研發及評估應用於骨組織工程之三維列印複合性生醫陶瓷支架

Abstract

前言： 齒槽骨缺損重建對於牙醫師一直是嚴峻的挑戰。即令是材料推陳出新及手術觀念的演進，迄今仍有許多不易克服的困難使骨重建充滿不可預期性(unpredictable)，例如：不易將骨材堆積成理想外型(ideal contouring)、骨材體積(graft volume)不易維持、或採用自體骨移植時易造成取骨區(donor site)傷口不適等問題。新近三維列印科技的發展，或許能為此等難題帶來可行的解決方式。 氫氧基磷灰石(HA，hydroxyapatite)(Ca10(PO4)6(OH)2)及三鈣磷酸鹽(β-TCP，beta-tricalcium phosphate)(Ca3(PO4)2)是牙科臨床最常使用的合成骨(alloplasts)的主要成分，，其主要組成為磷酸鈣(calcium phosphate) 具有骨引導能力(osteoconductivity)。我們期望以HA及β-TCP為主要材料，就現有數種主流三維列印技術做評估，找出用於骨組織工程的最可行的列印方式與配方，並驗證其物理性質、生物相容性(biocompatibility)、及促進骨生成能力 (osteopromotion)。 材料與方法： 首先進行回顧文獻，市面上的三維列印的原理與材料有許多種，就檢索分析結果而言，噴墨列印(inkjet printing)、機械手臂(robocasting)、雷射燒結(SLS，selective laser sintering)及熱熔擠壓成型(FDM，fused deposition modeling)為可能應用於骨組織工程的列印方式。進一步就這四種方式進行實地列印測試評估後顯示FDM較具有可行性，因此我們以FDM為技術，以磷酸鈣/聚己内酯(PCL，polycaprolactone)為材料，依據HA/β-TCP和PCL不同的組成比例設計以下四個組別：H7T3 (HA:35%，β-TCP:15%，PCL:50%)，H5T5 (HA:25%，β-TCP:25%，PCL:50%)，H3T7 (HA:15%，β-TCP:35%，PCL:50%)及PCL(PCL:100%)。列印出三維骨支架以進行後續物理化學性質測試，，包含以SEM下分析表面型態(surface topography)和孔洞大小(pore size)，以氦氣密度分析儀測試其孔洞性(porosity)，以萬用材料試驗機測試其抗壓強度(compressive strength)，以傅里葉轉換紅外光譜(FTIR，Fourier-transform infrared spectroscopy)確認其官能基，以X-射線繞射分析(XRD，X-ray diffraction)檢驗其晶格，將支架浸泡於磷酸鹽緩衝生理鹽水溶液(PBS，phosphate buffered saline)以量測其降解速率(degradation rate)。 接著，我們使用人類牙髓細胞(hDPCs，human dental pulp cells)進行三維支架生物相容性(biocompatibility)及促進骨生成能力 (osteopromotion) 的測試。於人類牙髓細胞培養的第1天、第3天、及第7天以阿爾瑪藍細胞活性測試(AlamarBlue cell viability assay)檢驗其生物相容性，並在第7天以掃描式電子顯微鏡(SEM，Scanning electron microscope)下分析細胞附著於支架之情況。同時，以材料萃取液培養hDPCs，在第7天、第14天、及第28天量測鹼性磷酸酶(ALP，Alkaline phosphatase)定性與定量表現、以及Alizarin red S stain分析鈣生成量，以作為成骨能力的探討。最後，我們將支架植入大鼠背部，於第3週、第7週犧牲，將支架及其周圍組織取下後，先以10%福馬林(formaldehyde)固定後，拍攝觀察放射線不透性(radiopacity)，接著酒精序列性脫水、石蠟包埋、組織切片、HE染色(hematoxylin and eosin stain)，在400倍下觀察其組織反應，並依據ISO 10993-6:2007標準評估其發炎反應。 結果： 噴墨列印和機械手臂難以達成符合我們需求的支架外型與機械強度，雷射燒結則是解析度較差，相較之下熱熔擠壓成型的支架成品強度與解析度均有較好的表現。 以下為FDM列印的磷酸鈣/PCL三維骨支架的物理測試結果。經由SEM觀察及氦氣密度分析儀量測，我們列印的支架具有適合細胞生長的孔洞大小(約450μm)，以及孔洞性(50~60%)。其抗壓強度(4~5MPa)接近鬆質骨(cancellous bone)的表現，符合臨床上骨材強度的要求。經由FTIR可確認三維支架的上具有磷酸根和 PCL的官能基。XRD也顯示經由FDM列印的磷酸鈣/PCL 三維支架不會改變HA和β-TCP結晶性和結構。此外，在一個月的降解實驗中沒有發現明顯的重量改變。 在生物相容性的實驗中，AlamarBlue檢驗顯示所有組別相對於度照組均有超過八成的細胞存活率，不論是第1天、第3天、及第7天的細胞存活率均以H3T7表現最佳，其次依序為H5T5及H7T3。SEM下可觀察到細胞伸出偽足貼附於支架之情形，我們發現細胞傾向於攀附於網格交錯之處。而促進骨生成能力的實驗當中，可觀察到在28天的期間內ALP濃度先增加、後減少，所有實驗組別均優於對照組，並以H3T7的表現最佳、並在第14天與對照組達統計上顯著差異(p<0.05)。Alizarin red S stain鈣生成量分析中，同時亦可觀察到H3T7有最多的鈣化物沉積、但未與對找組達統計上顯著差異。動物實驗中，我們可以觀察到PCL慢性發炎時間較長，其淋巴細胞之(lymphocyte)數量在第三週與第七週的觀察點仍居高不下；而H7T3、H5T5、H3T7三組，第三週出現之多型性白血球到第七週便完全消失，lymphocytes也是顯著下降。Fibrosis的現象在所有組別均可觀察到，同時均在第七週明顯增加。 結論： 以FDM列印含磷酸鈣/PCL的三維骨支架，具有良好的物理性質且能符合臨床期待。所有列印的支架當中，以含較高β-TCP濃度的H3T7的組別表現出最佳的生物相容性及促進骨生成能力。大鼠的實驗同時也驗證含磷酸鈣的支架在動物體內生物相容性比純PCL佳。本研究已研發出一種未來在齒槽骨組織工程的應用具有發展潛力與價值的三維列印方式及配方。

Introduction: The reconstruction of alveolar bone defect has possessed challenges to clinicians. There have been many difficulties such as graft contouring, graft loss, donor site morbidity in spite of the development of materials and techniques. The advancement of 3d printing may be a possible resolution for the bone tissue engineering. HA(hydroxyapatite) (Ca10(PO4)6(OH)2) and β-TCP(beta-tricalcium phosphate) (Ca3(PO4)2), the most common alloplasts in dentistry, which consist of calcium phosphate, have the capability of osteoconductivity. Our purpose was to find the most feasible formula in bone tissue engineering by HA and β-TCP among the several mainstream 3d printing method, and to evaluate the physical properties, biocompatibility, and osteopromotion of the printed scaffolds. Materials& Methods: There are many types of 3d printing in manufacturing process and materials. According to the literature, inkjet printing, robocasting, SLS(selective laser sintering), and FDM(fused deposition modeling) were the possible 3d printing methods in bone tissue engineering. Therefore, we invested and evaluated these four methods. The initial examination indicated that FDM was the most feasible method; therefore, we decided to print the 3d bone scaffolds from calcium phosphate/(PCL，polycaprolactone) by FDM for the further studies. We designed the following four experiment groups according to different ratio of HA/β-TCP and PCL: H7T3 (HA:35%, β-TCP:15%, PCL:50%), H5T5 (HA:25%, β-TCP:25%, PCL:50%), H3T7 (HA:15%, β-TCP:35%, PCL:50%) and PCL(PCL:100%). Physics and chemistry tests were then empolyedon these printed scaffolds. We analyzed the surface topography and pore size by SEM(scanning electron microscope), porosity by gas pycnometer, compressive strength by Instron Universal Testing Systems. Functional groups were detected by FTIR(Fourier-transform infrared spectroscopy), and the crystal structures were examined by XRD( X-ray diffraction). The degradation rate was measured by soaking the scaffolds in PBS(phosphate buffered saline) for 1 month. Biocompatibility and osteopromotion tests were carried out as well. We cultured hDPCs(human dental pulp cells) on these scaffolds to evaluate the biocompatibility of the scaffolds by AlamarBlue Cell Viability Assay on day 1, day 3, and day 7, and to observe the morphology of the cell attachment to the scaffolds by SEM on day 7. Meanwhile, we assessed the osteopromotion of the scaffold by performing the ALP qualification and quantification tests, and Alizarin red S stain for calcification deposition by scaffold extracts. Finally, we performed subcutaneous implantation into the rat back, and sacrificed after 3 weeks and 7 weeks respectively. These scaffolds with surrounding tissue were removed, fixed in 10% formaldehyde. X ray films were taken to evaluate the ability of conduction of mineralization. Then the samples were routinely processed, dehydrated, embedded in paraffin wax, and stained with haematoxylin and eosin (HE). ISO 10993-6 :2007 for biological evaluation of medical devices was employed for the assessment of the inflammatory response. Results: The scaffolds printed by the inkjet printing and robocasting could barely meet our requirement in mechanical strength, while those by SLS were too low-resolution to meet the clinial requirement. The FDM-printed scaffolds achieved a better performance in both mechanical strength and resolution. The result of the physical experiments of the FDM-printed calcium/PCL 3d scaffolds were as follows: The SEM and gas pycnometer revealed the scaffolds had the pore size of 450μm and porosity between 50%~60%, which were suitable for cell growth. The compressive strength (4~5MPa) which was closed to that of cancellous bone achieved the clinical requirement. The FTIR analysis indicated the presence of peaks ascribed to phosphate and PCL, and the XRD analysis also confirmed that the synthesis process of the CaP/PCL composite scaffolds by FDM did not alter the crystallinity and the structure of HA and β-TCP.Furthermore, all samples recorded no obvious weight changes in degradation test after 1 month. In biocompatibility study, AlamarBlue exhibited that the cells seeded on to the scaffolds had survival rate higher than 80% compared to that of the control group. H3T7 showed the highest cell survival rate throughout the experimental period, followed by H5T5 and H7T3. The SEM showed the cell spread out on the scaffolds; what’s more, we found the cells were prone to adhere to the pores we designed. In the evaluation of osteoconduction, we could observe the ALP increased and then declined during the period of 28 days. The value of ALP in all the experimental groups were higher than those in the control group, and was found to be highest in H3T7. A significant difference could be observed between H3T7 and the control group on day 14. The Alizarin red S stain also showed the highest calcification deposition in H3T7 but without significant difference compared to the control group. In animal study, we could found the inflammation persisted in PCL, with lymphocyte number remaining high from week 3 to week 7. In the groups of H7T3, H5T5, and H3T3, the leukocytes howed increased on week 3 and dramatic droped on week 7, and the number of lymphocytes also decreased significantly. The phenomenom of fibrosis could be observed in all groups and increased on week 7. Conclusion: The CaP/PCL 3d printed scaffold by FDM had good physical properties and met the clinical requirements. H3T7, which had a relatively higher concentration of β-TCP, showed the best biocompatibility and bioactivity, and could effectively induce osteogensis differentiation of hDPCs. The study on rat also proved the biocompatibility was better in the scaffold withs calcium phosphate than with pure PCL. In thie study, we have developedreliable and promsing technique and formula in the field of alveolar bone tissue engineering.