幾丁聚醣生醫材料於骨組織工程之應用

Chitosan-Based Biomaterials for Bone Tissue Engineering Applications

張根育(Ken-Yu Chang)

指導教授 ： 謝國煌

國立臺灣大學/工學院/化學工程學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01851

摘要

骨創傷的修復在臨床醫學上是個很重要的課題，藉由組織工程的發展，我們得以發展出能夠引導骨缺陷修復的人工植入材料。本論文的主軸乃是設計及開發以幾丁聚醣天然高分子為主之生醫材料，藉由不同形式之基材設計與生物活性分子之導入，欲發展出能夠取代現今骨修復之填充材料，使其具有臨床應用性之價值。本論文主要包括了以下之幾部分:
(1) 骨引導及骨誘導性之幾丁聚醣薄膜材料之開發:乃是利用化學固定骨形態生長因子於幾丁聚醣薄膜上，進行體外骨母細胞培養及大型骨缺陷之動物體內測試。研究結果發現經由化學固定能夠使生長因子發揮長效且專一作用於受傷部位之功能，且表面固定有生長因子之薄膜顯著提高細胞活性及分化，證實經過化學固定後骨生長因子仍保有生物活性。在植入兔子橈骨之大型骨缺陷後，本研究開發之表面固定生長因子之幾丁聚醣薄膜能不同於對照組與純幾丁聚醣薄膜，在短時間內(六週)即引導骨缺陷之修復癒合。
(2) 三維立體之多巨孔性幾丁聚醣支架之開發:乃是以溫度相轉換法為基礎，加以修正，以改良過之方式製造具有專一方向性之巨大孔洞分佈的多孔性基材。
(3) 幾丁聚醣支架於不同骨生長動物模型比較:前法製作出之幾丁聚醣支架分別進行兔子橈骨大型骨缺陷與老鼠門牙拔除後齒槽骨再生之體內測試。研究結果發現，純幾丁聚醣支架於植入橈骨缺陷中12週後仍未出現骨癒合的徵兆，只有在骨斷面新生出些許的骨痂；然而在植入牙齒拔牙後之傷口後一個月，不僅齒槽骨的再吸收明顯被抑制，且齒槽骨大量地再生而填充滿整個拔牙傷口。此研究結果代表幾丁聚醣支架的骨引導性明顯會隨不同的植入部位而有所變化，而此研究開發出之純幾丁聚醣支架在齒槽骨引導再生上有非常突出之表現。
(4) 骨引導及骨誘導性之多巨孔幾丁聚醣支架之開發:乃是沿用化學固定骨生長因子之方式，將骨型態生長因子固定於多孔性支架表面，製作出具長效型且易植入之骨填充材料。動物實驗中經由與純幾丁聚醣、表面物理吸附骨生長因子之幾丁聚醣支架之比較發現，化學固定骨生長因子之支架大大的提升了骨組織再生及癒合的速度與機率。
(5) 幾丁聚醣與氫氧基磷灰石複合多孔性支架之開發:藉由與陶瓷材料製作成複合基材，能夠提升支架之機械性質與骨引導性。研究結果發現複合支架仍保有專一方向性、多孔性及巨大孔洞之特徵。隨氫氧基磷灰石之導入，材料之機械性質上升而裂解速率與程度隨之下降，且於兔子橈骨模型中發現骨缺陷能在短時間內修復且重整為緻密骨之組織結構。

關鍵字

幾丁聚醣 ； 骨生長因子 ； 化學固定 ； 氫氧基磷灰石 ； 骨組織工程

並列摘要

Tissue engineering has shown great promise as a viable alternative to current bone graft solutions due to its use of biocompatible, biodegradable scaffolds as support systems for cellular attachment, proliferation, migration, and maintenance of normal phenotypic expression. The overall goal of this thesis was to design and develop biodegradable, biocompatible chitosan-based scaffolds that will be practical alternatives to current bone repair materials.
The first specific aim was to develop a new method to prepare an osteoinductive-osteoconductive bioimplant based on chitosan and recombinant human bone morphogenetic protein-2 (rhBMP-2). BMP-2 was chemically immobilized on the chitosan membrane in order to provide a bioactive surface that can enhance bone-regeneration capacity. Cellular evaluation demonstrated this novel rhBMP-2-immbilized membrane to be biocompatible and osteoionductive, with evidence of enhanced cellular proliferation and early alkaline phosphatase expression. Accelerated bone healing observed histologically and radiographically in the rabbit radius critical-sized defect indicated that it would seen to be applicable for inducing significant and localized bone formation in future guided tissue regeneration.
The next objective was to develop 3-dimensional chitosan scaffolds. The macroporous chitosan sponges were fabricated by the modified method based on thermally induced phase separation. A combination of solid-liquid phase separation/solvent-extraction/neutralization/freeze drying paths were successfully developed to fabricate highly anisotropic chitosan scaffolds with high porosities (>90%) and macropores (>100µm) which may favor cell growth, migrate, nutrient transportation and further bone tissue regeneration. When implanted in a segmental long bone defect, however, macroporous chitosan sponges did not show sufficient osteoconductivity for complete bone defect healing.
It was therefore another aim of this thesis to chemically immobilize rhBMP-2 on macroporous chitosan scaffolds in order to provide osteoinductivity for quick and promoted bone regeneration. Bone defect bridging and union was achieved within 4 weeks in 8 of 10 specimens. Such a high bone healing efficiency of rhBMP-2-immobilized chitosan sponges revealed that they are good candidate bioimplants for guided tissue regeneration application.
A trial related to implantation of pure chitosan sponge in the tooth extraction socket was also made in this thesis. Surprisingly, unlike long-bone defect model, the alveolar bone was preserved and regenerated when the extraction socket was implanted with pure chitosan sponge. It could be concluded that osteoconductivity of chitosan has much to do with the implanted site. Pure chitosan sponge has great potential to be used as a socket filler to prevent adsorption of alveolar bone after tooth extraction.
In spite of the promising performance of the chitosan scaffolds, they could not be applied in the load-bearing bone defects because polymer themselves are mechanically too weak. Consequently, a final aim was to prepare chitosan/hydroxyapatite composites designed to mimic the properties of bone, which itself is a composite. The mechanical properties were significantly improved as hydroxyapatite was incorporated. In vivo animal studies in male New Zealand white rabbits showed that composite scaffolds provided a suitable structure for new cellular infiltration throughout the scaffold pore structure. Composite scaffolds also supported the vascularization of new tissue within the defect site, as well as newly mineralized bone tissue at the margins of the defect.
In summary, this dissertation demonstrates the successful generation of a biomimetic scaffold capable of localizing growth factor delivery which indicates significant potentials in tissue engineering and regenerative medicine.

並列關鍵字

chitosan ； bone growth factor ； chemical immobilization ； hydroxyapatite ； tissue engineering

參考文獻

1. Weiss L. Cell and Tissue Biology, A Textbook of Histology. Baltimore, MD: Urban and Schwarzenberg; 1988.

2. Huiskes R, Ruimerman R, van Lenthe GH, Janssen JD. Effects of mechanical forces on maintenance and adaptation of form in trabecular bone. Nature 2000; 405:704-6.

4. Hart R. Bone Modeling and Remodeling: Theories and Computation. Cowin SC, editor. BoneBiomechanics Handbook. Boca Raton: CRC Press LLC; 2001; 31:1-42.

5. Brighton, Carl T. and Robert M. Hunt. Histochemical localization of calcium in the fracture callus with potassium pyroantimonate: possible role of chondrocyte mitochondrial calcium in callus calcification. Journal of Bone and Joint Surgery 1986;68:703-715.

6. Brighton, Carl T. and Robert M. Hunt, Early histologic and ultrastructural changes in medullary fracture callus. Journal of Bone and Joint Surgery 1992; 73-A : 832-847.