Use of Platelet-rich Fibrin (PRF) as Scaffold Seeded with Porcine Articular Cartilage Fragments for Tissue Engineering: an in vivo Autologous Transplantation Analysis

由於成熟關節軟骨本身並沒有血管分布，所以軟骨的修復並不容易。近年來因為關節退化而飽受疾病困擾的病患日進增加，發病的年齡也漸趨年輕化，關節軟骨的疾病已經不再是年老疾病。當關節軟骨受損引起退化性關節炎，會因為其不易修復甚至導致永久性破壞，因此組織工程再生在關節軟骨的研究就越來越重要。組織工程得領域中有三個要素，分別是細胞、細胞支架以及生長因子，這三者缺一不可。細胞支架本身是一個具有三度空間立體結構的材料，根據他的成份分為生物性及非生物性。要稱為良好的細胞支架必須具有良好生物降解性、低組織排斥性、可以幫助細胞移行及黏連、可促進細胞分化等多項優點。在關節軟骨的組織工程研究中，其中自體來源的纖維素不僅可作為良好的生物性材料，它同時具有低組織排斥性以及良好的生物降解性；而組織工程中細胞會因其來源有所不同，通常包括自體移植、異體移植以及異種移植。富血小板纖維素是一種第二代血小板衍生物，具有黃色凝膠樣的外觀，由於它是經過離心緩慢形成，所以在富血小板纖維素中同時含有許多生長因子。在我們的實驗中，是利用富血小板纖維素作為細胞支架同時混合自體來源的豬隻關節軟骨碎片進行關節軟骨組織工程再生。在經體內試驗六個月後，關節軟骨的外觀可以發現實驗組與對照組之間有明顯的差異（p<0.05）；除此之外同時在顯微鏡下評估經過組之工程手術的部位，可以發現在細胞排列、細胞外基質的生長以及分佈，實驗組與對照組織間都有顯著的差異（p<0.05）。而在關節軟骨彈力試驗測得的結果，發現正常的軟骨彈性大於實驗組的軟骨彈性，又大於對照組的彈性。根據Alcian blue以及Safarnin O的染色結果發現在實驗組中大分子醣類與醣蛋白的表現，無論是表現量或是分佈都與正常的軟骨相近，而對照組則是低表現量，此外成不均質樣分佈。免疫組織化學染色的結果，實驗組的第二型膠原蛋白的分布情形，也與正常關節軟骨相近。綜合以上結果發現在關節軟骨經過組織工程手術後，無論是在外觀形態表現、關節軟骨彈性的恢復、組織學下細胞生長量以及細胞外基質的再生，都有顯著的再生結果；此外利用富血小板纖維素作為細胞支架同時移植細胞，不僅可將細胞保留局現在手術部位，也可幫助軟骨組織的再生。Introduction Adult articular cartilage is characterized by a poor ability to repair damage spontaneously. In the study of tissue engineering, scaffold provides a physicochemical and biological three－dimensional microenvironment for cell growth. Platelet rich fibrin (PRF) was a second-generation platelet concentrate that has a gel-like construct with plenty of growth factors. It composed of fibrin fibrilla, fibronectin, glycanic chain, and platelet to form a network structure. bject Since most studies reported by means of transplantation, the aim of our study could be achieved by shifting this autologous cartilage shreds quickly during open articular surgery procedure. xperimental design, There were three experimental groups and control group (n=4), were established in our study. Experimental groups were separated into PRF group (implants with platelet-rich fibrin, n=4), CAR group (implants with cartilage fragments, n=4), and P/C group (cartilage fragments embedded with platelet-rich fibrin, n=4). The articular cartilages harvested from 16 Lan Yu pigs after implantation for 6 months.esult To inspect the defect margin of native and regenerated cartilage, the edge is almost invisible in P/C group and approximately visible present in control group. There are significantly different in the parameters of coverage color and defect margin (p>0.05). Matrix stiffness test is ranked as normal cartilage>experimental groups>control group; exactly, the stiffness test between three experimental groups are ranked as and was ranked as P/C group>PRF groups>CAR group (p<0.05). Under histological assessment, the gap of P/C group lefts only point historical remains and most of the repair cartilage is fusion with native cartilage. Besides, the surface of P/C group regenerates with mostly smooth surface, and result also equal to the gross appearance. The matrixes of P/C group augment with large partial of hyaline-like cartilage and the macromolecular of matrix displays homogenously under Alcian blue and Safranin O staining. Regeneration of P/C group under histological analysis was agreement to the cartilage compressive stiffness test. The repair matrix of P/C group was mostly near to native cartilage that could predict the well mechanical function in prognosis.onclusion In summary, platelet-rich fibrin was able to help chondrocytes regeneration and cartilage fragments were proficient to deliver cartilage matrix and chondrocytes. Because of suitable tissue scaffold and cartilage fragments, our results suggested that the tissue regeneration of P/C group was facility to regenerate cartilage micro and macro-structure, the mechanical function and weight-bearing restored nearly to the native cartilage.Chapter 1 Literature Review ……………………………………...1ection 1 Articular Cartilage……………………………….....1.1 Cartilage types…………………………………………………..1.2 Components and Structure of Articular Cartilage…………2.3 Cartilage Function……………………………………………….4.4 Development and Repair of Articular Cartilage………....5.4.1 Growth of cartilage……………………………………………5.4.2 Repair Mechanism of Cartilage…………………………….6ection 2 Tissue Engineering of Articular Cartilage………..7.1 Introduction …………………………………………….7.2 Growth Factors…………………………………………………….8.3 Scaffold……………………………………………………………9.4 Cell……………………………………………………………….10ection 3 Introduction of Platelet-Rich Fibrin…………….11.1 Introduction of Choukroun’s Pletelet-rich fibrin....11.2 Introduction of Vivostat PRF……………………….13ection 4 Achievement of Platelet-Rich Fibrin…………....14ection 5 Assessment……………………………………………...17.1 In Situ Compressive Stiffness Test…………………………17.2 Gross Assessment of Articular Cartilage…………………19.3 Histological Assessment of Cartilage…………………….19hapter 2 Introduction…………………………………………….21hapter 3 Materials and Methods…………………………………23ection 1 Materials………………………………………………..23.1 Solutions for Alcian blue staining………………………..23.2 Solutions for Safranin O Staining…………………………23.3 Preparation of Platelet-Rich Fibrin………………………23.4 Experimental Animals……………………………………………24ection 2 Methods……………………………………………………25.1 Anesthesia…………………………………………………...25.2 Surgical Procedures ………………………………………...26.3 Cartilage Harvest………………………………………………27.4 Assessment of Articular Cartilage…………………………27.4.1 Articular Cartilage Gross Assessment ………………27.4.2 Articular Cartilage In Situ compressive Stiffness Test…………………………29.5 Articualr Cartilage Histological Analyses……………..30.5.1 Biopsy Technique and Fixation ……………………………30.5.2 Histological Grading Scale ……………………………….30.6 Gaps Investigation under Histological Analysis…………32.7 Alcian Blue Stain Analyses……………………………………32.8 Safranin O Stain Analysis ……………………………………33.9 Immunohistochemistry Analysis ………………………………33ection 3 Statistical Analysis …………………………………34hapter 4 Result ………………………......................36.1 Surgical Procedure ……………………………………………36.2 Platelet-Rich Fibrin Preparation and Appearance………36.3 Gross Scaling Assessment …………………………….36.4 In Situ Compressive Stiffness Test ………………………37.5 Histological Assessment ………………………………………38.5.1 Gaps Investigation …………………………………………38.5.2 Analysis of Histological Scaling Assessment …………39.6 Outcome of Alcian Blue Stain ………………………………40.7 Outcome of Safranin O Stain ………………………………41.8 Outcome of Immunohistochemistry Stain ……………41hapter 5 Discussion ………………………………………………43hapter 6 References ……………………………………………52able ……………………………………………………………………65igure……………………………………………………………………7