聚乳酸甘醇酸/奈米級氫氧基磷灰石製備之微球體對人類骨母細胞貼附、增生及分化之影響

Abstract

骨相關疾病雖不及惡性腫瘤、心血管疾病等列為十大死亡原因之疾病，但卻是在人數與年齡都分布較廣的疾病，其中包括髖關節置換術、脊柱融合術、癌症造成骨損傷、骨折；其治療的方法輕則以鋼板或石膏固定損傷處，嚴重則需開刀進行骨移植手術或以人工代用骨填補損傷處，然而，過去的醫治方法大多無可避免開刀一途，所使用的人工代用骨只能達暫時骨引導和支持的功能，隨著組織工程的發展，許多研究也不斷提出更多改良的材料，欲達到更好的骨修復效果；發展至此，目前遇到的瓶頸包括仿生性高的材料製備使組織工程骨能具備更好促進骨增生及分化的能力、將支架改良成不需以開刀的方式進行骨損傷修補等。 本研究的目的在於使用FDA認可的聚乳酸甘醇酸(PLGA)與人體骨主要的無機成分奈米級氫氧基磷灰石(nano-Hydroxyapatite)，以不同比例混合，製備出可注射型微球體支架，作為骨細胞載體，並研究比較PLGA/nano-HA比例對於人類骨母細胞貼附、生長及分化之影響。為達此目的，必須先作出大小適合以注射方式植入的微球體，我們利用兩種多重乳化法(Solid-in-oil-in-water, S/O/W, Water-in-oil-in-water, W/O/W)的方式製備聚乳酸甘醇酸-奈米級氫氧基磷灰石(PLGA-nHA)微球體，其球體直徑分布在50-200微米，由於成骨母細胞大小大約在5-10微米，若直徑小於50微米，細胞貼附情形不好，球體過大會不利注射，故此微球體支架大小適合作為成骨母細胞的載體。另外，我們製備PLGA與n-HA不同比例混合的微球體，觀察其結構的差異以及與細胞培養後，人類成骨母細胞貼附、增生及骨誘導分化的情形。 研究顯示，以S/O/W方法製備的微球體，n-HA會平均分散在整顆實心球體，能使微球體支架性質更加穩定，其中又以1:1重量百分比混合的微球體結構較為完整；固定PLGA濃度與不同比例n-HA混合，發現當添加的n-HA達70%，已超過PLGA能攜帶的比例。為確認不同比例的PLGA/n-HA微球體對成骨細胞貼附、增生及分化的影響，我們將其支架與成骨母細胞混合培養，利用MTS測量細胞活性推算貼附於微球體的細胞數並觀察增生結果；另外，以成骨母細胞分泌的鹼性磷酸酶評估細胞分化的能力。結果證實，n-HA含量愈高，初期貼附細胞數較多，但n-HA在70%時，細胞貼附的穩定性較差，但就成骨母細胞在微球體上增生的情形而言，n-HA的添加有助於增生；而誘導分化時，發現貼附於PLGA-nHA以1:1重量百分比混合的微球體上的細胞，具有鹼性磷酸酶較高的活性表現。 因此藉由本實驗證明以S/O/W製備以聚乳酸甘醇酸/奈米級氫氧基磷灰石微球體適合用於注射的骨修補仿生支架，且n-HA添加的比例以1：1混合對於人類骨母細胞貼附、增生、分化有最佳的影響。

Decades, although bone related diseases are not as serious as malignant tumors and cardiovascular disease, which are listed in the top ten causes of death, they are problems for most people, either children or adults. These bone related diseases include hip replacement, spinal fusion, bone demage caused by cancer, fracture, and so on. The traditional treatment for fracture is cast immobilization, and for severe cases, patients must accept bone transplantation or implantation ofartificialbonesubstitute. However, surgery is inevitable, and the artificial bone substitute can only provide the function of osteo-conduction and temporary support. With the development of tissue engineering, recent studies have proposed many improved materials, and hope to achieve better results. How to make more biocompatible and injectable scaffolds and enhancing osteoblast proliferation and differentiation are the goals of bone tissue engineering. The aim of this study was to fabricate injectable, different ratio composite microsphere scaffold by FDA-approved PLGA and nano-hydroxyapatite, applied the PLGA/n-HA microspheres to enhancing human osteoblasts’ adhesion, proliferation and differentiation, and figured out which ratio was better for mimicking the microenvironment in human body. To achieve the goals, solid different PLGA/n-HA ratio microspheres were fabricated in solid-in oil-in-water single emulsion method, with a mean size of approximately 50-200μm. This size range was suit for needle injection and human osteoblast adhesion. On the other hand, we also compared the effect of different preparation of microspheres with their morphology, structure and mechanical stress. This study suggested that by single emulsion method, nano-hydroxyapatite would be well-despersed in microspheres, but double emulsion doesn’t, and help stabilize dynamic equilibrium during the formation of microspheres. The PLGA/n-HA-5/5 micorspheres had relatively complete and firm structure. Moreover, when adding nano-hydroxyapatite up to 70%, PLGA failed to completely package all nano-hydroxyapatite. After the fabrication of three types of PLGA/n-HA microspheres, PLGA, 5/5, 3/7, we cocultured microspheres with human osteoblasts, and observed the effect of adding different weight ratio of nano-hydroxyapatite on adhesion, proliferation, and differentiation of human osteoblasts by MTS and alkaline phosphatase assay. Either adhesion or proliferation, PLGA/n-HA-5/5 had the best result comparing PLGA and PLGA/n-HA-3/7, and so did the expression of alkaline phosphatase which an indicator of early osteoblast differentiation. Hence in comparison of two of methods, the PLGA/n-HA biomimicking, injectable microsphere scaffold made in single emulsion is more suitable for bone tissue engineering, and the 50 weight percent of nano-hydroxyapatite is better for adhesion, proliferation ,and differentiation than other ratio.