以3D列印羥基磷灰石支架結合功能化人造可調韌性基質促進齒槽骨再生

3D-printed hydroxyapatite scaffold with peptide-functionalized tunable substrate stiffness for alveolar ridge regeneration

林芷婕(Zhi-Jie Lin)

指導教授 ： 張博鈞

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202003663

摘要

目的
目前，開發一種同時具有巨觀環境和微觀條件的生醫材料來再生缺損的齒槽骨，是一項重大挑戰。這項研究旨在結合一個能提供穩定的巨觀環境且具有活化界面的3D羥基磷灰石支架（3DPHS）和一種可調韌性功能化基質（PFS），進行微觀條件的仿造細胞外基質特性，以此結合的材料促進齒槽骨再生。
材料與方法
透過加入RGD序列和過氧化鈉來使藻膠分別具有細胞黏附力和降解力。可調韌性的PFS分別與不同濃度的硫酸鈣溶液進行交聯作用後，產生具有不同程度的機械力和降解力。以90%羥基磷灰石和10% PLGA聚合物所組成的生物性墨水進行3D列印產生3DPHS，具有均勻分佈且垂直排列的300 µm孔洞。將PFS和3DPHS結合為一的材料，並植入大鼠下顎的大型缺損中，經過四周後，以微斷層掃描（micro-CT）和組織染色來評估材料的治療潛力。
結果
使用12 mM和48 mM硫酸鈣分別交聯無氧化PFS後，得到楊氏係數為2.61±0.73 kPa（低韌性）和25.42±7.66 kPa（高韌性）。而96 mM和192 mM硫酸鈣交聯氧化PFS後，分別得到2.91±0.31 kPa和25.04±4.02 kPa。與無氧化PFS相比，氧化PFS表現出較小的膨脹率以及更快速的降解現象。正交型孔洞的3DPHS，平均孔徑大小為0.420±0.028 mm x 0.328±0.005 mm，且大多數的3DPHS都能與實驗鼠的下顎缺損處吻合。動物實驗四周後，從micro-CT圖像顯示有3DPHS處理的部位，都比未填充的對照組具有更明顯的礦化的組織。3DPHS結合有無氧化PFS的實驗組別，都能看到更多的新生骨形成，特別是有氧化PFS的實驗組結果更加明顯。無氧化PFS的組別互相比較，發現高韌性的組別有顯著增加新生骨的形成。組織染色的結果圖中，在無氧化PFS的組別中，新生骨會明顯沉積在PFS上，尤其在高韌性的PFS組中發現大量且大塊的PFS。然而有氧化PFS的組別中，發現低韌性和高韌性的PFS都只有殘留些許，並且新生骨有明顯朝向缺損處中心生長的趨勢。
結論
以硬度25 kPa左右且具有降解力的PFS，搭配3DPHS所形成的生醫材料，會促進骨生長作用，將有潛力用於齒槽骨的再生。

關鍵字

骨再生 ； 3D列印 ； 組織支架 ； 羥基磷灰石 ； 仿細胞外基質

並列摘要

Objective
Developing a biologic with macro- and micro-scopic biological mechanical properties appropriate for alveolar ridge regeneration remains a major challenge. This study aimed at integrating a 3D-printed hydroxyapatite scaffold (3DPHS), which provides bioactive interface and macroscopic stable environment, and a peptide-functionalized substrate matrix (PFS) with tunable stiffness, which mimics extracellular matrix and provides osteogenic micromechanical environment, to promote alveolar ridge regeneration.
Materials Methods
Non-oxidized (NO) and oxidized (O) alginate were functionalized by adding RGD sequence without and with sodium peroxide, respectively. Tunable PFS was further formulated by crosslinking with calcium sulfate and was characterized in terms of the mechanical and degradable properties. The 3DPHS with homogeneous orthogonal 300-µm pores was digitally designed and fabricated by an extrusion-based 3D bioprinter using a bio-ink composed of 90% hydroxyapatite and 10% bio-derived polymer (polylactic-co-glycolic acid). The therapeutic potential of PFS and 3DPHS integration was evaluated by delivering integrated PFS and 3DPHS into large-sized mandibular defects of rats, and the results were evaluated by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and Masson’s trichrome staining after 4 weeks of treatment.
Results
The Young’s moduli were 2.61±0.73 kPa (low-stiff (LS)) and 25.42±7.66 kPa (high-stiff (HS)) for the NO-PFS crosslinked with 12 mM and 48 mM calcium sulfate, respectively, and were 2.91±0.31 kPa and 25.04±4.02 kPa for the O-PFS crosslinked with 96 mM and 192 mM calcium sulfate, respectively. Compared with NO-PFS, the O-PFS exhibited a lesser swelling and a more rapid degradation pattern. The 3DPHS revealed orthogonal interconnected pores with mean pore size of 0.420±0.028 mm by 0.328±0.005 mm, and most scaffolds fitted the defects well. At 4 weeks, compared with the unfilled control, micro-CT images showed that the mineralized tissue was significantly greater in sites treated by 3DPHS, and the supplement of PFS resulted in greater new bone (NB) formation, specifically when O-PFS was supplied. In sites supplied by the NO-PFS, compared with LS-NO-PFS, significantly greater NB formation was achieved by HS-NO-PFS. Histologically, in sites with NO-PFS, NB was evidently deposited on the NO-PFS, specifically on the HS-NO-PFS, whereas bulks of HS-NO-PFS were still noted. In sites with O-PFS, fewer O-PFS remained, and ingrowth of NB was evident, regardless of the stiffness of PFS.
Conclusions
Integration of degradable PFS with stiffness around 25 kPa and 3DPHS was feasible for promoting osteogenesis and could be a potential biologic to improve alveolar ridge regeneration.

並列關鍵字

bone regeneration ； three-dimensional printing ； tissue scaffold ； hydroxyapatite ； synthetic extracellular matrix

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