富含血小板血浆应用于骨组织工程的研究
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摘要
由肿瘤、创伤和感染等原因造成的骨缺损,给患者的生存质量带来很大的影响。常用的修复方法如自体、异体骨移植、人工替代物等,都有一定的局限性。所以,骨缺损的修复和治疗一直是困扰临床医生的一个难题。而骨组织工程技术的出现和发展,为解决这些问题提供了可能。
骨组织工程技术研究领域主要包括种子细胞、支架材料、生长因子三方面内容。经过近20年的研究,骨组织工程研究已经取得了长足的进展。在长期的骨组织工程研究过程中,人们逐渐认识到无创、微创的重要性,以及应该尽量避免使用异体、异种材料,并已经在进行积极的研究和探索。
富含血小板血浆(platelet rich plasma, PRP),是全血通过离心、浓集而得到的成分血液制品。其特点是其中血小板浓度高,并含有多种生长因子以及丰富的纤维蛋白网络;通过与凝血酶复合,纤维蛋白彼此铰链,它还可以形成凝胶。
本课题充分利用PRP的这些特点,制各及检测PRP,并以PRP为支架、用注射方式构建组织工程骨,使PRP同时成为骨组织工程研究的支架材料和生长因子的提供者;而且,在构建组织工程骨的过程中,可以实现无创或微创的目标。
第一部分 富含血小板血浆的制备及检测
实验一 富含血小板血浆的制备及血小板、TGF-β浓度检测
Bone defects caused by tumor, trauma and infection diseases are very familiar. The ordinary reconstructive ways, such as autograft, allograft and artifical grafts can solve these problems in a way, but have their limitations. Autogenous bone grafting, thought to be an ideal form, cannot be used to reconstruct extensive bone defect because of limitation of bone that can be harvested. This method also has the drawback of causing damage to normal bone and soft tissue. Allogeneic bone can be obtained in sufficient quantities, but does not always show satisfactory results due to immunological rejection. In recent years, the development of tissue-engineered bone shows us a potential way to solve this problem.The research of tissue-engineered bone includes cells, scaffolds and growing factors. During the sutuies, researchers found that we'ed better use autogenous tissue and the limitation elimination of trauma is very important.Platelet rich plasma (PRP) is drawn from the whole blood by centrifugation. It contains not noly high concentration platelets, but also many kinds growing factors. It also contains abundant fibers. PRP can be turned into gel when it is mixed with thrombin.Thses studies apply PRP in bone tissue engineering. We use PRP as the scaffold of injectalbe tissue-engineered bone. All these studies were carried out in a micro-trauma ways.Chapter 1: The Manufacture and Examination of PRP Experiment 1: The Manufacture of PRP and the Concentration of Platelet and TGF-β
[Abstract] Objective: To find a simple and effective way to prepare and examine PRP. Methods: Whole blood was drawn from rabbit, after two times of centrifugation, red blood cells and platelet poor plasma was separated with PRP. Platelet count was carried out and the concentration of TGF- P examined. Results: PRP was prepared successfully and the platelet count was 5.49 times of that of the whole blood and the concentration of TGF- 3 increased 2.93 times comparing with the whole blood. Conclusion: A convenient two-step centrifugation method of concentrating platelets was established. PRP produced by this technique was eligible.Experiment 2: The Observe of Platelet Rich Plasma Gel with Scanning Electron Microscope[Abstract] Objective: To observe the construe of platelet rich plasma gel, and estimate whether it can be used as a scaffold of tissue engineering. Methods: Platelet rich plasma was prepared with centrifuge and was cross-linked with bovine thrombin. Observe it with scanning electron microscope. Results: Platelet rich plasma gel was interweaved by many fibers and there was many cavum in it. Conclusion: Platelet rich plasma gel can be used as a scaffold of tissue engineering.Chapter 2: The Examination of Biological Characteristic of PRPExperiment 1: Study of Cellular Biocompatibility of PRP [Abstract] Objective: to observe the biocompatibity of PRP. Methods: Bone marrow stromal cells (BMSCs) were separated and cultured, in the same time, autologous PRP was prepared. The BMSCs was mixed with PRP with final cellular density of 5xlO6/ml. The BMSCs/PRP composites were turned into gel when it was mixed with bovine thrombin. The gel was cultured in the standard condition for 48 hours. After that, the gel was freeze-dryed and examined with scanning electron microscope (SEM). Results: The BMSCs could attach, spread and proliferate well on the surface and pore of the PRP scaffold. Conclusion: PRP had good cell biocompatibility to BMSCs and could be used as scaffold of tissue-engineered bone.Experiment 2: The study of PRP on multiplication of BMSCs in vitro.
[Abstract] Objective: To explore the effects of PRP on the multiplication of BMSCs. Methods: PRP were extracted from autologous blood and then dissolved into DMEM medium. BMSCs were stimulated with PRP in different dilutions (5%~50%). 24h, 72h, 120h later, a MTT test was performed to measure the cellular proliferation rate. Results: The proliferation rate of the BMSCs was (concentration-dependent) increased up to a certain plateau by adding PRP. Further stimulation led to a slight decrease in the proliferation rate. The statistical analysis of the extinction measurements showed significant differences in the cell proliferation rates between mostly all PRP concentration groups (pO.Ol). Conclusion: This study shows that the proliferation rate of BMSCs can be stimulated in vitro by concentration-dependent platelet concentrates. This in vitro result supports the currently discussed assumption that the clinical use of PRP might increase bone regeneration.Chapter 3: The Application of PRP to Bone Tissue Engineering Experiment 1: Injectable Tissue-engineered Bone Regeneration using BMSCs and PRP[Abstract] Objective: The study investigates the utilization of PRP carrier for delivering osteoblasts and creating new bone tissue in nude mice model via injection. Methods: BMSCs harvested from iliac bone of New Zealand rabbits was cultured and harvested. The BMSCs were mixed with PRP solution to generate BMSCs/PRP composites with final cellular density of 5 X 106/ml. Bovine thrombin was used as cross-linking agent to gel BMSCs/PRP composites. The composites were injected into the dorsal subcutaneous tissue of nude mice. Results: 8 weeks after injection, the hard knobbles were easily palpated under the dorsal skin of animals. X-ray photograph showed that the knobbles has calcified image with more density than sounding soft tissue. In histological analysis, new bone formation was observed in the BMSCs/PRP composites. The osteogenesis was in association with regenerated hematopoietic bone marrow. Conclusion: These results revealed that new bone tissue could be created through the injection of PRP mixed with BMSCs.Experiment 2: Repair of Skull Defects of Rabbit with Cultured BMSCs Combined with PRP
骨组织工程技术研究领域主要包括种子细胞、支架材料、生长因子三方面内容。经过近20年的研究,骨组织工程研究已经取得了长足的进展。在长期的骨组织工程研究过程中,人们逐渐认识到无创、微创的重要性,以及应该尽量避免使用异体、异种材料,并已经在进行积极的研究和探索。
富含血小板血浆(platelet rich plasma, PRP),是全血通过离心、浓集而得到的成分血液制品。其特点是其中血小板浓度高,并含有多种生长因子以及丰富的纤维蛋白网络;通过与凝血酶复合,纤维蛋白彼此铰链,它还可以形成凝胶。
本课题充分利用PRP的这些特点,制各及检测PRP,并以PRP为支架、用注射方式构建组织工程骨,使PRP同时成为骨组织工程研究的支架材料和生长因子的提供者;而且,在构建组织工程骨的过程中,可以实现无创或微创的目标。
第一部分 富含血小板血浆的制备及检测
实验一 富含血小板血浆的制备及血小板、TGF-β浓度检测
Bone defects caused by tumor, trauma and infection diseases are very familiar. The ordinary reconstructive ways, such as autograft, allograft and artifical grafts can solve these problems in a way, but have their limitations. Autogenous bone grafting, thought to be an ideal form, cannot be used to reconstruct extensive bone defect because of limitation of bone that can be harvested. This method also has the drawback of causing damage to normal bone and soft tissue. Allogeneic bone can be obtained in sufficient quantities, but does not always show satisfactory results due to immunological rejection. In recent years, the development of tissue-engineered bone shows us a potential way to solve this problem.The research of tissue-engineered bone includes cells, scaffolds and growing factors. During the sutuies, researchers found that we'ed better use autogenous tissue and the limitation elimination of trauma is very important.Platelet rich plasma (PRP) is drawn from the whole blood by centrifugation. It contains not noly high concentration platelets, but also many kinds growing factors. It also contains abundant fibers. PRP can be turned into gel when it is mixed with thrombin.Thses studies apply PRP in bone tissue engineering. We use PRP as the scaffold of injectalbe tissue-engineered bone. All these studies were carried out in a micro-trauma ways.Chapter 1: The Manufacture and Examination of PRP Experiment 1: The Manufacture of PRP and the Concentration of Platelet and TGF-β
[Abstract] Objective: To find a simple and effective way to prepare and examine PRP. Methods: Whole blood was drawn from rabbit, after two times of centrifugation, red blood cells and platelet poor plasma was separated with PRP. Platelet count was carried out and the concentration of TGF- P examined. Results: PRP was prepared successfully and the platelet count was 5.49 times of that of the whole blood and the concentration of TGF- 3 increased 2.93 times comparing with the whole blood. Conclusion: A convenient two-step centrifugation method of concentrating platelets was established. PRP produced by this technique was eligible.Experiment 2: The Observe of Platelet Rich Plasma Gel with Scanning Electron Microscope[Abstract] Objective: To observe the construe of platelet rich plasma gel, and estimate whether it can be used as a scaffold of tissue engineering. Methods: Platelet rich plasma was prepared with centrifuge and was cross-linked with bovine thrombin. Observe it with scanning electron microscope. Results: Platelet rich plasma gel was interweaved by many fibers and there was many cavum in it. Conclusion: Platelet rich plasma gel can be used as a scaffold of tissue engineering.Chapter 2: The Examination of Biological Characteristic of PRPExperiment 1: Study of Cellular Biocompatibility of PRP [Abstract] Objective: to observe the biocompatibity of PRP. Methods: Bone marrow stromal cells (BMSCs) were separated and cultured, in the same time, autologous PRP was prepared. The BMSCs was mixed with PRP with final cellular density of 5xlO6/ml. The BMSCs/PRP composites were turned into gel when it was mixed with bovine thrombin. The gel was cultured in the standard condition for 48 hours. After that, the gel was freeze-dryed and examined with scanning electron microscope (SEM). Results: The BMSCs could attach, spread and proliferate well on the surface and pore of the PRP scaffold. Conclusion: PRP had good cell biocompatibility to BMSCs and could be used as scaffold of tissue-engineered bone.Experiment 2: The study of PRP on multiplication of BMSCs in vitro.
[Abstract] Objective: To explore the effects of PRP on the multiplication of BMSCs. Methods: PRP were extracted from autologous blood and then dissolved into DMEM medium. BMSCs were stimulated with PRP in different dilutions (5%~50%). 24h, 72h, 120h later, a MTT test was performed to measure the cellular proliferation rate. Results: The proliferation rate of the BMSCs was (concentration-dependent) increased up to a certain plateau by adding PRP. Further stimulation led to a slight decrease in the proliferation rate. The statistical analysis of the extinction measurements showed significant differences in the cell proliferation rates between mostly all PRP concentration groups (pO.Ol). Conclusion: This study shows that the proliferation rate of BMSCs can be stimulated in vitro by concentration-dependent platelet concentrates. This in vitro result supports the currently discussed assumption that the clinical use of PRP might increase bone regeneration.Chapter 3: The Application of PRP to Bone Tissue Engineering Experiment 1: Injectable Tissue-engineered Bone Regeneration using BMSCs and PRP[Abstract] Objective: The study investigates the utilization of PRP carrier for delivering osteoblasts and creating new bone tissue in nude mice model via injection. Methods: BMSCs harvested from iliac bone of New Zealand rabbits was cultured and harvested. The BMSCs were mixed with PRP solution to generate BMSCs/PRP composites with final cellular density of 5 X 106/ml. Bovine thrombin was used as cross-linking agent to gel BMSCs/PRP composites. The composites were injected into the dorsal subcutaneous tissue of nude mice. Results: 8 weeks after injection, the hard knobbles were easily palpated under the dorsal skin of animals. X-ray photograph showed that the knobbles has calcified image with more density than sounding soft tissue. In histological analysis, new bone formation was observed in the BMSCs/PRP composites. The osteogenesis was in association with regenerated hematopoietic bone marrow. Conclusion: These results revealed that new bone tissue could be created through the injection of PRP mixed with BMSCs.Experiment 2: Repair of Skull Defects of Rabbit with Cultured BMSCs Combined with PRP
引文
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