Primate dentine extracellular matrix induces bone differentiation in heterotopic sites of the baboon (Papio ursinus)

In rodents, demineralized dentine matrix induces local differentiation of endochondral bone. This study investigated the osteoinductive potential of primate dentine matrices when implanted extraskeletally in allogeneic recipients. Demineralized dentine cylinders prepared from adult baboon incisors and demineralized dentine matrix pulverized to a particle size of 74-420 microns were implanted into the rectus abdominis of 4 subadult male baboons (Papio ursinus). Specimens were harvested 30 and 90 d after implantation. Histological analysis on serial sections showed bone differentiation in demineralized dentine cylinders after partial resorption of the external demineralized layer, and in resorption lacunae and excavation chambers within the matrix. Implants of demineralized dentine matrix of 74-420 microns particle size showed no osteoinductive activity as determined biochemically (alkaline phosphatase activity) and histologically. The demonstration of bone induction by primate dentine prepared from fully erupted tooth matrix suggests that putative osteogenic proteins may be conserved after dentinogenesis and embryonic tooth development, and may play a role during healing after surface demineralization of exposed root surfaces during regenerative procedures in humans.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblast differentiation and suppresses matrix metalloproteinase-1 production in human bone cells isolated from mandibulae

Bone morphogenetic protein (BMP), a member of the transforming growth factor superfamily, is one of the most potent growth factors that stimulate osteoblast differentiation and bone formation. We investigated the effects of recombinant human BMP-2 (rhBMP-2) on osteoblast differentiation and matrix metalloproteinase-1 (MMP-1) production in human bone cells (HBC) isolated from mandibulae of 3 adult patients. rhBMP-2 at concentrations over 50 ng/ml significantly stimulated alkaline phosphatase activity and parathyroid hormone (PTH)-dependent 3′, 5′-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in HBCs. rhBMP-2 (500 ng/ml) also enhanced the level of PTH/PTH related-peptide receptor mRNA expression in HBCs. Although neither HBCs untreated nor treated with rhBMP-2 produced measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D₃ [l,25(OH)₂D₃] induced ostocalcin mRNA expression and its protein synthesis in these cells. rhBMP-2 inhibited l,25(OH)₂D₃-induced osteocalcin synthesis in HBCs at both the mRNA and protein level. rhBMP-2 also significantly suppressed MMP-1 production and MMP-1 mRNA expression at concentrations over 500 ng/ml. These results suggest that rhBMP-2 exerts anabolic effects on human osteoblastic cells derived from mandibulae by stimulation of osteoblast differentiation and down-regulation of MMP-1 synthesis.     

脱细胞脱钙人牙用于大鼠颅盖骨缺损修复的实验研究

目的: 探讨脱细胞脱钙牙作为骨支架材料的可行性和诱导大鼠颅盖骨缺损成骨能力。方法: 离体人牙24颗,分别进行脱钙和脱钙脱细胞处理。实验1—取6只10~12周龄雄性SD大鼠,在大鼠腹部皮下同时包埋脱钙牙、脱细胞脱钙牙,4周后处死大鼠,进行H-E染色,观察2种材料周围炎症反应情况。实验2—取12只10~12周龄雄性SD大鼠,制备双侧颅盖骨区域骨缺损,在左侧骨缺损处植入脱细胞脱钙牙,以未植入任何材料的右侧作为空白对照,4周、8周时分别处死6只,获取完整标本,进行Micro-CT扫描,从大体形态学上比较成骨量。取4周大鼠进行H-E染色,观察周围炎症反应,免疫组织化学观察新生成骨程度。采用SPSS 23.0软件包进行重复测量方差分析。结果: 实验1—4周时H-E染色显示,脱细胞脱钙牙与周围组织炎症反应程度显著小于脱钙牙。实验2—取4周和8周的大鼠颅盖骨进行Micro-CT扫描和骨缺损处新生骨量定量分析,显示植入脱细胞脱钙牙的骨缺损处成骨量显著多于空白对照组量（P<0.05）。4周时H-E染色结果显示,脱细胞脱钙牙周围的炎症反应程度与空白对照处相似。免疫组织化学染色显示,脱细胞脱钙牙周围新生的成骨细胞显著多于空白对照组。结论: 脱细胞脱钙人牙是一种具有良好生物相容性、成骨诱导能力和可塑性的生物支架材料。     

Guided bone regeneration using demineralized allogenic bone matrix with calcium sulfate: case series

PURPOSE

The purpose of this case series was to evaluate the effect of guided bone regeneration using demineralized allogenic bone matrix with calcium sulfate.

MATERIALS AND METHODS

Guided bone regeneration using Demineralized Allogenic Bone Matrix with Calcium Sulfate (AlloMatrix™, Wright. USA) was performed at the time of implant placement from February 2010 to April 2010. At the time of the second surgery, clinical evaluation of bone healing and histologic evaluation were performed. The study included 10 patients, and 23 implants were placed. The extent of bony defects around implants was determined by measuring the horizontal and vertical bone defects using a periodontal probe from the mesial, distal, buccal, and lingual sides and calculating the mean and standard deviation of these measurements. Wedge-shaped tissue samples were obtained from 3 patients and histologic examination was performed.

RESULTS

In clinical evaluation, it was observed that horizontal bone defects were completely healed with new bones, and in the vertical bone defect area, 15.1% of the original defect area remained. In 3 patients, histological tests were performed, and 16.7-41.7% new bone formation was confirmed. Bone graft materials slowly underwent resorption over time.

CONCLUSION

AlloMatrix™ is an allograft material that can be readily manipulated. It does not require the use of barrier membranes, and good bone regeneration can be achieved with time.     

犬脱钙骨基质复合骨髓间充质干细胞的体外培养

目的:制备犬脱钙骨基质(demineralized bone matrix,DBM),研究DBM作为骨组织工程支架材料的可行性。方法:杂种犬的股骨行脱脂、脱钙、脱非胶原蛋白、冷冻干燥、灭菌制成DBM,与骨髓间充质干细胞(mesenchymal stemcells,MSCs)复合,应用倒置相差显微镜、扫描电镜观察脱钙骨基质的结构及细胞在材料表面的生长情况。结果:犬脱钙骨基质具有三维立体网孔结构,平均孔隙直径为(254.39±88.71)μm,孔隙率约为70%。与MSCs复合培养后,细胞黏附其上,上架率高,生长状态良好,增殖迅速,并分泌大量细胞外基质。结论:DBM具有良好的生物相容性。     

组织工程骨修复9例颅骨缺损畸形初步报道

目的：探讨利用自体骨髓间充质干细胞（BMSC）作为种子细胞的组织工程骨修复外伤后颅骨缺损畸形的可行性。方法：运用自体BMSC组织工程骨技术,对9例外伤术后颅骨缺损患者行颅骨修复手术。抽取患者自体骨髓,密度梯度离心法分离BMSC,经体外扩增和成骨诱导后,接种部分脱钙骨支架材料,构建组织工程骨。细胞材料复合物体外共培养1周后,手术植入颅骨缺损区。分别于术后1周和3、6、12个月进行临床外形和三维CT检查,随访治疗效果。结果：术后1周三维CT显示,颅骨缺损区被所植入的组织工程骨填充,头颅外形明显改善。术后3至12个月CT显示,组织工程骨形成并修复颅骨缺损,新生骨与骨缺损断端融合。9例患者中2例术后失访,2例高龄患者及大面积骨缺损患者发生不同程度的组织工程骨吸收现象,其余均良好地修复了颅骨缺损。结论：通过选择适宜的病例,以自体BMSC作为种子细胞,运用组织工程技术可以在人体内形成稳定的组织工程化骨并修复颅骨缺损。     

Histomorphometrical analysis of bone formed in human maxillary sinus floor elevations grafted with OP-1 device, demineralized bone matrix or autogenous bone. Comparison with non-grafted sites in a series of case reports

Bone morphogenetic proteins have proven to be effective bone inductors in animals and are therefore promising as inductors of bone formation in humans. In the present study we investigated the tissue formed after grafting osteogenic protein 1 on a collagen carrier (OP-1-device) in the human sinus floor elevation procedure. Three patients were grafted with OP-1 device. For comparison 3 groups of 3 patients were included in the study receiving respectively, autogenous bone, human freeze-dried demineralized bone matrix (DBM) or no graft. This last group had a sufficient alveolar bone height for dental implantation. Six months after grafting, at the time of implantation, biopsies were taken from the grafted area and/or the future dental positions. Undecalcified sections were used for histological and histomorphometrical analysis. All grafted sinuses showed an increased osteoid percentage when compared to non-grafted sinuses. Autogenous bone grafts all showed lamellar bone formation. In the DBM grafts mostly woven bone had been formed, predominantly by what appeared to be osteo-conduction. The OP-1 device gave rise to bone formation in 2 of the 3 patients. After 6 months implants could only be placed in 1 out of the 3 patients treated with OP-1 device. This patient showed mature lamellar bone formation, comparable to autogenous bone grafts. In the second patient all bone found was woven and the presence of a high osteoid percentage and large osteocyte lacunae indicated that this was recently-formed bone. Remnants of the collagen carrier were rare and new bone was never found against them, suggesting that this bone was formed by osteo-induction. In the third patient no new bone had been formed. The device had been encapsulated with fibrous tissue and inflammatory reaction was present. We conclude that in the human sinus floor elevation OP-1 has potential bone inductive capacity, but that results in the 3 patients tested with the current OP-1 device are inconsistent.     

脱矿牙本质基质材料在颌骨骨缺损修复的应用

颌骨缺损会导致颌面部的功能及形态的异常。所以术后的骨缺损修复是必要的。为了避免自体骨移植所带来的二次创伤,寻找优良的骨替代材料是目前的研究热点。牙本质材料因为其易获取,制备简单,成骨效果良好而获得许多关注。并且学者发现脱矿后的牙本质成骨能力更为显著。该文将对脱矿牙本质材料的成骨基质及临床应用作一综述。     

The effect of Emdogain on ectopic bone formation in tubes of rat demineralized dentin matrix

BACKGROUND: Emdogain (EMD) is made from enamel matrix proteins (EMPs) from the tooth germ of swine and propylene glycol alginate (PGA) as a matrix. The function of EMD is known to differentiate cells of the dental follicle into cementoblasts. However, little is known about the effect of EMD on mesenchymal cells in other tissue. OBJECTIVE: The purpose of this study was to investigate whether EMD has the ability to induce hard tissue when applied with or without demineralized dentin matrix. METHODS: Half of the dentin tubes prepared from rat incisors were demineralized by treatment with 0.6 N hydrochloric acid for 3 h. EMD or PGA was injected into the demineralized or non-demineralized dentin tubes, which were then transplanted into rectus abdominis muscles. Untreated dentin tubes were also transplanted as a control. Animals were killed at 7, 14 and 21 days after the implantation. RESULTS: Non-demineralized dentin tubes with or without EMD or PGA did not form any hard tissue. In the demineralized group, chondrogenesis in the PGA groups occurred earlier than in the EMD groups. The expression of vascular endothelial growth factor (VEGF) mRNA in the demineralized group with PGA at day 14 was the highest. The expression of osteopontin and osteocalcin mRNAs was higher in all groups at 21 days compared with 7 or 14 days. CONCLUSION: These results suggest that neither EMD nor PGA has the ability to induce hard tissue and that EMPs contained within EMD might aggregate on the dentin surface and inhibit the effect of the demineralized dentin matrix.     

Denaturation of demineralized bone matrix significantly reduces bone formation by guided tissue regeneration

AIM: To examine in a discriminating capsule model whether denaturation of demineralized bone matrix (DBM) by heating may influence bone formation. MATERIALS AND METHODS: DBM was produced from the long bones of rats. Half the portion of DBM was denatured by heating in distilled water for 20 min at temperatures between 70 degrees C and 90 degrees C. Prior to the study, the destruction of the osteoinductive properties of the DBM was confirmed in three rats following intramuscular implantation. Thirty, 4-month-old, male albino rats of the Wistar strain were used in the study. Following surgical exposure of the mandibular ramus, a hemispherical Teflon capsule (internal diameter = 5.0 mm) was placed, with its open part facing the lateral aspect of the ramus. On one side (test side), the capsule was loosely packed with denatured DBM, while on the contralateral side, serving as control, the capsule was loosely packed with the same amount of non-denatured DBM. After healing periods of 30, 60, and 120 days, groups of 10 animals were killed and 40-70 microm thick undecalcified sections of the capsules were produced. Three sections from each specimen, representing the mid-portion of the capsule, were subjected to histological analysis and computer-assisted planimetric measurements. RESULTS: Increasing amounts of newly formed bone were observed in both test and control capsules during the experimental period. At 4 months, the new bone formed in the control capsules occupied 46.7% of the cross-sectional area of the capsules, while it was only 19.1% in the test capsules (P<0.05). CONCLUSION: Denaturation of DBM by heating significantly reduces bone formation by guided tissue regeneration.     

糖尿病对大鼠牙槽骨缺损修复中骨形态发生蛋白-2表达影响的研究

目的 观察糖尿病(diabetes mellitus,DM)对实验性大鼠牙槽骨缺损修复过程中不同时期骨形态发生蛋白-2(bonemorphogenetic protein-2,BMP-2)表达的影响。方法 将48只雄性SD大鼠随机分为DM组和对照组,每组24只,DM组大鼠经腹腔注射链脲佐菌素造成DM大鼠模型,建模成功后行大鼠牙槽骨骨缺损制备,2组均分别于骨缺损制备后1、2、4、8周各取6只大鼠处死,取术区组织。苏木精-伊红染色(hematoxylin-eosin staining,HE染色)镜下观察缺损区内新生骨样组织形成情况;用免疫组化法检测术后1、2、4、8周缺损区内BMP-2的表达情况,比较各组的平均光密度。结果 HE染色观察显示DM组成骨较对照组减少。术后1周、2周免疫组化观察对照组新骨BMP-2的表达强于DM组,二者之间差异有统计学意义(P<0.05),术后4周、8周对照组BMP-2表达较之前弱,但与DM组二者之间差异无统计学意义(P>0.05)。结论 DM影响了大鼠牙槽骨缺损的修复,可能是DM使BMP-2形成减少,从而抑制了未分化间充质向成骨细胞的转化,从而影响种植体骨结合,降低种植初期稳定性。?     

珊瑚、异体脱钙骨、自体骨髓复合可注射骨替代物异位成骨的研究

目的 :检验珊瑚 (NC)、异体脱钙骨基质 (DBM )、自体骨髓 (AM )复合可注射骨替代材料的异位诱导成骨能力。方法 :将 2 4只新西兰兔随机分为 4组 ,分别在背部皮下注射NC/DBM /AM、NC/DBM、NC/AM、NC ,术后 2、4周取材 ,通过X线、组织学检查、碱性磷酸酶 (ALP)测定 ,进行比较观察。结果 :X线、组织学检查结果均表明NC/DBM /AM组有较多新骨形成 ,NC/AM组诱导出少量骨 ,NC/DBM组仅见微量软骨和骨新生 ,NC组未见新生骨。NC/DBM /AM组ALP水平较NC/DBM组、NC/AM组、NC组高 ,统计学上具有显著性差异 (P <0 .0 1)。结论 :NC/DBM /AM复合可注射骨替代材料具有良好的骨诱导性和临床应用价值     

牙本质胞外基质对人血管内皮细胞增殖的影响

目的：探讨牙本质胞外基质成分(dentine extracellular matrix components，DM)对血管内皮细胞增殖的影响。方法：用100g／L EDTA，在pH7．2和蛋白酶抑制剂存任的条件下，从健康人牙中提取DM。第3代人脐静脉内皮细胞(human umbilical vein endothelial cell，HUVEC)，存含有不同浓度DM培养液中培养8d，每2天计数1次，绘制生长曲线。并对第8天对照(不含DM培养)与实验组的细胞数进行Mann—Whitney统计分析。结果：HUVEC在DM浓度为0.0001、0．001和0．01mg／mL的培养液中培养时较对照分别增长了49％(P=0．0006)、21％和9％，在DM浓度为0.1和1mg／mL时，减少22％和74％(P=0．0003)。结论：DM对血管内皮细胞增殖具有计量依赖型影响效应。低浓度DM可刺激内皮细胞的增殖，而高浓度DM则可抑制该细胞的增殖。提示：这一计量依赖型效应可能正是牙髓损伤后造成局部不同的血管形成方式，并最终导致不同修复结果的原因。     

原核表达重组人骨形态发生蛋白-7对牙槽骨修复再生的实验研究

目的研究原核表达重组人骨形态发生蛋白- 7（rhBMP- 7）对牙槽骨修复再生的影响。方法采用新西兰白兔建立动物模型,以明胶海绵作为载体,将大肠杆菌原核表达的rhBMP- 7与之复合后植入即刻拔除牙齿的牙槽窝内进行干预治疗,术后2、4、8及12周处死动物,取标本进行大体观察、扫描电镜分析、碱性磷酸酶（ALP）活性测定。结果大体观察结果显示,实验组和对照组牙槽嵴高度的吸收差异有显著性;扫描电镜观测显示,实验组的骨创愈合比对照组大约提前4～6周;ALP活性测定显示实验组均明显高于对照组,其差异有统计学意义（P<0.05）。结论原核表达rhBMP- 7具有促进新骨修复再生、防止牙槽骨吸收的作用。     

The effect of enamel matrix proteins and deproteinized bovine bone mineral on heterotopic bone formation

To evaluate the osteoinductive potential of deproteinized bovine bone mineral (DBBM) and an enamel matrix derivative (EMD) in the muscle of rats. Sixteen rats were used in this study. The animals were divided in three groups. Group A: a pouch was created in one of the pectoralis profundis muscles of the thorax of the rats and DBBM particles (Bio-Oss) were placed into the pouch. Healing: 60 days. Group B: a small pouch was created on both pectoralis profundis muscles at each side of the thorax midline. In one side, a mixture of EMD (Emdogain) mixed with DBBM was placed into one of the pouches, whereas in the contralateral side of the thorax the pouch was implanted with DBBM mixed with the propylene glycol alginate (PGA--carrier for enamel matrix proteins of EMD). Healing: 60 days. Group C: the same procedure as group B, but with a healing period of 120 days. Qualitative histological analysis of the results was performed. At 60 days, the histological appearance of the DBBM particles implanted alone was similar to that of the particles implanted together with EMD or PGA at both 60 and 120 days. The DBBM particles were encapsulated into a connective tissue stroma and an inflammatory infiltrate. At 120 days, the DBBM particles implanted together with EMD or PGA exhibited the presence of resorption lacunae in some cases. Intramuscular bone formation was not encountered in any group. The implantation of DBBM particles alone, combined with EMD or its carrier (PGA) failed to exhibit extraskeletal, bone-inductive properties.     

Activated adult human alveolar bone cells: a new model of matrix mineralization

Activated adult human alveolar bone cells were isolated from 2-wk-old osteogenic tissue recuperated from dental implant surgeries following a two-step procedure. Osteogenic tissues were cultured as explant for 2 months. Cells began to migrate in the first 3 d and were confluent in 3–4 wk. However, adjacent to the explants, multicellular cell layers began to form in 10 d, and matrix mineralization was observed by 4 wk in these areas. These formations enlarged and by the end of the culture period, large diffuse matrix mineralization areas were observed. Light and electron microscopic observations confirmed the presence of a collagen matrix undergoing a mineralization process but showing important differences with the mineralized matrix tissue formed with a rat embryo calvaria bone cell system. This new model, using activated human alveolar bone cells, may provide a tool to investigate alveolar bone development and physiology and to set up new therapeutic approaches.     

Clinical application of autogenous partially demineralized dentin matrix prepared immediately after extraction for alveolar bone regeneration in implant dentistry: a pilot study

The aim of this study was to examine the efficacy and safety of autogenous partially demineralized dentin matrix (APDDM) prepared onsite, for clinical application in bone regeneration procedures related to implant dentistry, including socket preservation, alveolar ridge augmentation, and maxillary sinus floor augmentation. In this study, 16 patients underwent dental implant placement using APDDM transplantation. There were no systemic or local complications (including surgical site infection) in any of the cases, and oral rehabilitation using dental implants was successful in all cases for at least 2 years after attachment of the suprastructure. This report describes the clinical application of APDDM prepared immediately after tooth extraction to bone augmentation, taking advantage of the relatively short preparation time due to partial demineralization. APDDM, as introduced in this study, is an efficient, safe, and reasonable bone substitute. Consequently, this material has the potential to become one of the options as a bone substitute in implant dentistry.     

Histologic comparison of healing after tooth extraction with ridge preservation using mineralized versus demineralized freeze-dried bone allograft

Background: Allografts, such as demineralized freeze‐dried bone allograft (DFDBA) and mineralized freeze‐dried bone allograft (FDBA) are commonly used by clinicians for ridge preservation procedures. The primary objective of this study is to histologically evaluate and compare the healing of non‐molar extraction sockets grafted with DFDBA versus FDBA for ridge preservation. The secondary aim of this study is to compare dimensional changes in ridge height and width after grafting with these two materials. Materials: Forty patients were randomly divided into two groups of 20. Extraction sockets were filled with either FDBA or DFDBA. To minimize variables associated with the organ donor and with tissue processing, all of the graft material was procured from a single donor; the only difference in the two materials was the percentage mineralization of the final bone graft. A 2‐mm‐diameter core biopsy was taken from each grafted site ≈19 weeks after grafting. Histomorphometric analysis was performed to determine percentage of vital bone, residual graft particles, and connective tissue (CT)/other non‐bone components. Results: There were no significant differences when comparing changes in alveolar ridge dimensions of the two groups. There was no significant difference in percentage CT/other between groups. DFDBA had a significantly greater percentage of vital bone at 38.42% versus FDBA at 24.63%. The DFDBA group also had a significantly lower mean percentage of residual graft particles at 8.88% compared to FDBA at 25.42%. Conclusion: This study provides the first histologic and clinical evidence directly comparing ridge preservation with DFDBA versus FDBA in humans and demonstrates significantly greater new bone formation with DFDBA.