Choukroun富血小板纤维蛋白修复兔颅骨骨缺损的实验研究
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摘要
种植义齿植入过程中牙槽骨的缺损及骨量的不足影响了种植义齿适应症的选择和术后的美观效果,加大了手术失败的风险。利用自体血制备的血小板浓缩制品解决骨量不足的方法简单经济,取材方便,利于推广。Choukroun富血小板纤维蛋白( Choukroun’s platelet-rich fibrin , Choukroun’s PRF)被称为新一代血小板浓缩制品,它克服了以往血小板浓缩制品的诸多弊病,有望在临床得到推广应用。但是其相关研究尚处于起步阶段,报道文献仅见于法国和印度,在我国尚无报告,未引起广泛的临床关注。
本研究的目的在于采用Choukroun’s PRF修复标准化的动物骨缺损模型的实验,以验证Choukroun’s PRF具有的良好的成骨能力,观察其潜在的不良反应,并以此为基础,提供将其推广应用的临床指导。
本研究在利用Choukroun’s PRF修复大耳白兔颅骨缺损的实验观察中发现:在骨愈合期间,Choukroun’s PRF可以有效的促进骨缺损区纤维网架的形成及修复相关细胞的迁移、增殖和分化,显著促进新骨的形成与改建。此外,Choukroun’s PRF可以独立作为移植材料修复骨缺损,并且在一定程度上减轻炎症反应引起的水肿等症状。
Background:
Oral implantology initiates a brand-new era of dentition defect restoration and broadens the domain of stomatology. Nowadays, oral implant has already became a clinical tendency of dentition defect restoration. However, lots of limits confine the application of the dental implant during the clinical operations. One of the most distinct problems is the bone defect or inadequate bone quantities found during operations. According to the physical and pathological process of the bone regeneration, there appears lots of clinical methods to these clinical problems, of which the most outstanding one is the application of bone grafts. Attributed to the decades of research, many technologies have already been applied in order to remedy the bone defect, like bone transplantation and platelet concentrates. Whereas these methods are available, some disadvantages like secondary operation site、operation trauma and immuno-reject reaction restrict their utilization. Varieties of platelet concentrates catch dentists’eyes in dental implant clinic for many years. Choukroun’s platelet-rich fibrin (Choukroun’s PRF) that is known as the second generation platelet-concentrate was first developed by Choukroun in French in 2000. According to literatures, there are several advantages as follows: Simple preparation that can be manipulated before or during the surgery without any additives like anti-coagulate agents or blood preparation will not trigger any cross contamination; It is nothing but the centrifuged blood that is regarded as economical and convenient. The preparation process simulates the natural process of coagulation from which we can harvest the fibrin similar with that in natural coagulate clot. Choukroun’s PRF is rich in platelets and kinds of cytokines that can enhance healing ability. Inflammation factors and regenerative mediators released by immunocytes have the ability of regulation of inflammation reactions and the ability of anti-infection. Literatures about the establishment of standard bone defect animal model are seldom, and the delineation of histological process is not completed. Considering the potential unstable factors, we make the rabbits as the objects and research on the bone regeneration ability of Choukroun’s PRF in order to confirm it.
Objective:
We are aimed to reveal the therapeutic effect of Choukroun’s PRF on bone defect and explore the possible bone regeneration mechanisms initiated by Choukroun’s PRF.
Methods:
9 healthy rabbits that are 3 months old are chosen regardless of sex and the average weight is between 2.5-3.0Kg. They are divided into 4 weeks group、6 weeks group and 8 weeks group at random and there were 3 rabbits in each. Firstly expose and separate the jugular vein, draw 5ml blood with 10ml syringe, and then transfer the blood into a sterile tube rapidly under general anesthesia. Secondly, centrifuge the tube and then drive the serum to shape the glue. It requires noting additives, including anti-coagulate agent. Drill 4 standard defects(8mm×5mm×2mm)on each animal’s skull and then place the PRF in the defect sites on one side as the experimental group and place noting on the another side as control group zoned by middle cranial suture. 4、6、8 weeks later, animals are respectively sacrificed and samples are collected. Soft x-ray and histological stain are applied to the tissue blocks. The photography films are analyzed by Image-Pro Plus 6.0 software to measure the new bone area. And then, we manage the data with statistical software SPSS 13.0. Make the assessment of new bone in defect area according data.
Results:
Soft x-ray shows that the new bone area is significantly larger in experimental group than in control group in 4 weeks、6 weeks and 8 weeks (p<0.05). 4 weeks group shows the best effect (p<0.01). HE staining shows that bone heals better in experimental group than in control group in 4 weeks、6 weeks and 8 weeks. Central area of bone defect disappeared in experimental groups within 6 weeks compared with nearly 8 weeks in control groups. The density and shape of bone trabacula、volume and structure of new bone and volume of osteoblasts are all better in experimental groups than that in control groups. Modified Gomori staining shows that fibrous component of new bone is less and less and the extent of bone mineralization is more and more significant. Besides, though the structure of new bone inclines to mature lamellar bone both in experimental groups and control groups, experimental groups show a better maturation.
Discussion:
The mechanisms referred to the bone regeneration can be described as bone healing mechanisms: formation of coagulate clot、fibro-callus formation and osseo-callus formation and remodeling of callus. This experiment reveales that experimental groups show significant bone regeneration effect after the PRF placement in bone defect on animal’s skull by means of soft X-ray and histological stain. During the healing process, fibrin network provides histocytes a shelter of migration、reproduction and differentiation. Varieties of platelets and circulatory molecules are enmeshed in the 3-dimentional network, like TGF-β1、PDGF-AB、VEGF and IGF-Ⅰ. During the degradation, the concentrated platelets release kinds of cytokines which regulate and accelerate healing by specific effect and synergistic effect. Known as a kind of blood- derived product, Choukroun’s PRF has a degradation period, during which the bone regeneration effect can exist persistently. Because of the degradation, the bone regeneration effect will be weaker and weaker and then disappear. Compared with PRP, the bone regeneration effect keeps longer which shows the property of long acting. There appears no symptom of infection and edema of all animals during the experiment. It means that Choukroun’s PRF has the capacity of regulating inflammatory reaction to remedy the symptoms and decrease the infection with enhancing the anti-infection ability. Choukroun’s PRF can be used in the procedure as graft without anything accessory. It means that Choukroun’s PRF can not only accelerate the healing of bone but also induce the bone regeneration. None of the objective animals appears adverse reactions like hypersensitivity、intoxication, which means the safety and feasibility of Choukroun’s PRF can be confirmed. The data drew from our experiment can guide the clinical application to some extent. Take the extent of bone regeneration initiated by Choukroun’s PRF、effective period and method of assessing level of platelets and cytokines as examples, whereas the available conclusions, there are still something to be done to achieve the related researches.
Conclusion:
Through the experimental study, we initiated the study on Choukroun’s PRF in oral implantology domain in China. In addition, we described the histological process of bone regeneration by use of Choukroun’s PRF, and drew
conclusions as follows:
1、Choukroun’s PRF can enhance the bone regeneration;
2、Choukroun’s PRF can be applied alone as graft;
3、Choukroun’s PRF can act best at the early stage of the bone healing;
4、Choukroun’s PRF can remedy the inflammatory symptoms and ace- lerate the healing process of wound.
Taking all into account, we draw a conclusion that Choukroun’s PRF has the ability of enhancing the bone regeneration, which shows great potential in clinical utilization and extensive exploitation prospect.
本研究的目的在于采用Choukroun’s PRF修复标准化的动物骨缺损模型的实验,以验证Choukroun’s PRF具有的良好的成骨能力,观察其潜在的不良反应,并以此为基础,提供将其推广应用的临床指导。
本研究在利用Choukroun’s PRF修复大耳白兔颅骨缺损的实验观察中发现:在骨愈合期间,Choukroun’s PRF可以有效的促进骨缺损区纤维网架的形成及修复相关细胞的迁移、增殖和分化,显著促进新骨的形成与改建。此外,Choukroun’s PRF可以独立作为移植材料修复骨缺损,并且在一定程度上减轻炎症反应引起的水肿等症状。
Background:
Oral implantology initiates a brand-new era of dentition defect restoration and broadens the domain of stomatology. Nowadays, oral implant has already became a clinical tendency of dentition defect restoration. However, lots of limits confine the application of the dental implant during the clinical operations. One of the most distinct problems is the bone defect or inadequate bone quantities found during operations. According to the physical and pathological process of the bone regeneration, there appears lots of clinical methods to these clinical problems, of which the most outstanding one is the application of bone grafts. Attributed to the decades of research, many technologies have already been applied in order to remedy the bone defect, like bone transplantation and platelet concentrates. Whereas these methods are available, some disadvantages like secondary operation site、operation trauma and immuno-reject reaction restrict their utilization. Varieties of platelet concentrates catch dentists’eyes in dental implant clinic for many years. Choukroun’s platelet-rich fibrin (Choukroun’s PRF) that is known as the second generation platelet-concentrate was first developed by Choukroun in French in 2000. According to literatures, there are several advantages as follows: Simple preparation that can be manipulated before or during the surgery without any additives like anti-coagulate agents or blood preparation will not trigger any cross contamination; It is nothing but the centrifuged blood that is regarded as economical and convenient. The preparation process simulates the natural process of coagulation from which we can harvest the fibrin similar with that in natural coagulate clot. Choukroun’s PRF is rich in platelets and kinds of cytokines that can enhance healing ability. Inflammation factors and regenerative mediators released by immunocytes have the ability of regulation of inflammation reactions and the ability of anti-infection. Literatures about the establishment of standard bone defect animal model are seldom, and the delineation of histological process is not completed. Considering the potential unstable factors, we make the rabbits as the objects and research on the bone regeneration ability of Choukroun’s PRF in order to confirm it.
Objective:
We are aimed to reveal the therapeutic effect of Choukroun’s PRF on bone defect and explore the possible bone regeneration mechanisms initiated by Choukroun’s PRF.
Methods:
9 healthy rabbits that are 3 months old are chosen regardless of sex and the average weight is between 2.5-3.0Kg. They are divided into 4 weeks group、6 weeks group and 8 weeks group at random and there were 3 rabbits in each. Firstly expose and separate the jugular vein, draw 5ml blood with 10ml syringe, and then transfer the blood into a sterile tube rapidly under general anesthesia. Secondly, centrifuge the tube and then drive the serum to shape the glue. It requires noting additives, including anti-coagulate agent. Drill 4 standard defects(8mm×5mm×2mm)on each animal’s skull and then place the PRF in the defect sites on one side as the experimental group and place noting on the another side as control group zoned by middle cranial suture. 4、6、8 weeks later, animals are respectively sacrificed and samples are collected. Soft x-ray and histological stain are applied to the tissue blocks. The photography films are analyzed by Image-Pro Plus 6.0 software to measure the new bone area. And then, we manage the data with statistical software SPSS 13.0. Make the assessment of new bone in defect area according data.
Results:
Soft x-ray shows that the new bone area is significantly larger in experimental group than in control group in 4 weeks、6 weeks and 8 weeks (p<0.05). 4 weeks group shows the best effect (p<0.01). HE staining shows that bone heals better in experimental group than in control group in 4 weeks、6 weeks and 8 weeks. Central area of bone defect disappeared in experimental groups within 6 weeks compared with nearly 8 weeks in control groups. The density and shape of bone trabacula、volume and structure of new bone and volume of osteoblasts are all better in experimental groups than that in control groups. Modified Gomori staining shows that fibrous component of new bone is less and less and the extent of bone mineralization is more and more significant. Besides, though the structure of new bone inclines to mature lamellar bone both in experimental groups and control groups, experimental groups show a better maturation.
Discussion:
The mechanisms referred to the bone regeneration can be described as bone healing mechanisms: formation of coagulate clot、fibro-callus formation and osseo-callus formation and remodeling of callus. This experiment reveales that experimental groups show significant bone regeneration effect after the PRF placement in bone defect on animal’s skull by means of soft X-ray and histological stain. During the healing process, fibrin network provides histocytes a shelter of migration、reproduction and differentiation. Varieties of platelets and circulatory molecules are enmeshed in the 3-dimentional network, like TGF-β1、PDGF-AB、VEGF and IGF-Ⅰ. During the degradation, the concentrated platelets release kinds of cytokines which regulate and accelerate healing by specific effect and synergistic effect. Known as a kind of blood- derived product, Choukroun’s PRF has a degradation period, during which the bone regeneration effect can exist persistently. Because of the degradation, the bone regeneration effect will be weaker and weaker and then disappear. Compared with PRP, the bone regeneration effect keeps longer which shows the property of long acting. There appears no symptom of infection and edema of all animals during the experiment. It means that Choukroun’s PRF has the capacity of regulating inflammatory reaction to remedy the symptoms and decrease the infection with enhancing the anti-infection ability. Choukroun’s PRF can be used in the procedure as graft without anything accessory. It means that Choukroun’s PRF can not only accelerate the healing of bone but also induce the bone regeneration. None of the objective animals appears adverse reactions like hypersensitivity、intoxication, which means the safety and feasibility of Choukroun’s PRF can be confirmed. The data drew from our experiment can guide the clinical application to some extent. Take the extent of bone regeneration initiated by Choukroun’s PRF、effective period and method of assessing level of platelets and cytokines as examples, whereas the available conclusions, there are still something to be done to achieve the related researches.
Conclusion:
Through the experimental study, we initiated the study on Choukroun’s PRF in oral implantology domain in China. In addition, we described the histological process of bone regeneration by use of Choukroun’s PRF, and drew
conclusions as follows:
1、Choukroun’s PRF can enhance the bone regeneration;
2、Choukroun’s PRF can be applied alone as graft;
3、Choukroun’s PRF can act best at the early stage of the bone healing;
4、Choukroun’s PRF can remedy the inflammatory symptoms and ace- lerate the healing process of wound.
Taking all into account, we draw a conclusion that Choukroun’s PRF has the ability of enhancing the bone regeneration, which shows great potential in clinical utilization and extensive exploitation prospect.
引文
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[2] Assoian RK, Grotendorst GR, Miller DM, Sporn MB. Cellular transformation by coordinated action of three peptide growth factors from human platelets [J]. Nature, 1984,309:804-806.
[3] Wrotniak M, Bielecki T, Gazdzik TS. Current opinion about using the platelet-rich gel in orthopaedics and trauma surgery [J]. Ortopedia Traumat- ologia Rehabilitacja, 2007,9(3):227-238.
[4] Man D, PloskerH, Winland-Brown JE. The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibringlue) in cosmetic surgery [J]. Plast Reconstr Surg, 2001,107(1):229-237.
[5] Mooren RE, Merkx MA, Bronkhorst EM, et al. The effect of platelet- rich plasma on early and late bone healing: an experimental study in goats [J]. Oral Maxillofac Surg, 2007,36(7):626-631.
[6] Plachokova AS, van den Dolder J, Stoelinga PJ, Jansen JA. The bone regenerative effect of platelet-rich plasma in combination with an osteoconductive material in rat cranial defects [J]. Clinical Oral Implants Research, 2006,17(3):305-311.
[7] Gerard D, Carlson ER, Gotcher JE, Jacobs M. Effects of platelet-richplasma on the healing of autologous bone grafted mandibular defects in dogs [J]. Oral Maxillofac Surg, 2006,64(3):443-451.
[8] Jensen SS, Broggini N, Weibrich G, Hjorting-Hansen E, Schenk R, Buser D. Bone regeneration in standardized bone defects with autografts or bone substitutes in combination with platelet concentrate: a histologic and histomorphometric study in the mandibles of minipigs [J]. International Journal of Oral & Maxillofacial Implants, 2005,20(5):703-712.
[9] Vasconcelos, Gurgel BC, Goncalves PF, Pimentel SP, et al. Platelet-rich plasma may not provide any additional effect when associated with guided bone regeneration around dental implants in dogs [J]. Clin Oral Implants Res, 2007,18(5):649-654.
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