of periodontal tissues, whereas periodontal regeneration was rare in the HA/TCP group. Conclusions: Allogeneic SPDs can effectively repair hard and soft tissue loss brought about by periodontitis in a swine model. Allogeneic SHEDs, which are easily accessible, may be applied to treat periodontitis in clinics in the future.     

3D-CT evaluation of mandibular morphology after mandibular outer cortex osteotomy in young miniature pigs: The role of the periosteum

AimThe purpose of this study was to evaluate the role of periosteum on the healing and growth of mandible after mandibular outer cortex osteotomy using three-dimensional computed tomography.MethodsEighteen 3-month-old miniature pigs were randomized into three groups. The mandibular outer cortex osteotomy was performed on both sides in group I, and on the left side in group II. In groups I and II, the local periosteum on the left side was resected. In group III, no operation was performed. The evaluation of mandibular morphology of all the animals was performed based on multiple spiral CT data before and after surgery.ResultsThe bone defects healed well when the periosteum was preserved, whereas they healed poorly with residual bone defects when the periosteum was resected after surgery. When the periosteum was resected, the decrease in the mean thickness of the mandibular body was more than that of the contralateral side after surgery. In group I, about 66.7% of the animals exhibited mandible deviation at 24 weeks after surgery. The median point of mentum was inclined toward the side that the periosteum was preserved. In groups II and III, no mandible deviation was observed.ConclusionThe periosteum plays an important role in bone growth and fracture healing. Mandibular outer cortex osteotomy inhibited the mandibular development and resulted in postoperative mandibular deviation in young miniature pigs. The simultaneous periosteum resection may offset the phenomenon of mandibular deviation to a certain extent.     

Introduction of a Mixture of β‐Tricalcium Phosphate Into a Complex of Bone Marrow Mesenchymal Stem Cells and Type I Collagen Can Augment the Volume of Alveolar Bone Without Impairing Cementum Regeneration

Background: The purpose of this study is to evaluate whether β‐tricalcium phosphate (β‐TCP) could be a promising modality to help augment alveolar bone in periodontal tissue regeneration by bone marrow mesenchymal stem cells (BMMSCs). Methods: Expanded BMMSCs and atelocollagen (Col) were mixed together (MSC/Col). A combination of β‐TCP with MSC/Col was also prepared (MSC/Col/TCP). MSC/Col/TCP or MSC/Col was transplanted into experimental periodontal Class III furcation defects that had been exposed to inflammation in beagle dogs. Periodontal tissue regeneration was evaluated by histologic and morphometric analyses at 4 and 8 weeks after transplantation. Results: MSC/Col and MSC/Col/TCP enhanced periodontal tissue regeneration compared to Col and TCP/Col according to hematoxylin and eosin staining. The percentage of new cementum length in the MSC/Col/TCP group was not significantly different from that in the MSC/Col group at 4 and 8 weeks. On the other hand, the percentage of new bone area in the MSC/Col/TCP group was much higher than that in the MSC/TCP group at 4 weeks. However, at 8 weeks, no significant difference in new bone area was found between the two groups. In the MSC/Col/TCP group, β‐TCP was surrounded by newly formed bone. Multinucleated cells, which were positive for osteopontin and tartrate‐resistant acid phosphatase, were present in the interconnected macropores of β‐TCP. Conclusion: These findings suggest that β‐TCP is applicable as a scaffold for BMMSCs transplantation and helps augment alveolar bone without impairing regeneration of cementum.     

人骨髓间质干细胞的培养及成骨功能研究

目的　体外扩增人骨髓间质干细胞 (humanbonemarrow derivedmesonchymalstemcells,hBMMSCs) ,研究其生物学特征及体内、外成骨功能。方法　分离培养hBMMSCs,并对其形态、增殖动力学、碱性磷酸酶 (alkalinephosphatase,ALP)表达及体内、外成骨功能进行研究。结果　hBMMSCs可在体外培养扩增 ,群体倍增时间约为 3 5d。ALP检测表明 ,未经矿化诱导的hBMMSCs可表达少量ALP ,诱导后其表达量明显增高。vonkossa染色证实在矿化诱导液作用下hBMMSCs在体外可形成钙结节。体内成骨实验证实 ,1× 10 5个hBMMSCs接种到 30mm3 块状HA/TCP载体移植BALB/C裸小鼠皮下 3个月 ,可观察到层板状骨组织生成。结论　hBMMSCs是一种具有成骨潜能的前体细胞 ,经培养、扩增、诱导后 ,在体外可形成矿化结节 ,体内可在载体表面形成骨组织。     

Sinus floor augmentation with recombinant human growth and differentiation factor-5 (rhGDF-5): a histological and histomorphometric study in the Goettingen miniature pig

Aim: The aim of this study was to test the hypothesis that recombinant human growth and differentiation factor‐5 (rhGDF‐5) enhances bone formation in sinus floor augmentations in miniature pigs. Material and methods: The maxillary sinus floors in 12 adult female Goettingen minipigs were augmented with β‐tricalcium phosphate (β‐TCP) on one side. The contralateral test side was augmented using two concentrations of rhGDF‐5 (400 μg rhGDF‐5/g β‐TCP; 800 μg rhGDF‐5/g β‐TCP) delivered on β‐TCP (six animals each). One dental implant was inserted into each sinus floor augmentation. After 4 and 12 weeks, histological and histomorphometric assessment of non‐decalcified histological specimens was performed. Results: The results showed significantly higher mean values of volume density (VD) of newly formed bone using the concentration of 400 μg/g β‐TCP (22.8%) compared with the respective control (8%) after 4 weeks (P=0.05). The bone‐to‐implant contact rates were also significantly enhanced after 4 weeks between test sites (400 μg: 41.9%; 800 μg: 40.6%) and control sites (400 μg: 7.8%; 800 μg: 16.4%) (400 μg: P=0.024; 800 μg: P=0.048). Conclusion: It is concluded that rhGDF‐5 delivered on β‐TCP significantly enhanced early bone formation compared with β‐TCP alone in sinus lift procedures in miniature pigs.     

The effect of therapeutic radiation on canine alveolar ridges augmented with hydroxylapatite.

The purpose of this investigation was to evaluate the effect of radiation on hydroxylapatite (HA) implanted subperiosteally for alveolar ridge augmentation in dogs. All bicuspids and molars were extracted from 16 dogs. After 6 weeks, nonporous HA granules were implanted subperiosteally on the alveolar ridge. Following 4 months of healing, 12 dogs (experimental group) underwent therapeutic radiation therapy (Co60, 4,000 rad [40 Gy]) to the head and neck region. Four dogs were not irradiated and served as controls. Four animals (three experimental and one control) were killed at 5,6,7, and 8 months after HA augmentation. Light microscopic evaluation showed that approximately 25% of HA granules were encased by bone while the others were surrounded by fibrous connective tissue. Dissolution of the HA was observed. Microparticles of HA were phagocytized as part of a granulomatous inflammatory reaction. This reaction decreased significantly as time elapsed after implantation. Osteoclastic activity was seen at the junction of HA and periosteum and as part of bone remodeling. Dissolution of the HA granules and the granulomatous inflammatory reaction were not significantly increased by therapeutic radiation. The radiation did not cause development of dehiscence or osteonecrosis.     

Mouse mandible contains distinctive mesenchymal stem cells

Although human orofacial bone-marrow-derived mesenchymal stem cells showed differentiation traits distinctly different from those of mesenchymal stem cells (MSCs) derived from long bone marrow (BMMSCs), mouse MSCs derived from orofacial bone have not been isolated due to technical difficulties, which in turn precludes the use of mouse models to study and cure orofacial diseases. In this study, we developed techniques to isolate and expand mouse orofacial bone/bone-marrow-derived MSCs (OMSCs) from mandibles and verified their MSC characteristics by single-colony formation, multi-lineage differentiation, and in vivo tissue regeneration. Activated T-lymphocytes impaired OMSCs via the Fas/Fas ligand pathway, as occurs in BMMSCs. Furthermore, we found that OMSCs are distinct from BMMSCs with respect to regulating T-lymphocyte survival and proliferation. Analysis of our data suggests that OMSCs are a unique population of MSCs and play an important role in systemic immunity. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; HA/TCP, hydroxyapatite/tricalcium phosphate; OMSC, orofacial mesenchymal stem cell; OVX, ovariectomized.     

Osteogenesis of hydroxyapatite and tricalcium phosphate used as a bone substitute.

Hydroxyapatite (HA) and tricalcium phosphate (TCP) are useful for grafting and augmentation of bone tissue. In this study, conventional and histochemical transmission electron microscopy were used to study osteogenic events at the interface between the implanted materials and adjacent tissue from 1 to 4 weeks postoperatively. The microscopic results indicated that TCP was resorbed more rapidly than HA after implantation, with a notable breakdown of material and replacement by mesenchymal cells with ultrastructural features resembling osteoprogenitor cells and collagen up to 4 weeks postoperatively. Alkaline phosphatase and acid phosphatase reactivity in the tissues helped to identify and differentiate the histologic differences observed between HA and TCP.