去蛋白脱钙皮质骨基质微颗粒用于骨髓基质干细胞体外培养的相关研究

目的 比较牛去蛋白脱钙皮质骨基质(bDpDBM)微颗粒和牛脱钙皮质骨基质(bDBM)微颗粒用于体外干细胞培养的优缺点,探讨其作为微载体制备候选材料的可行性. 方法 冷冻粉碎牛四肢皮质骨,筛选出直径为200 ～ 500μm的微颗粒,按照常规方法进行脱脂、脱钙、脱蛋白处理.设bDpDBM和bDBM微颗粒为研究对象,用于静态条件下骨髓基质干细胞(BMSCs)的培养,比较两组微颗粒对干细胞增殖及功能保持的影响,分析其用于BMSCs培养的可行性. 结果 两组微载体进行培养第14天后显示bDpDBM组细胞扩增较bDBM组快,差异有统计学意义(P＜0.05).经bDBM组培养的BMSCs的ALP定量及胞内钙含量明显较bDpDBM组多,差异有统计学意义(P＜0.05).而bDpDBM颗粒培养的BMSCs经诱导可向成骨、成脂及成软骨三系分化. 结论 bDpDBM由于去除了脱钙骨基质中的活性诱导成分,培养增殖效率增高,可较好地保持干细胞表型,适宜用作体外大量扩增BMSCs的微载体候选材料.     

自体骨髓联合脱钙牙基质治疗骨折不愈合

2006年3月～2008年12月,我科采用自体骨髓移植联合脱钙人牙基质材料(骨又生,decalcified dentin matrix,DDM)治疗骨折延迟愈合或不愈合患者19例,取得了良好的疗效.     

管型与非管型脱钙骨基质支架材料成骨活性差别的实验研究

目的探讨管型与非管型骨缺损修复材料成骨活性的差别。方法以兔同种异体尺桡骨脱钙骨基质为原料,制成管型(A组)及非管型(B组)修复材料各30只,随机植入30只兔双侧桡骨中上段1.5 cm骨缺损中,分别于术后1、2、4、8、12周随机处死6只取材进行X线、组织学切片、大体观察、单位湿重Ca值等检查,结果进行统计学处理。结果早期(术后2周)A组具有更多的骨痂生成(P<0.05)、更高的单位湿重Ca值(P<0.05);中期(术后4周)A组编织骨具有更高的孔隙率(P<0.05);后期(术后8、12周)A组表现为更薄的板层骨、更好的髓腔再通率、更好的塑形、更成熟的骨髓形成(P<0.05)、更高的单位湿重Ca值(P<0.05)。结论实验显示管型脱钙骨基质较非管型具有更好的节段性骨缺损修复能力,提示管型人工骨治疗节段性骨缺损具有美好的前景。     

髓内脂肪细胞对骨髓基质细胞成骨能力的影响

目的探讨在骨髓腔中,来源于骨髓基质细胞的脂肪细胞对骨髓基质细胞成骨能力的影响。方法将骨髓基质细胞以1×106个/ml浓度接种于24孔培养板中,共分为5组,分别加入0、103、104、105、106/ml浓度的脂肪细胞悬液,建立脂肪细胞和骨髓基质细胞共培养体系。共培养12d,每4d取细胞样本,检测细胞内碱性磷酸酶活性,利用原位杂交方法检测I型胶原mRNA表达,3H脯氨酸掺入实验检测骨髓基质细胞胶原合成能力。结果经过12d培养,不同时段的共培养体系中,随着脂肪细胞浓度上升,骨髓基质细胞内碱性磷酸酶相对活性下降,各实验组明显低于对照组(P<0.05或P<0.01);I型胶原mRNA表达减弱,对照组染色最深,实验组着色较淡;各实验组3H脯氨酸掺入实验min-1值均低于同期对照组(P<0.05或P<0.01)。结论髓内脂肪细胞干扰了骨髓基质细胞的成骨能力,可能与原发性骨质疏松症的发病有关。     

The bone inductive capacity of decalcified bone matrix modified by diphenylhydantoin

Decalcified bone matrix was prepared from cortical bones of rats premedicated with I) Diphenylhydantoin (DPH), II) DPH + Vitamin D3, III) Vitamin D3 or IV) no premedication for 10 days. In the donor animals, DPH lowered the serum calcium level, caused a weight loss of 10 per cent, and stopped the growth of the long bones. Vitamin D3 supplementation normalized the serum calcium concentration but had no effect on the other parameters. Vitamin D3 alone caused hypertrophy of the growth cartilage, while the bone growth and structure was normal. The bone inductive capacity of decalcified bone matrix was highest in the DPH group, and the DPH + D3 group also showed significantly higher values than the D3, and control groups. The results of the present study show that the bone inductive capacity of the decalcified bone matrix is independent of Vitamin D3 metabolism.     

脱钙皮质骨基质的拉伸力学性能研究

目的探讨脱钙皮质骨基质厚度与拉伸力学性能的关系,为将其作为组织工程支架材料奠定实验基础。方法取市售新鲜小牛胫骨,常规快速脱钙制备脱钙皮质骨基质,大体观察其颜色、质地等物理性状,并进行脱钙检测。将脱钙皮质骨基质沿径向纵切成不同厚度的片材,并根据厚度分为4组(n=16),分别为A组100～300μm,B组300～500μm,C组500～700μm,D组700～1 000μm;对每组标本进行拉伸性能和组织学观测。结果大体观察显示脱钙皮质骨基质经H2O2处理后呈乳白色,柔韧性好,富有弹性。脱钙皮质骨基质的脱钙率达97.6%。A组强度及弹性模量明显小于B、C、D组(P<0.05),B、C、D组间比较差异均无统计学意义(P>0.05);脱钙皮质骨基质刚度随厚度增加呈逐渐增大趋势,A组刚度显著低于B、C、D组(P<0.05),B、C组低于D组(P<0.05),B、C组间差异无统计学意义(P>0.05);各组极限应变差异均无统计学意义(P>0.05)。组织学观察各组均可见典型骨单位结构,其最大直径范围为102～325μm,平均最大直径为182μm。结论骨单位的完整性对脱钙皮质骨基质的力学性能有重要影响;脱钙皮质骨基质作为组织工程支架材料,在厚度>300μm时可保持其拉伸力学性能。     

The use of decalcified granulated homologous cortical bone matrix in the correction of diaphyseal bone defect

Summary In order to investigate an alternative way to correct diaphyseal bone defects, granulated decalcified homologous cortical bone was used as a graft.Because of the suitable anatomic arrangement, the forearm of rabbits was chosen as an experimental model of bone defect. A 2 cm long bone cylinder was removed from the diaphysis of both radii preserving the periosteum. The artificial bone defect was filled with granular decalcified bone on the right side. The left side was used as control and kept empty or filled with undecalcified granular bone.The 18 animals were sacrificed in batches, 3, 6, and 9 weeks after the operation.New bone formation was followed by X-rays, routine histology and incorporation of calcein blue, xylenol orange and tetracycline.In the decalcified granular bone grafts new bone formation was already detected at the first week and 9 weeks after the graft operation there was a well developed cylindric ossicle, in 89% of the cases. In some cases a medullary canal was present.No bone formation was found neither in the empty defects nor in the ones filled with undecalcified granular bone grafts.

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Zusammenfassung Zur Untersuchung einer Alternative für die Auffüllung diaphysärer Knochendefekte wurde homologe Kortikalis als entkalktes Knochengranulat implantiert.Wegen der günstigen anatomischen Bedingungen wurde der Vorderlauf des Kaninchens als experimentelles Modell gewählt. Ein 2 cm langes Knochensegment wurde aus den Diaphysen beider Speichenknochen unter Erhaltung des Periosts reseziert. Am rechten Vorderlauf wurde der künstliche Knochendefekt mit entkalktem Knochengranulat aufgefüllt. Auf der linken Seite wurde zur Kontrolle der Defekt leer gelassen oder mit unentkalktem Knochengranulat aufgefullt.Die Tiere wurden gruppenweise 3, 6 und 9 Wochen nach der Operation getötet.Die Knochenneubildung wurde im Röntgenbild, durch Standard-Histologie und durch Markierung mit Kalzein-Blau, Xylenol-Orange und Tetracyclin überprüft.In den Implantaten aus entkalktem Knochengranulat wurde bereits in der ersten Woche Knochenneubildung festgestellt und nach 9 Wochen fand sich in dem Defekt ein gut entwickeltes zylindrisches Knochenregenerat in 89% der Fälle. In einigen Fällen war sogar eine Markhöhle vorhanden.Aber auch in den Defekten, die leer gelassen oder mit unentkalktem Knochengranulat aufgefüllt worden waren, ließ sich Knochenbildung erkennen.

     

The osteoninductive property of decalcified bone matrix. An experimental study,.

Demineralised homologous bone-matrix implant was used to bridge a large circumferential osteoperiosteal gap in the diaphysis of the ulna of rabbits. Periodic observations of the graft were made clinically, radiologically, histologically and by tetracycline fluorescence up to forty-two weeks. By the twelfth week after operation 81 per cent of the animals revealed bone formation in the implant and complete bridging of the gap. The new bone was laid on the surface and in the substance of the matrix, suggesting that the inductive principle was acting locally. The bone, once formed, remodelled to the texture of a mature tubular bone and did not undergo absorption during a long follow-up period. Demineralise bone-matrix proved to be a highly osteoinductive and readily osteoconductive material. The graft did not evoke any appreciable local foreign-body or immunogenic reaction. The high degree of success in bridging massive bone defects justifies further serious studies and hopes for a useful substitute for massive autologous bone grafts.     

The clinical use of autologous marrow to improve osteogenic potential of bone grafts in pediatric orthopedics

The high osteogenic potential of living autologous marrow cells can be combined with foreign bone to enhance new bone formation. Xenogeneic bone was combined with autologous red marrow and used in 23 patients aged 5-17 years. Kiel bone was impregnated with marrow aspirated from the iliac crest and, apart from one case of lesion recurrence, gave excellent results in all patients under conditions covering a wide range of indications for bone grafting. Unlimited supplies of xenograft bone and other bone substitutes can be rendered osteogenic by a simple procedure. Combining fresh autologous red marrow with other types of bank bone allograft or xenograft, or even with biological or synthetic biocompatible material that favors the induction of new bone, may provide even better results.     

成型脱钙骨基质移植重建完全半月板缺损的实验研究

[目的]探索成型脱钙骨基质作为完全半月板缺损后替代假体的可行性，并进行移植假体引导自体组织再生效果的评价。[方法]脱钙骨基质按照Urist方法制备，并依据半月板的形状预制成型。48只新西兰大白兔随机分为脱钙骨基质移植组和脱细胞同种异体移植（冻融处理）对照组，并将两组按照三个不同时期分为3个配伍组。将各组动物分作关节功能评价，大体观察，组织学和免疫学观察；并进行统计分析。[结果]脱钙骨基质组回缩较小，细胞组织的增殖活性更高；两组总体和各期在移植体回缩率，免疫组织化学检查均有统计学意义。[结论]成型脱钙骨基质作为半月板假体较脱细胞同种异体半月板具有更好的性能结构和增殖活性。     

同种异体脱钙骨复合骨髓间充质干细胞在兔膝关节腔内培养组织工程软骨的研究

目的 比较同种异体脱钙骨和细胞因子诱导骨髓间充质干细胞(BMSC)制成细胞-支架复合物在兔膝关节腔内环境较体外培养出组织工程软骨的优点.方法 BMSC向软骨细胞诱导后与同种异体脱钙骨支架复合分别在体外培养(A组)和成年雄性新西兰白兔(15只)左侧膝关节腔内(B组)培养,右侧膝关节腔内(C组)培养单纯脱钙骨支架做空白对照.每4、8、12周各组标本分别取材,制石蜡切片行苏木素-伊红(HE)染色、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学等组织学观察,并通过形态学分析软件计算免疫组织化学平均光度(A)值.结果 标本石蜡切片HE染色:4周时A组标本见软骨细胞散在分布于支架表面,内部基本未观察到细胞.B组标本支架内软骨细胞数量明显较多,软骨陷窝形成,陷窝周围基质深染,可见由单个细胞分裂形成的同源细胞群.8周时B组标本以成熟软骨细胞为主,细胞数量多,出现柱状排列的同源细胞群,细胞周围分泌着色均一的玻璃样基质,且渗入支架结构内部,脱钙骨支架有部分被吸收.12周时各组支架结构明显被吸收,A组支架内填满软骨细胞,部分已纤维化,但结构排列紊乱.B组支架呈透明软骨样,软骨细胞充分渗透进入支架内,呈一定应力方向排列.Ⅱ型胶原免疫组织化学A值统计学分析比较发现:第4,8,12周B组Ⅱ型胶原免疫组织化学的A值均高于A组,差异有统计学意义(P＜0.01).两组间A值随时间的变化趋势差异有统计学意义(P＜0.01).结论 同种异体脱钙骨支架复合经细胞因子诱导的BMSC,在膝关节腔内进行培养,利用关节腔内低氧、多种生长因子的微环境以及关节活动时的应力刺激等优势,较体外培养可以培养出组织学特点更好的工程软骨.

Abstract：

Objective To compare the superiority of cultivating tissue engineered cartilage by homologous decalcified bone matrix combined with bone-marrow mesenchymal stem cells (BMSCs) in rabbits' knee cavity with culture in vitro. Methods Archiogeneration BMSCs were isolated and purified by adhering to the culture glassware wall from neonatal New Zealand white rabbits. The third generation BMSCs were induced to chondrocytes by transforming growth factor TGF-β1, insulin-like growth factor IGF-1 and vitamin C. Seven days later, the cells were bond to homologous decalcified bone matrix and cultured in vitro (group A) , cultured in rabbits' left knee cavity (group B) , or bond to homologous decalcified bone matrix and cultured in rabbits' right knee cavity ( group C ). Every 4 weeks after cellular transplant, all rabbits in groups B and C were sacrificed and paraffin-embedded sections were made from the specimens.All the section were subjected to H-E stain, toluidine blue stain and type Ⅱ collagen immunohistochemical staining with chromogen diaminobenzidine ( DAB) . Immunohistochemical absorbance ( A) values were calculated by morphology analysis software. Measurement data were expressed as mean ± standard, and satistical analysis was performed by t test, one-way ANOVA using SPSS 15. 0 software. Results After cultivation for 4 weeks, H-E stain showed diffuse distribution of chondrocytes on scaffold' s surface in group A.In group B, there were a great quantity of chondrocytes in the scaffold, and cartilage lacuna, matrix anachromasis and isogenous group were group. At the 8th week, in group B maturated chondrocytes and isogenous groups arranged as column, surrounded by a pond of cellular matrix infiltrating into the scaffolds. The decalcified bone matrix was absorbed partly. At the 12th week, all the scaffolds were absorbed obviously.Chondrocytes filled into the scaffolds in group A, but arranged irregularly. In group B chondrocytes arranged in the stress orientation. At 4th, 8th, and 12th week, A values of type Ⅱ collagen in group B was significantly higher than in group A with the different being significant. Conclusion Compound of Homologous decalcified bone matrix-induced BMSCs cultured in rabbits' knee cavity could yield better histological feature tissue engineered cartilage.     

地塞米松对骨髓间充质细胞增殖及成骨、成脂分化的效应

目的 探讨不同浓度地塞米松(dexamethasone,DEX)作用不同时间对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的增殖及成骨、成脂分化的影响.方法 体外分离培养大鼠BMSCs,将状态良好的第三代细胞接种于96孔板中,随机分组为对照组(不加入DEX)及不同浓度(1 nmol/L、10 nmol/L、0.1μmol/L、1μmol/L)DEX作用组,培养1、3、5、7、9 d后,分别用细胞增殖检测(CCK-8)法检测细胞增殖状况.将BMSCs接种于6孔板中,随机分为对照组及不同浓度(1 nmol/L、10 nmol/L、0.1μmol/L、1μmol/L)DEX作用组,培养7、14 d后分别用荧光定量PCR方法检测成骨、成脂相关基因的表达,培养14 d后用Western blot检测成骨、成脂相关蛋白的表达.培养5 d后进行碱性磷酸酶(alkaline phosphatase,ALP)染色,培养14 d后进行油红"O"染色、茜素红染色.结果 较低浓度的DEX促进BMSCs增殖,而较高浓度DEX抑制BMSCs增殖.干预早期,较低浓度的DEX促进BMSCs成骨分化而高浓度DEX抑制其成骨分化;干预晚期,各浓度DEX均促进BMSCs成骨指标的表达,但是不能诱导BMSCs钙质沉积.DEX浓度依赖性地促进BMSCs成脂相关指标基因和蛋白的表达,并诱导BMSCs细胞内脂质沉积.结论 DEX可直接影响BMSCs增殖及向成骨、成脂方向分化,这种效应存在浓度和干预时间依赖性.     

Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow.

Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age-related decrease in bone mass could reflect decreased osteoblasts secondary to an age-related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1-L5) from 41 donors (16 women and 25 men) of various ages (3-70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 x 106 cells/10-cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM-derived cells from donors of various ages was 5 x 106 cells/10-cm dish. BM-derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU-F/ALP+ present in the BM of the younger donors (3-36 years old) was 66.2 +/- 9.6 per 106 cells (mean +/- SEM), but only 14.7 +/- 2.6 per 106 cells in the older donors (41-70 years old). With longer-term culture (4-5 weeks) of these BM cells in medium containing 10 mM beta-glycerophosphate and 100 microg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU-F/ALP+) decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age-related osteoporosis.     

Nature of bone morphogenetic protein (BMP) from decalcified rabbit bone matrix

Rabbit bone morphogenetic protein (BMP) from demineralized and defatted rabbit bone matrix was partially purified. BMP activity was examined by the implantation of fractionated materials into the thigh muscle pouch of the mouse. Rabbit BMP was solubilized by both 4M guanidine hydrochloride (GuHCl) and 6M urea solutions. Crude BMP had isoelectric point precipitation at pH 3 in 6M urea and showed bone morphogenesis. Fractions eluted with 0 and 0.2 N NaCl in DEAE CL-6B ion exchange chromatography showed bone morphogenesis in each individual pH of pH 4 to pH 7 but the fraction eluted with 1.0 N NaCl did not show any activity. Sephadex G-75 filtration separated the crude material into three peaks and the peak of about 23,000 showed bone morphogenesis. In sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing, rabbit BMP was thought to be an acidic protein having a molecular weight of 24,000 with an isoelectric point around 4.85.     

The effect of noncoherent red light irradiation on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising for use in regenerative medicine. Low-level light irradiation (LLLI) has been shown to modulate various processes in different biological systems. The aim of our study was to investigate the effect of red light emitted from a light-emitting diode (LED) on bone marrow MSCs with or without osteogenic supplements. MSCs both with and without osteogenic supplements were divided into four groups, and each group was irradiated at doses of 0, 1, 2 and 4 J/cm². Cellular proliferation was evaluated using WST-8 and 5-ethynyl-2′-deoxyuridine (EdU) fluorescence staining. The alkaline phosphatase activity, mineralization, and expression of osteoblast master genes (Col1α1, Alpl, Bglap and Runx2) were monitored as indicators of MSC differentiation towards osteoblasts. In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of the MSCs was not enhanced. In groups with osteogenic supplements, red light increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of Bglap and Runx2, but decreased cellular proliferation. In conclusion, nonconherent red light can promote proliferation but cannot induce osteogenic differentiation of MSCs in normal media, while it enhances osteogenic differentiation and decreases proliferation of MSCs in media with osteogenic supplements.     

人骨髓间充质干细胞的生物学特性及成骨诱导分化的研究

[目的]探讨成人骨髓间充质干细胞分离、纯化、培养及鉴定的方法,观察其成骨分化过程中Runx2基因的动态表达以及生物学特性。[方法]取自人股骨近端骨髓标本,利用联合密度梯度离心和差异贴壁法分离骨髓间充质干细胞,体外扩增和传代培养,流式细胞仪检测细胞表面标记,诱导向成骨细胞分化,并采用RT-PCR和Western blot方法检测Runx2的动态表达。[结果]原代和传代细胞呈纺锤状外观,生长增殖能力良好,骨髓间充质干细胞的生长曲呈成"S"形,细胞表面标记物CD90阳性表达,CD34和CD45阴性表达。经定向诱导分化后,细胞分别呈现成骨细胞的表型特征,随着诱导时间的增加,Runx2的表达也明显增加,与对照组相比有统计学差异(P<0.05)。[结论]该方法能从人骨髓中高效分离和扩增MSCs,生物学性状稳定,具有成骨分化潜能,为骨组织工程提供理想的种子细胞,同时证实Runx2在成骨分化中起到重要的调控作用。     

The relationship between metabolic status and endochondral calcification in the process of bone development following decalcified bone matrix implantation

Experimental bone formation induced by decalcified bone matrix is an excellent model in vivo for studying endochondral bone formation. This study investigated the relationship between metabolic status and the onset of initial calcification in a decalcified matrix implanted subcutaneously in rat to characterize changes at discrete phases after implantation. Morphological evaluation and analyses of acid soluble mineral contents revealed that calcification took place on day 12 in regions of hypertrophic cartilage which were newly produced in the bone matrix. Levels of inorganic phosphate ions increased from day 6 and plateaued on day 9. Alkaline phosphatase activities peaked on day 12, and thereafter decreased with the progression of calcification. Energy charge ratio (ECR), which was considered to be an index reflecting the energy status of cells, was found to have shifted to lower values with the onset of calcification. These data support the hypothesis that the metabolic status of cells is closely associated with the beginning of calcification.     

冻干骨对间充质干细胞成骨能力的影响

目的：研究人冻干骨(FDB)对间充质干细胞(MSCs)成骨能力的影响。方法：通过体外细胞培养模型，以扫描电镜观察MSCs在FDB上的附着情况：将在体外孵育不同时间(2h、1、10d)的MSCs-FDB复合物植入裸鼠体内，分别在2、4和8周，对植入物进行组织学和形态计量学检查。结果：FDB表面及内部都有MSCs附着生长，孵育了不同时间的MSCs-FDB复合物，在裸鼠体内都有明显的成骨，骨量无明显差别。结论：FDB与MSCs有良好的相容性，MSCs在冻干骨上孵育不同的时间(10d内)，对其植入体内后的成骨能力没有明显影响。