PRP复合间充质干细胞在颌骨组织工程领域的应用

组织工程骨修复骨缺损是目前研究的热点,种子细胞、生长因子以及生物支架材料是构建组织工程骨的三大要素。由于间充质干细胞（mesenchymal stem cells,MSCs）具有多向分化潜能,能向成骨细胞分化,取材容易,体外易扩增,免疫原性低等优点,是理想的种子细胞。富血小板血浆（platelet rich plasma,PRP）中富含大量自体源性生长因子,能诱导MSCs增殖分化,促进骨再生。PRP复合MSCs行组织工程化骨具有一定可行性,已应用于颌骨缺损的修复、牙周组织再生以及种植外科等领域的研究。本文就PRP复合MSCs在口腔颌面骨组织工程领域的应用现状作一综述。     

骨收集器收集自体骨充填种植体周围骨缺损的实验研究

目的:研究利用植入钻孔时骨收集器收集的骨碎末修复种植体周围骨缺损的可行性。方法:以5只家犬双侧下颌骨为实验对象,拔除双侧各3颗前磨牙3月后,行种植术,每侧植入4颗种植钉。种植时人为造成种植体颊侧2mm×2mm大小骨缺损,在同体同侧的4个骨缺损中的3个,分别植入自体骨屑、Bio-Oss骨粉、二者1∶1混合骨末,剩余1处不植入任何材料作为对照。9周后进行各组间比较。结果:植入的自体骨碎屑修复了一定的骨缺损,自体骨混合Bio-Oss组骨缺损恢复最佳。各组与对照组比较均存在统计学差异。结论:植入钻孔时收集的自体骨碎屑对于修复种植体周围少量的骨缺损,是完全可行的。当供骨量不足时,自体骨碎末可以混合适当的Bio-Oss颗粒植入,效果更为理想。     

Healing of Fresh Frozen Bone Allograft with or without Platelet‐Rich Plasma: A Histologic and Histometric Study in Rats

Purpose: This study histomorphometrically analyzed the effect of autogenous platelet‐rich plasma (PRP) on healing of fresh frozen bone allograft (FFBA) in bony defects in rat calvaria. Materials and Methods: A 5 mm–diameter defect was created in the calvarium of 30 rats. Animals were divided into three groups: C (defect was filled by blood clot only), FFBA (defect was filled with 0.01 mL of FFBA), and FFBA/PRP (defect was filled with 0.01 mL of FFBA combined with 100 µL of PRP). All animals were euthanized at 30 days postoperatively. Histomorphometry and histology analyses were performed. Data were statistically analyzed (analysis of variance, Tukey, p < .05). Results: FFBA had a statistically smaller new bone area than groups FFBA/PRP and C. No statistically significant differences were observed between groups FFBA and FFBA/PRP with regard to remaining bone graft particle area. Conclusion: It can be concluded that (1) PRP improved the incorporation of FFBA, increasing the amount of new bone formed; (2) PRP has not influenced the resorption of nonviable particles of the FFBA; and (3) presence of remaining FFBA particles might have accounted for the smaller amount of new bone observed in group FFBA when compared with control group.     

Bone healing in critical-size defects treated with platelet-rich plasma activated by two different methods. A histologic and histometric study in rat calvaria

Background and Objective: The purpose of this study was to analyze histologically the influence of platelet‐rich plasma (PRP) coagulated with two different activators on bone healing in surgically created critical‐size defects (CSD) in rat calvaria. Material and Methods: Forty‐eight rats were divided into three groups: C, PRP‐C and PRP‐T. An 8 mm diameter CSD was created in the calvarium of each animal. In group C, the defect was filled by a blood clot only. In groups PRP‐C and PRP‐T, the defect was filled with PRP activated with either calcium chloride or thromboplastin solution, respectively. Each group was divided into two subgroups (n = 8 per subgroup) and killed at either 4 or 12 weeks postoperatively. Histologic and histometric analyses were performed. The amount of new bone formed was calculated as a percentage of the total area of the original defect. Percentage data were transformed into arccosine for statistical analysis (analysis of variance, Tukey’s post hoc test, p < 0.05). Results: No defect completely regenerated with bone. Group PRP‐C had a statistically greater amount of bone formation than groups C and PRP‐T at both time points of analysis. No statistically significant differences were observed between groups C and PRP‐T. Conclusion: It can be concluded that the type of activator used to initiate PRP clot formation influences its biological effect on bone healing in CSD in rat calvaria.     

Marginal Bone‐Level Alterations at Implants Installed in Block versus Particulate Onlay Bone Grafts Mixed with Platelet‐Rich Plasma in Atrophic Maxilla. A Prospective 5‐Year Follow‐Up Study of 15 Patients

Background: Extensive atrophy of the alveolar process may require a bone‐grafting procedure prior to implant treatment. Autogenous bone grafts from the iliac crest, used as onlay block and particulate bone, have been used together with sinus‐lift procedure in order to rehabilitate patients with extremely resorbed maxillae. However, there are to our knowledge no 5‐year follow‐up studies evaluating the extent of bone‐level change in patients treated with respectively block and particulate autogenous bone grafts. Purpose: The purpose of this prospective clinical study was to conduct a 5‐year follow‐up analysis with focus on bone‐level alteration in block versus particulate onlay bone grafts. Material and Methods: Fifteen out of originally 19 patients who were treated with iliac bone grafts and oral implants in the maxilla have been followed through the first 5 postoperative years. In a first study conducted on 19 patients, the role of platelet‐rich plasma in conjunction with autogenous bone was evaluated. In this 5‐year follow‐up study, the marginal bone alterations have been documented at base line, 1 year and 5 years of loading to the nearest 0,1 mm at mesial and distal surfaces of the implants. Two implants were installed on each side of the midline in either block or particulate bone grafts giving test and control sides in each patient. Additionally, two implants on each side were installed in residual bone/grafted sinus floor. Result: Marginal bone alteration in the anterior maxilla appeared larger at the side augmented by block bone at baseline, and after 1 and 5 years of loading, but the change was not statistically significant. Moreover, there was a significantly higher degree of marginal alteration during the first year of loading, compared with the examinations after 5 years. Conclusion: The present follow‐up study showed that there is no significant difference in the extension of resorption between block‐ and particulate autogenous bone grafts over a 5‐year period. Most of the resorption occurred during the first year in function.     

钛膜和胶原膜联合应用引导种植体周骨缺损骨再生的临床研究

目的：评价钛膜与胶原膜联合应用引导种植体骨缺损骨再生的临床效果。方法：将34颗种植体植入30例患者的狭窄形牙槽嵴或唇颊骨壁缺损拔牙窝,所有种植体的唇、颊侧面部分暴露,种植体周骨缺损空间维持能力较差。测量种植暴露部分的最大长度,将羟基磷灰石珊瑚骨粉置于骨缺损处,采用钛膜覆盖稳定骨移植材料,然后将胶原膜覆盖于钛膜表面,无张力缝合伤口,术后6个月行Ⅱ期手术,取下钛膜,检查骨缺损骨再生的状况,再次测量种植暴露部分的最大长度。结果：2例患者于手术3个月左右因钛膜局部暴露,将钛膜取出。钛膜暴露率为6.6%。术后6个月Ⅱ期手术时见,所有种植体暴露部分完全被再生骨覆盖,种植体暴露部分长度为0。结论：在空间维持能力较差的骨缺损处,钛膜和胶原膜联合应用引导骨再生可获得理想结果。钛膜和胶原膜联合应用可显著降低钛膜的暴露率,延迟发生膜暴露的时间,从而使引导骨再生的结果更加具有可预测性。     

BMSCs复合PRP修复牙种植体周围骨缺损的实验研究

目的:探讨骨髓基质细胞(bone marrow stromal cells,BMSCs)复合富血小板血浆(platelet-rich plasma,PRP)修复牙种植体周围骨缺损的可行性。方法:拔除6只Beagle犬双侧下颌8颗前磨牙,术后1个月抽取犬骨髓,采用全骨髓贴壁法培养BMSCs,并向成骨细胞诱导,将BMSCs与PRP、β-磷酸三钙(β-tricalcium phosphate,β-TCP)分别复合形成组织工程化复合物。术后2个月,在实验犬每侧下颌缺牙区分别植入BLB4mm×11mm种植体4颗,同时在每个种植体的近中制造4mm×4mm×5mm大小的骨缺损,分别植入BMSCs-PRP复合凝胶、自体骨、BMSCs-β-TCP复合物以及空白对照,分别于种植术后4、8、12周各处死2只犬,行大体、环境扫描电镜及组织学检查,观察各个时期各组骨缺损区的成骨情况以及新生骨与种植体间骨结合情况。结果:3个实验组在术后4、8、12周均有不同程度的骨组织再生,而空白对照组只有少量骨组织再生。各个时期内扫描电镜观察显示BMSCs-PRP组新生骨与种植体间的骨结合较其他三组多。结论:BMSCs-PRP复合凝胶是一种良好的骨修复材料,具有促进新骨形成及种植体骨结合的效应。     

Relative Contributions of Osteogenic Tissues to New Bone Formation in Periosteal Distraction Osteogenesis: Histological and Histomorphometrical Evaluation in a Rat Calvaria

Background: The relative contributions of different, potential factors to new bone formation in periosteal distraction osteogenesis are unknown. Purpose: The aim of the present study was to assess the influence of original bone and periosteum on bone formation during periosteal distraction osteogenesis in a rat calvarial model by means of histology and histomorphometry. Methods: A total of 48 rats were used for the experiment. The contribution of the periosteum was assessed by either intact or incised periosteum or an occlusive versus a perforated distraction plate. The cortical bone was either left intact or perforated. Animals were divided in eight experimental groups considering the three possible treatment modalities. All animals were subjected to a 7‐day latency period, a 10‐day distraction period and a 7‐day consolidation period. The newly formed bone was analyzed histologically and histomorphometrically. Results: New, mainly woven bone was found in all groups. Differences in the maximum height of new bone were observed and depended on location. Under the distraction plate, statistically significant differences in maximum bone height were found between the group with perforations in both cortical bone and distraction plate and the group without such perforations. Conclusions: If the marrow cavities were not opened, the contribution to new bone formation was dominant from the periosteum. If the bone perforations opened the marrow cavities, a significant contribution to new bone formation originated from the native bone.     

Five‐Year Results Evaluating the Effects of Platelet‐Rich Plasma on the Healing of Intrabony Defects Treated With Enamel Matrix Derivative and Natural Bone Mineral

Background: Regenerative periodontal surgery using the combination of enamel matrix derivative (EMD) and natural bone mineral (NBM) with and without addition of platelet‐rich plasma (PRP) has been shown to result in substantial clinical improvements, but the long‐term effects of this combination are unknown. Methods: The goal of this study was to evaluate the long‐term (5‐year) outcomes after regenerative surgery of deep intrabony defects with either EMD + NBM + PRP or EMD + NBM. Twenty‐four patients were included. In each patient, one intrabony defect was randomly treated with either EMD + NBM + PRP or EMD + NBM. Clinical parameters were evaluated at baseline and 1 and 5 years after treatment. The primary outcome variable was clinical attachment level (CAL). Results: The sites treated with EMD + NBM + PRP demonstrated a mean CAL change from 10.5 ± 1.6 to 6.0 ± 1.7 mm (P <0.001) at 1 year and 6.2 ± 1.5 mm (P <0.001) at 5 years. EMD + NBM–treated defects showed a mean CAL change from 10.6 ± 1.7 to 6.1 ± 1.5 mm (P <0.001) at 1 year and 6.3 ± 1.4 mm (P <0.001) at 5 years. At 1 year, a CAL gain of ≥4 mm was measured in 83% (10 of 12) of the defects treated with EMD + NBM + PRP and in 100% (all 12) of the defects treated with EMD + NBM. Compared to baseline, in both groups at 5 years, a CAL gain of ≥4 mm was measured in 75% (nine of 12 in each group) of the defects. Four sites in the EMD + PRP + NBM group lost 1 mm of the CAL gained at 1 year. In the EMD + NBM group, one defect lost 2 mm and four other defects lost 1 mm of the CAL gained at 1 year. No statistically significant differences in any of the investigated parameters were observed between the two groups. Conclusions: Within their limits, the present results indicate that: 1) the clinical outcomes obtained with both treatments can be maintained up to a period of 5 years; and 2) the use of PRP does not appear to improve the results obtained with EMD + NBM.     

煅烧骨复合PRF对牙周骨缺损的实验研究

目的:研究比较异种煅烧骨和Bio-oss骨分别与富血小板纤维蛋白(platdet-rich fibrin,PRF)联合,及其各自单独使用对牙周骨缺损修复的效果。方法:制备煅烧骨,PRF凝胶及其复合物。选54只新西兰大白兔,制备牙周组织缺损模型,随机分成空白对照组、PRF凝胶组、煅烧骨组、Bio-oss骨组、煅烧骨/PRF凝胶组、Bio-oss骨/PRF凝胶组,将这些材料植入牙周缺损区,每组9只兔子。术后4周、8周和l2周处死动物,获取完整标本,观察影像学和组织学。行组织学染色检测,测量新生牙周组织的高度。结果:X线结果表明:4周、8周和l2周煅烧骨/PRF 组和Bio-oss/PRF 组平均密度均明显高于对照组且缺损处骨组织连续。组织学观察:实验组均在牙根面可见新生牙骨质样组织、牙周膜和牙槽骨样组织的形成,随着时间的推移逐渐成熟、增多。12周时,煅烧骨/PRF凝胶、Bio-oss骨/PRF凝胶较空白对照组明显提高,较其他3组差异有统计学意义(P<0.05)。结论:煅烧骨/PRF凝胶、Bio-oss骨/PRF凝胶复合物可明显促进牙周组织的再生。     

自体髂骨游离移植同期种植体植入修复骨量不足区牙缺失的动物实验研究

目的:利用自体髂骨游离移植一期植入种植体的动物实验研究,揭示自体髂骨游离移植同期植入种植体的愈合过程,为临床工作提供理论依据和借鉴。方法:选择16只健康成年雄性日本大耳白兔,在双侧髂骨制备髂骨缺损模型,游离移植自体髂骨,并同期分别在双侧髂骨植骨区及非植骨区植入自制圆柱状羟基磷灰石种植体。随机分成4组,每组4只。术后2周、4周、8周、12周各处死一组动物,切取标本。进行大体标本观察,放射线、组织学(脱钙HE染色)、扫描电镜检查、力学测试。结果:大体标本观察见各组种植体和髂骨结合紧密。肉眼见种植体和髂骨之间多为骨性结合,仅2周标本及4周实验组标本一些区域可见一薄层纤维样组织。放射线检查:2周标本可见有放射线透射区,实验组密度稍低于对照组密度;余各组种植体周围均未见明显的放射线透射区;4周标本实验组密度稍低于对照组;8周标本、12周标本实验组密度与对照组密度基本无差别。组织学及扫描电镜检查实验组与对照组种植体均与骨质形成不同程度的骨结合;对照组种植体比实验组形成更好的骨结合;各组种植体4周、8周、12周依次形成更好的骨结合。力学测试(反向推出实验)显示2周、4周、8周、12周骨结合实验组最大负荷分别是66.3N/cm2、143.9N/cm2、194.6N/cm2、248.3N/cm2。对照组最大负荷分别是108.6N/cm2、229.2N/cm2、307.1N/cm2、377.6N/cm2。结论:自体髂骨游离移植同期种植修复骨量不足区牙缺失是切实可行的修复方法。     

Clinical and radiographic evaluation of the efficacy of platelet-rich plasma combined with hydroxyapatite bone graft substitutes in the treatment of intra-bony defects in maxillofacial region

Objective. The aim of this study is to evaluate the efficacy of platelet-rich plasma (PRP) clinically and radiographically when combined with bovine derived hydroxyapatite (HA) bone grafting materials and resorbable collagen membranes for the treatment of intra-bony defects frequently seen at the distal aspect of mandibular second molars following the surgical extraction of fully impacted mandibular wisdom teeth. Study design. Eighteen patients were scheduled for post-operative visits at 1, 3 and 6 months post-operatively, probing depths were measured and digital panoramic radiographs were taken. Results. There were no significant differences on probing depths among two groups. Radiographic assessment also showed no significant difference among groups at 1st and 6th month intervals, while 3 months post-operatively the amount of radiographic density at the PRP side was significantly higher. Conclusion. Combined use of PRP and bovine-derived HA graft materials for the treatment of intra-bony defects might be an appropriate approach when the main goal is providing earlier bone regeneration.     

Effect of Doxycycline in Gel Form on Bone Regeneration: Histomorphometric and Tomographic Study in Rat Calvaria

Background: This study aims to investigate the effects of the local application of doxycycline in the form of natrosol‐based gel on bone regeneration by examining critical defects in rat calvaria. Methods: Twenty‐four rats were randomly divided into three groups with eight animals each, according to the treatment received: BC (untreated, filled with blood clot), NAT (natrosol gel alone), and DOX (10% doxycycline gel). Four animals from each group were euthanized at 4 and 8 weeks postoperatively. In tomographic analysis, mean density in the region of the defect was calculated as a percentage relative to the native bone density. In histomorphometric analysis, the newly formed bone area was calculated as a percentage of the total area. The values obtained underwent analysis of variance and Tukey testing (P <0.05). Results: The mean percentage of tomographic density in the region of the defect at the end of 8 weeks was higher for DOX (44.37%), and statistical differences in this period were observed between DOX and NAT (33.10%) and DOX and BC (32.43%). Regarding new bone formation, at the end of 8 weeks, DOX (61.11%) also had the highest mean bone formation, and statistical differences were observed between DOX and NAT (34.61%) and DOX and BC (23.11%). Conclusion: It was observed that 10% doxycycline gel had a good effect on bone regeneration regarding the filling of critical defects in rat calvaria.     

Combination of Controlled Release Platelet‐Rich Plasma Alginate Beads and Bone Morphogenetic Protein‐2 Genetically Modified Mesenchymal Stem Cells for Bone Regeneration

Background: Platelet‐rich plasma (PRP) consists of platelet‐derived growth factor and transforming growth factor‐β that increase proliferation of mesenchymal stem cells (MSCs), whereas bone morphogenetic protein‐2 (BMP2) promotes osteogenic differentiation of MSCs. However, the high degradation rate of fibrin leads to the dissociation of cytokines even before the process of bone regeneration begins. To the best of the authors’ knowledge, this is the first study to examine the combined effect of sustained release of PRP from alginate beads on BMP2‐modified MSC osteogenic differentiation in vitro and sustained release of PRP alone on a fracture defect model ex vivo as well as its effect on calvarial suture closure. Methods: After optimizing the alginate concentration for microspheres, the combined osteogenic and mineralization effect of PRP and BMP2 on MSCs was studied. Self‐setting alginate hydrogel carrying PRP was tested on a femur defect model ex vivo. The effect of PRP at day 15 on the closure of the embryonic mouse calvaria sutures ex vivo was also studied. Results: Increase of PRP concentration promoted proliferation of MSCs, and 2.5% to 10% of PRP gradually increased alkaline phosphatase (ALP) activity in the cells in a dose‐dependent manner. Sustained release of PRP and BMP2 demonstrated significantly higher ALP and mineralization activity (P <0.05). Radiographs of alginate hydrogel with PRP‐treated bone demonstrated nearly complete healing of the fracture, and histologic sections of the embryonic calvaria revealed that PRP leads to suture fusion. Conclusion: Sustained release of PRP along with BMP2‐modified MSCs can significantly promote bone regeneration.