脱钙骨基质/磷酸钙复合骨水泥骨诱导活性观察

目的:观察磷酸钙骨水泥(CPC)中加入脱钙骨基质(DBM)后形成的复合骨水泥的成骨诱导活性.方法:将DBM/CPC复合骨水泥分别植入兔背肌肌袋内,于不同时间取材,通过组织学切片、ALP等手段观察异位诱导成骨情况.结果:术后2周,DBM/CPC复合骨水泥组可见间充质细胞增殖、聚集并包绕DBM骨粒.4周时,DBM已有部分吸收,并软骨样细胞和软骨样组织包裹.8周,DBM进一步吸收,软骨细胞和软骨组织逐渐成熟,新骨形成.12周,DBM吸收并被新骨组织部分或大部分代替且相互连接成片.ALP测定结果与组织学观察的新骨形成情况基本一致.结论:DBM/CPC复合骨水泥有较强的异位诱导成骨能力,诱导新骨的形成伴随着材料的降解,可以有效弥补单纯使用CPC时降解速度太慢和无骨诱导能力的不足.     

脱钙骨基质/磷酸钙复合骨水泥骨诱导活性观察

目的：观察磷酸钙骨水泥（CPC）中加入脱钙骨基质（DBM）后形成的复合骨水泥的成骨诱导活性。方法：将DBM/CPC复合骨水泥分别植入兔背肌肌袋内，于不同时间取材，通过组织学切片、ALP等手段观察异位诱导成骨情况。结果：术后2周，DBM/CPC复合骨水泥组可见间充质细胞增殖、聚集并包绕DBM骨粒。4周时，DBM已有部分吸收，并软骨样细胞和软骨样组织包裹。8周，DBM进一步吸收，软骨细胞和软骨组织逐渐成熟，新骨形成。12周，DBM吸收并被新骨组织部分或大部分代替且相互连接成片。ALP测定结果与组织学观察的新骨形成情况基本一致。结论：DBM/CPC复合骨水泥有较强的异位诱导成骨能力，诱导新骨的形成伴随着材料的降解，可以有效弥补单纯使用CPC时降解速度太慢和无骨诱导能力的不足。     

三种骨移植材料的骨诱导活性对比实验研究

[目的]通过对人工骨硫酸钙(CS)、同种异体和异种脱矿骨(DBM)3种骨移植材料异位诱导成骨效应的观察,比较其骨诱导活性优劣,为临床选择合适的骨移植材料提供参考依据.[方法]采用大鼠股部肌袋内埋植实验.选择成年斯普拉大鼠共36只,随机分成A、B 2组,每组18只.A组左侧植入cs(A1),右侧植入同种异体DBM(A2);B组左侧植入异种DBM(B1),右侧不植入任何材料行空白对照(B2).通过异位诱导成骨实验,在植入后2、4、6周取材作大体、组织形态学观察及碱性磷酸酶(ALP)和钙离子生化测定,对不同材料的骨诱导活性进行评价.[结果]术后2周,2种DBM被纤维组织包裹,可观察到DBM骨片周围间充质细胞聚集;4周时可见软骨细胞、成骨细胞形成;6周时有类似软骨基质样物质形成;ALP和钙离子含量在各时间段均高于对照组,说明2种DBM具有骨诱导活性,其结果差异具有供体依赖性.而CS降解吸收快,炎症反应轻,未见诱导成骨现象.[结论]2种DBM具有骨诱导活性,同种异体DBM较好,异种DBM次之,供体的差异性和DBM的制备可影响其在体内的骨诱导活性.CS生物相容性较好,是可供选择的骨修复填充材料.     

一种新型生物活性人工骨的制备及成骨活性的研究

目的：研制CPC/BMP复合人工骨,检测其成骨活性。方法：制备CPC/BMP及CPC骨块,扫描电子显微镜观察表面结构。用小鼠肌袋植入实验观察材料的成骨活性。结果：BMP在CPC中呈微球状均匀分布。CPC植入小鼠肌袋内不能诱导,CPC/BMP植入后1周有软骨细胞出现,2周有编织骨,4周以后小梁骨生成,16周出现成熟的板层骨。同时材料出现降解迹象。有机质含量、碱性磷酸酶浓度在CPC/BMP组出现升高,扫描电镜结果同样证实有新骨形成。结论：CPC/BMP生物活性人工骨可异位诱导成骨,可望成为新型的骨缺损修复材料。     

一种新型可塑形骨填充材料的制备及骨修复能力研究

目的 制备一种黏性载体和成骨基质颗粒均来源于人骨的可塑形骨填充材料，通过动物实验评价其安全性和骨诱导能力。方法 取自愿捐赠的人体长骨，采用粉碎、清洗、脱矿等方法制备脱钙骨基质（demineralized bone matrix,DBM）颗粒，再将DBM颗粒经温浴法制备骨基质明胶（bone matrix gelatin,BMG），将BMG及DBM颗粒混合制备成可塑形骨填充材料（实验组）；以单纯DBM颗粒作为对照组。取15只6～9周龄健康雄性无胸腺裸小鼠，于两侧臀中肌与臀大肌间制备肌间空隙，均植入实验组材料，术后1、4、6周处死动物，行HE组织学染色评价异位成骨效果。取8只9月龄日本大耳兔，于两侧后肢髁骨处制备直径6 mm骨缺损，左、右侧分别填充实验组和对照组材料，术后12、26周处死动物，行Micro-CT及HE组织学染色评价骨缺损修复效果。结果 异位成骨实验HE染色示，术后1周可观察到大量软骨细胞，术后4、6周可观察到明显新生软骨组织。兔髁骨缺损修复实验HE染色示，术后12周，部分材料被吸收，实验组及对照组均可观察到新生软骨；术后26周，大部分材料被吸收，对照组可观察到大量新生骨，实验...     

磷酸钙水泥降解成骨机制的研究

目的: 研究磷酸钙水泥 (CPC) 降解吸收成骨的机制。方法: 制备羊胫骨平台压缩性骨折模型, 植入CPC填充骨缺损。3个月时拍X线片、取材制作不脱钙切片, 观察CPC的组织学变化。结果: CPC吸收迅速, X线片可见 2只动物几乎吸收完毕, 另 3只已大部被吸收。组织学观察可见到许多骨水泥吸收骨形成单位, 吸收的骨水泥由新生骨填充, 未见纤维组织形成。结论: CPC吸收成骨机制与正常骨重建过程相类似, 骨水泥吸收与新生骨形成同步协调进行。     

胶原支架增强自固化磷酸钙骨水泥的力学及成骨性能研究

[目的]研究胶原支架（CS）对磷酸钙骨水泥（CPC）的力学及其在体内成骨的影响。[方法]试验分CPC／CS及CPC两组，三点弯曲试验测试材料的强度和弹性模量；组织学观察材料植入兔股骨22及54周的成骨状况。[结果]CPC／CS比CPC的弯曲强度、韧性强度分别提高了64．2％、3933．3％，弹性模量降低了45．7％；组织学显示22周CPC／CS内的胶原支架已完全被新骨替代，CPC只在边缘有少量成骨及材料降解而内部无成骨；54周CPC／CS已大部分降解孔化，孔内充满大量新骨及髓样组织，而CPC边缘区的成骨及材料降解虽比22周时明显，但其内部仍未见成骨。[结论]胶原支架既可改善CPC的力学性能，又能促进新骨长入CPC／CS复合材料内部，因此，CPC／胶原支架是较好的骨缺损修复材料。     

可注射磷酸钙骨水泥填充固定羊胫骨平台骨折的体内生物力学研究

目的 比较磷酸钙骨水泥(CPC)与松质骨移植在修复羊外侧胫骨平台压缩性骨折中的体内生物力学特点.方法 成年健康山羊10只,双侧后肢制成外侧胫骨平台压缩性骨折模型,一侧以CPC填充(CPC组),对侧用自体骨填充(自体骨组).术后3个月和6个月各处死5只动物,取双侧胫骨观察关节面塌陷程度,摄X线片观察骨水泥降解情况,测定骨折部位抗压刚度,制备不脱钙切片观察关节面组织学改变.结果 自体骨组10个标本中的8个发生了骨折块的中重度下陷,CPC组只有3个发生了轻度下陷.X线片示3个月时骨水泥降解吸收被分割成几块,外形变模糊,6个月时大部分骨水泥被降解吸收.CPC组和自体骨组抗压刚度在3个月时分别为(226.89±62.81)N/mm和(172.97±79.82)N/mm,差异无统计学意义(t=1.369,P=0.243),6个月时分别为(471.27±212.89)N/mm和(371.05±207.98)N/mm,差异无统计学意义(t=0.948,P=0.397).组织学观察显示自体骨组关节面塌陷较严重,修复的软骨组织中纤维组织偏多;CPC组关节面软骨修复过程中对位较好,由纤维软骨形成相连接,纤维组织较少.结论 CPC的支撑效果好于自体骨,可在早期提供一定的抗压强度,防止胫骨平台关节面塌陷移位,有利于术后早期活动.     

硫酸钙骨水泥降解成骨性能的实验研究

目的通过对硫酸钙骨水泥（CSC）、磷酸钙骨水泥（CPC）及同种异体骨三种常用骨替代材料成骨性能的比较，研究CSC在兔松质骨内降解成骨的性能。方法16只成年白兔随机均分为A、B两组，A组兔的左、右侧髂骨内分别植入CSC、CPC，B组兔的左、右侧髂骨内分别植入CSC、同种异体骨；采用影像学、组织学及扫描电镜等方法在不同时间点（2、6、10、16周）观察和评价上述移植物在松质骨内的吸收降解和成骨情况。结果CSC置人6周被部分吸收，10周时完全吸收，新骨生成；CPC吸收速度缓慢，至植人体内16周时仅小部分被吸收；部分同种异体骨植骨区域出现强烈的免疫反应。结论CSC生物相容性较好，植入兔松质骨后的吸收速度快于CPC，生物降解与新骨生成同步进行，是一种良好的骨移植替代物。     

磷酸钙骨水泥作为骨形成蛋白载体修复节段性骨缺损及相关研究

目的 制备CPC/BMP复合人工骨，通过动物实验研究其对骨缺损的修复作用及相关问题，探讨临床应用的可能性。方法 参考有关文献方法合成CPC，并将其作为BMP的载体制成CPC/BMP复合物，植入兔桡骨15mm骨缺损处，术后不同时间处死动物。通过生物力学测定，组织学染色分析，电镜扫描及X射线电子能谱分析。X线摄片，无机质含量测定以及骨密度测定等手段观察新骨形成和材料降解情况。同时以单纯的CPC及空白组作为对照研究。综合评价CPC/BMP对骨缺损的修复能力及对机体的影响。结果 术后CPC/BMP和CPC两组动物均无毒性反应。随着时间的延长，血清中碱性磷酸酶浓度逐渐升高，尤以CPC/BMP组显著，提示CPC/BMP复合物和单纯的CPC均可以促进新骨形成，前者新骨形成量大，骨修复能力明显好于后者。CPC/BMP植入2周时可见大量间充质细胞分化，在材料与骨端之间出现一层软骨细胞。4周时软骨细胞向编织骨分化，16周时板骨层骨长人材料并与之相互分割包裹，24周时骨缺损初步修复，新骨密度明显高于CPC组，说明BMP的加入不仅有效地促进了新骨的形成，同时也加速了新骨的钙化。24周组标本生物力学测定结果表明，新骨形成的同时伴随材料的降解，CPC组材料降解速度缓慢，CPC/BMP组降解速度优于CPC组，但24周时仍有部分材料残存。在新骨形成和材料降解过程中可出现血清钙浓度的一过性升高。结论 CPC是BMP的理想载体。CPC/BMP生物活性人工骨对骨缺损有较强的修复能力，可望成为新型的骨缺损修复材料。     

兔自体骨髓间充质干细胞体内复合移植的成骨研究

目的 观察兔自体骨髓间充质干细胞（MSCs)体内复合移植的成骨能力，以寻求理想的MSCs载体。方法将10只新西兰大白兔随机分成A、B两组，从自体骨髓中分离出MSCs，体外培养并扩增后分别与相同大小的小牛脱钙骨（DBCB），自固化磷酸钙人工骨（CPC）复合移植于两组大白兔左侧骶棘肌中，同时右侧骶棘肌分别植入相同大小的DBCB，CPC作空白对照；16周后取出标本，观察成骨情况并行组织学检查。结果 A组5例：MSCs DBCB侧均发现有新骨组织形成，单纯DBCB侧3例被完全吸收，降解，2例大部分吸收，降解；B组5例；MSCs CPC侧和单纯CPC侧均未见骨组织形成，植入物也无明显吸收，结论 自体骨髓MSCs在适合载体负载下可在体内自动分化成骨，DBCB是MSCs的良好载体之一。     

Effects of Pore Size on the Osteoconductivity and Mechanical Properties of Calcium Phosphate Cement in a Rabbit Model

Calcium phosphate cement (CPC) porous scaffold is widely used as a suitable bone substitute to repair bone defect, but the optimal pore size is unclear yet. The current study aimed to evaluate the effect of different pore sizes on the processing of bone formation in repairing segmental bone defect of rabbits using CPC porous scaffolds. Three kinds of CPC porous scaffolds with 5 mm diameters and 12 mm length were prepared with the same porosity but different pore sizes (Group A: 200–300 µm, Group B: 300–450 µm, Group C: 450–600 µm, respectively). Twelve millimeter segmental bone defects were created in the middle of the radius bone and filled with different kinds of CPC cylindrical scaffolds. After 4, 12, and 24 weeks, alkaline phosphatase (ALP), histological assessment, and mechanical properties evaluation were performed in all three groups. After 4 weeks, ALP activity increased in all groups but was highest in Group A with smallest pore size. The new bone formation within the scaffolds was not obvious in all groups. After 12 weeks, the new bone formation within the scaffolds was obvious in each group and highest in Group A. At 24 weeks, no significant difference in new bone formation was observed among different groups. Besides the osteoconductive effect, Group A with smallest pore size also had the best mechanical properties in vivo at 12 weeks. We demonstrate that pore size has a significant effect on the osteoconductivity and mechanical properties of calcium phosphate cement porous scaffold in vivo. Small pore size favors the bone formation in the early stage and may be more suitable for repairing segmental bone defect in vivo.     

Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: I. Differences in the Cellular Response to Demineralized Bone Matrix Implanted in Calvarial Defects and in Subcutaneous Sites

The cellular and biochemical sequences of osteogenesis induced by implanting demineralized bone matrix (DBM) in rat cranial defects and in subcutaneous sites have been studied by histological, histochemical, and biochemical techniques from days 2 to 28 after implantation. In subcutaneous sites, allogenic DBM induced cartilage cells and matrix for approximately the first 10 days which were subsequently resorbed and replaced by bone with little evidence for the classical endochondral sequence of ossification. In sharp contrast, the first cells that differentiated from the mesenchymal stem cells in the cranial defects were alkaline phosphatase (ALP) positively stained osteoblasts that appeared 3 days after implantation followed by synthesis of bone matrix which calcified shortly thereafter. A few clusters of cartilage cells were observed beginning at days 6–7 which were spatially distinct from the new bone and later resorbed. By day 28 the tissue induced in both the subcutaneous and cranial sites consisted almost solely of bone; however, the total amount of new bone in the subcutaneous implants was significantly less than the mass of bone formed in the calvarial defects. Bovine DBM induced bone formation in rat cranial defects to a very much lesser extent than allogenic DBM. A few cartilage cells were induced by bovine DBM in subcutaneous sites and rapidly resorbed and not replaced with bone. These results clearly indicate that the cellular sequence induced by allogenic and xenogenic DBM and the repair tissues synthesized are distinctly different in the cranial defects from those induced in the subcutaneous sites. Received: 1 September 1998 / Accepted: 15 January 1999     

The Effect of Poloxamer 407-Based Hydrogel on the Osteoinductivity of Demineralized Bone Matrix

Background

Demineralized bone matrix (DBM) is used for bone healing due to its osteoinductivity, but it requires a carrier for clinical application. Here, we report the effects on the osteoinductivity of DBM by use of a poloxamer 407-based hydrogel as the carrier, compared to sterile water.

Methods

DBM-W and DBM-H represent 27 wt% of DBM with sterile water and DBM with a poloxamer 407-based hydrogel, respectively. Both of the compositions were applied to human mesenchymal stem cell (MSC) cultures, and monitored for alkaline phosphatase (ALP) staining and ALP activity. Six 10-week-old athymic nude rats were used for abdominal muscle grafting with either DBM-W or DBM-H, and were tested by plane radiography, microfocus X-ray computed tomography (CT), and decalcified histology to evaluate ectopic bone formation.

Results

The DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, and significantly higher ALP activity at 7 and 14 days of treatment, compared to the DBM-H group. Plane radiography could not confirm the radio-opaque lesions in the rat ectopic bone formulation model. However, ectopic bone formation was observed in both groups by micro-CT. Compared to the DBM-H group, the DBM-W group showed higher bone volume, percent bone volume and trabecular number, and the difference in percent bone volume was statistically significant. Decalcified histology found bony tissue with lamellation in both groups.

Conclusions

Our results suggest that poloxamer 407-based hydrogel has efficacy as a DBM carrier since it shows ectopic bone formation, but its effects on the quality and quantity of osteoblastic differentiation in rat abdominal ectopic bone and MSC are considered negative.     

温敏性高黏度几丁聚糖水凝胶复合脱钙骨基质修复兔膝全层软骨缺损的初步研究

目的将高黏度几丁聚糖水凝胶(high viscous chitosan/glycerol phosphate,HV-C/GP)与异体脱钙骨基质(demineralized bone matrix,DBM)颗粒复合,评估修复兔膝全层软骨缺损的效果。方法将HV-C/GP与DBM按2∶1(W/W)复合,制备HV-C/GP-DBM复合物。取成年健康34周龄新西兰大白兔24只,体重3.5～4.5kg,于两侧股骨髁间窝制备直径为3.5mm、深度为3mm的软骨全层缺损区。右侧缺损区植入HV-C/GP-DBM复合物作为实验组;左侧不作处理,作为对照组。术后4、8、16周,分别处死动物取材,行大体及组织学观察。根据国际软骨修复协会组织学评分(International Cartilage Repair Society Histological Scoring,ICRS)标准对第16周标本评分,评估软骨修复效果。结果大体及组织学观察:术后4、8周,实验组缺损区大部分被透明软骨样组织修复,DBM颗粒部分吸收;对照组缺损区可见少量纤维组织及软骨细胞;术后16周,实验组6例关节表面平滑,缺损区已基本被透明软骨样软骨组织修复,缺损区与周围软骨整合较好,新骨形成活跃,部分DBM颗粒未被爬行替代;对照组仍存在凹陷,修复组织为纤维组织。术后16周ICRS评分结果示,实验组的软骨细胞生成以及与周围软骨整合等6个方面均优于对照组,差异有统计学意义(P<0.05)。结论HV-C/GP-DBM复合物组织相容性好,可降解,可注射,是一种较好的软骨修复材料。     

Bone marrow enriched graft,modified by self-assembly peptide,repairs critically-sized femur defects in goats

Purpose

This study focuses on nanoscale self–assembly peptides (SAP) modified demineralized bone matrix (DBM) which provided a more effective osteogenesis and regeneration for critically-sized femur defects in goats using the selective cell retention (SCR) strategy.

Methods

RADA16–I peptide was used to modify DBM and formed a composite scaffold (SAP/DBM). The morphological change and dynamic expression of osteogenic genes of mesenchymal stem cells (MSCs) derived from marrow in SAP/DBM was observed. The cells and factors in bone marrow were enriched into SAP/DBM by technology of selective cells retension (SCR). The construct was transplanted into 20-mm femur defects in goats and their osteogenesis was evaluated.

Results

The SAP/DBM scaffold formed a three-dimensional interweaving nanofiber in pores of DBM. MSCs exhibited better morphology in SAP/DBM than that in only DBM, and the levels of expression of ALP ,OCN and Runx2 gene in SAP/DBM samples was significantly higher than that of DBM at 14 days in vitro (P?P?Conclusion Our study may not only have a significant impact on the construction method of tissue engineering but also provide a viable, simple and effective method for clinical bone construction.     

Use of a guanidine extract of demineralized bone in the treatment of osteochondral defects of articular cartilage

In order to evaluate the ability of a guanidine extract of demineralized bone to repair osteochondral defects in articular cartilage, plugs made of this extract were implanted into defects in rabbit knees. The repair tissue was examined macroscopically, histologically, and immunohistochemically at 4, 8, 12 and 30 weeks. Controls (defects that were left empty) showed no cartilage formation. Four weeks after implantation of a guanidine extract plug, histological examination showed a nonhomogeneous metachromatically stained region extending from the surface of the repair tissue down to cancellous bone. This region also was labeled by an anti-type-II collagen antibody, indicating that cartilage-like tissue had been induced. At 8 weeks, the newly formed cartilage in the subchondral and cancellous bone had been partially replaced by bone. At 12 weeks, the thickness of the newly formed cartilage layer had decreased, and most of the newly formed cartilage in the subchondral and cancellous bone had been replaced by bone. In addition, a tidemark was observed. At 30 weeks, the repair tissue was a mixture of cartilage and fibrocartilage, and there was severe degeneration of the cartilage surrounding the repaired defects. These findings indicate that osteochondral defects of articular cartilage can be partially repaired by the implantation of a guanidine extract and that the newly formed cartilage-like tissue is not permanent.