熔融沉積成型之三維列印聚乳酸支架結構對於類骨細胞生長與礦化之影響

Abstract

過去研究指出骨組織工程支架的最適孔徑為100~350 μm，近期則另有文獻指出類骨細胞被培養於孔徑為700 μm的支架中與被培養於孔徑為350 μm的支架中七天後的增生情形無統計上顯著差異。然而以往文獻中採用的支架大部分為多孔性支架，而較缺乏探討具有狹長形溝槽狀構造且其溝槽寬度在200 μm以下的支架其最適於骨細胞生長與沉積骨質的溝槽寬度各為何。本研究的目的是探討當類骨細胞被培養於具有狹長形溝槽狀構造且其溝槽寬度範圍在200 μm以下的支架上時，最適於類骨細胞生長與成骨分化的溝槽寬度值各為何。本實驗中藉由熔融沉積成型之三維列印機列印具有「支柱(strut)-間隙(gap)-支柱(strut)-間隙(gap)」重複出現之結構的支架(scaffolds)。我們設計了三種具有不同間隙寬度(gap width)的溝槽式結構支架，其間隙寬度的預設值分別為100、150和200 μm，並依序給予簡稱為G100組、G150組和G200組，至於支柱寬度(strut width)之預設值則皆為270 μm。我們也另外製作了一組具有完全平坦表面的全平型組支架(flat type scaffolds)作為正控制組。當支架列印完成後，我們首先於光學顯微鏡下檢視支架其內部結構的實際尺寸；也進行了支架體外靜態降解實驗、利用示差掃描量熱儀(DSC)分析列印前與列印後聚乳酸材料的熱性質變化以及利用凝膠滲透層析儀(GPC)測量列印前後材料的分子量與分子量分布的變化。最後一部分則是將MG-63類骨細胞培養於各組支架上經過1, 4, 7天後，再透過MTT試驗比較各組細胞增生情形的差異；另外也透過ARS試驗比較當類骨細胞被培養於各組支架上並受到成骨分化誘導後經過4, 7, 10天後，其鈣沉積量是否各有差異。 實驗結果顯示，G100組、G150組和G200組各組實際的間隙寬度分別為87.8±23.7、142.5±20.0、188.3±23.5 μm，而各組實際的支柱寬度分別為279.4±15.6、274.8±14.0和276.5±14.1 μm。本研究所製備之聚乳酸支架經過22週的體外降解期程後，並未觀察到有明顯的重量損失現象，而降解液也未有因聚乳酸降解而導致的較明顯的酸鹼值下降現象。DSC分析顯示經過列印後的聚乳酸其玻璃轉移溫度(Tg)在圖形上變得不明顯，此乃因列印後的聚乳酸的結晶度升高所致。GPC結果顯示列印後聚乳酸的分子量下降、分子量分布則變窄，顯示列印過程會導致較大分子的聚乳酸裂解為較小的分子。MTT試驗結果顯示，當MG-63類骨細胞被培養於各組支架上七天後，細胞於G100組支架上的增生量有顯著差異地高於G200組和全平型組，但與G150組相比則無顯著差異；至於細胞於TCPS上的增生量乃是所有組別中最高的且與其餘四組相比皆有統計上顯著差異。由ARS結果得知，於第四天和第七天時，皆以G100組和G150組其上細胞的鈣沉積量較高；於第十天時，則以G150組和G200組其上細胞的鈣沉積量較高；然而於第四天和第十天時，細胞在TCPS上的鈣沉積量仍顯著地高於各組支架。 本實驗成功製作出尺寸精度屬於可接受的三維列印支架，並釐清了當類骨細胞被培養在一系列設計有特定溝槽寬度的狹長形溝槽狀構造的支架上經過7天(觀察細胞增生)和10天(觀察細胞經成骨誘導後的鈣沉積量)後，最適於其增生與成骨分化的溝槽寬度約落在142.5±20.0 μm (即本實驗中的G150組支架)，期望本研究之成果能作為未來骨再生相關研究的參考。

Previous studies pointed out that the optimum pore size for bone tissue engineering scaffolds was in the range of 100-350 μm. Recently another study found out that the proliferation of osteosarcoma cells cultured in scaffolds with pore size of 700 μm has no significant difference with that of the cells cultured in scaffolds with pore size of 350 μm for 7 days. However, most of the experiments described in the literature mainly investigated the effects of pore sizes of scaffolds on bone cell proliferation through fabrication of several types of porous scaffolds with various pore sizes, and so in the literature there is a lack of understanding of the effect of the structure of scaffolds with narrow groove-like configuration and groove widths of 200 μm or less designed within them on the bone cell proliferation and differentiation. The purpose of this study is to investigate the optimal groove width between struts within the scaffolds for the growth and osteogenic differentiation of osteoblast-like cells when they are cultured on scaffolds with narrow groove-like configuration and groove widths of 200 μm or less. In this experiment, scaffolds having a structure in which a basic unit that consists of a strut and an adjacent gap reappears ('strut-gap-strut-gap' structure) was printed by a fused deposition modeling type 3D printer. We have designed three kinds of scaffolds with different preset gap widths of 100, 150 and 200 μm and referred to as G100 scaffolds, G150 scaffolds and G200 scaffolds, respectively, and the preset value of the strut width in all three groups was 270 μm. We also created a kind of flat type scaffold with fully flat surfaces as the positive control group. When the printing of the scaffolds was completed, we at first examined the actual widths of the struts and gaps within the scaffolds under an optical microscope. The in vitro static degradation experiment of the scaffolds was also carried out, and the pre-printing and post-printing materials were analyzed by differential scanning calorimetry (DSC) to explore the changes of the thermal properties caused by the printing process. The change in molecular weight and molecular weight distribution of the materials before and after printing was measured by gel permeation chromatography (GPC). In the last part, MG-63 osteoblast-like cells were cultured on each scaffold group for 1, 4, and 7 days, and then the MTT assay was performed to compare the differences between each group in cell proliferation. In addition, the ARS assay was performed to compare whether the amount of calcium deposited by osteoblast-like cells cultured on each group of scaffolds and induced by osteogenic induction medium for 4, 7, 10 days was different between each group. The experimental results show that the actual gap widths within the G100, G150, and G200 scaffolds are 87.8±23.7, 142.5±20.0, 188.3±23.5 μm, respectively, and the actual strut width within each group of scaffolds are 279.4±15.6, 274.8±14.0 and 276.5±14.1 μm, respectively. After the 22-week in vitro degradation period of the 3D-printed polylactic acid scaffolds prepared in this study, no obvious weight loss was observed, and the degradation fluid did not show a significant decrease in acid-base value due to degradation of polylactic acid. DSC analysis showed that the Tg (glass transition temperature) of the printed polylactic acid became inconspicuous in the pattern due to an increase in crystallinity of the printed polylactic acid. The GPC results showed that the molecular weight of the polylactic acid decreased and the molecular weight distribution became narrower after the 3D-printing procedure. The results of MTT assay showed that after MG-63 osteoblast-like cells were cultured in each group for seven days, the proliferation of cells on the G100 scaffolds was significantly higher than that of cells on the G200 scaffolds and on the flat-type scaffolds, but there was no significant difference when the proliferation of cells on the G100 scaffolds was compared with that of cells on the G150 scaffolds; as for the amount of proliferation of cells on TCPS, it was the highest in all groups and there was a statistically significant difference compared with the other four groups. According to the ARS results, on the 4th and the 7th days after osteogenic induction started, the cells cultured on the G100 and the G150 scaffolds had higher calcium deposition results; on the 10th day, the cells cultured on the G150 and the G200 scaffolds had higher calcium deposition results; however, on the 4th and 10th day, the calcium deposition results of cells on TCPS were still significantly better than that of cells on all the other four groups. In this experiment, we successfully fabricated three-dimensional printed scaffolds with acceptable dimensional accuracy, and we discovered the optimum gap width for the proliferation and osteogenic differentiation of MG-63 osteoblast-like cells when they were cultured on scaffolds with narrow groove-like configuration for a short-term culture days (7 days for cell proliferation and 10 days for osteogenic differentiation) is between 142.5±20.0 μm. (The scaffold type which resulted in the best osteoblast-like cells’ proliferation performance within 7 days and the best osteoblast-like cells’ osteogenic differentiation performance within 10 days is G150 scaffolds in our experiment.) We hope that our experimental results can attribute to the advancement of the field of bone tissue engineering.