明膠-雙相磷酸鈣複合凍膠支架促進齒槽骨生成之 成效探討

Abstract

重建缺損的齒槽骨是現今植牙治療的一大挑戰。本研究旨在發展一種創新的治療策略，意即製作明膠/雙相磷酸鈣凍膠支架(gelatin/biphasic calcium phosphate cryogel scaffold)促進齒槽骨組織再生。 複合凍膠支架是由經鍛燒牛骨後萃煉之雙相磷酸鈣顆粒與明膠混合後於低溫交聯（crosslinking）形成。動物實驗驗證分成兩部分，第一部分是透過大鼠齒槽骨上骨再生（supra-alveolar osteogenesis）模式探討明膠/雙相磷酸鈣凍膠支架促進齒槽骨垂直方向骨再生以及與植體骨整合之表現。先將老鼠雙側下顎第一大臼齒拔除並等待四周傷口癒合，再將直徑5 mm、高度2.5 mm的圓柱形支架使用迷你鈦植體（mini-titanium implant）固定在拔牙區的顎骨頰側。為最佳化再生效果，除了未使用任何支架的控制組（Group Control-1）、單純明膠/雙相磷酸鈣凍膠支架組（Group HAP）外，亦將第二型骨成型蛋白質 (Bone morphogenetic protein 2 ; BMP-2 )以直接注射（Group BMPi）或以靜電噴霧造粒技術（coaxial electrohydrodynamic atomization technology）製作含BMP-2之聚乳酸-羟基乙酸共聚物( poly(lactic-co-glycolic acid) ; PLGA)微球體（microspheres）嵌入明膠/雙相磷酸鈣凍膠支架（Group BMPm）。動物於四周後犧牲，再生成效使用微電腦斷層成像（micro-computed tomography；micro-CT）、硬組織切片染色以及骨生成螢光染色檢測。 第二部分是透過大鼠齒槽骨內缺損(intra-bony defect)模式探討雙相磷酸鈣顆粒尺寸對於骨再生效果之影響。大鼠雙側的上顎第一大臼齒拔除後等待四周的傷口癒合，於原先拔牙區齒槽脊手術製作直徑2 mm、深1 mm的圓柱形缺損，並於缺損中分別填入含有顆粒直徑50-100 µm雙相磷酸鈣（Group HAPs）、250-1000 µm雙相磷酸鈣（Group HAPr）、及250-1000 µm未經鍛燒之牛骨顆粒（Group DBBM）與明膠交聯的凍膠支架以及不放支架的控制組(Group control-2)。動物於四週及八週犧牲，並透過微電腦斷層成像（micro-CT）以及組織切片進行骨再生的比較。 明膠/雙相磷酸鈣凍膠支架的孔洞直徑（pore size）為439±56 µm、孔隙率（porosity）為81.7±1.2 %。微球體的直徑為9.29±1.89 µm、包覆效率（encapsulation efficiency）為64.9%±4.59，並且在四週內有穩定長效的釋放。在第一部份的動物實驗中，micro-CT影像顯示所有放置凍膠支架的組別於二週及四週後均有顯著的齒槽骨增寬，其中BMPi組和BMPm組有較顯著的骨生成螢光標定，頭兩週的骨新生量BMPm組相較於HAP組亦顯著提高，且齒槽骨上的新生骨和植體的骨整合以及骨小樑結構只有在四週於BMPm組才能被觀察到。在第二部分的動物實驗中，在四週及八週的micro-CT影像檢查下各組間並沒有達到顯著的差異，然而HAPs組相較於HAPr組及DBBM組仍有較為穩定及多量的骨新生的趨勢。 本實驗的結論是結合明膠/雙相磷酸鈣凍膠支架可增進齒槽骨增厚，嵌入含BMP-2的PLGA微球體能進一步促進骨新生及植體骨整合；使用小顆粒(50-100µm)的雙相磷酸鈣製作凍膠支架可能可以提供更穩定及較佳的骨新生效果。

The reconstruction of supra-alveolar ridge deficiencies is a major challenge for dental implants. We aimed at developing a gelatin/hydroxyapatite/beta-tricalcium phosphate (gelatin/HA/ß-TCP) cryogel scaffold as a novel treatment strategy for promoting alveolar ridge regeneration. HA/ß-TCP particles were extracted by calcining bovine bone matrix and were crosslinked with gelatin in subzero temperature to form a gelatin/HA/ß-TCP cryogel scaffold. Two animal models were utilized. The first model was to investigate the osteogenic potential of the gelatin/HA/ß-TCP cryogel scaffold in a rat supra-alveolar regeneration model. Bilateral mandibular first molars were extracted, and the scaffold was fixed on the buccal plate of the edentulous ridge using a mini-titanium implant. Specimens were divided into four groups, including no scaffold (Group Control-1), gelatin/HA/ß-TCP cryogel scaffold alone (group HAP), bone morphogenetic protein-2 (BMP-2) infused gelatin/HA/ß-TCP cryogel scaffold (group BMPi), and BMP-2 loaded microspheres encapsulated gelatin/HA/ß-TCP cryogel scaffold (group BMPm). The microspheres were composed of poly(D,L-lactide-co-glycolide) (PLGA) and were fabricated by electrohydrodynamic atomization technology. Bone fluorochromatic agents were injected after 3 and 14 days of scaffold implantation, and the animals were sacrifice after 4 weeks. Therapeutic efficiency was evaluated by microcomputed tomographic (micro-CT) imaging, bone fluorochromatic signals and histology. The second model was to evaluate the effect of the particle size of HA/ß-TCP in a rat intra-alveolar repair model. Acute intra-alveolar defects were surgically created after 4 weeks of maxillary first molars extractions and were filled with gelatin/HA/ß-TCP cryogel scaffold with regular size (250-1000 µm) of uncalcined bovine bone matrix（Group DBBM）, regular size (250-1000 µm) of HA/ß-TCP(Group HAPr) , smaller size (50-100 µm) of HA/ß-TCP(Group HAPs), or unfilled with any scaffold (Group Control-2). Animals were sacrifice at 8 weeks, and the therapeutic efficiency was evaluated by micro-CT imaging and histology. The gelatin/HA/ß-TCP cryogel scaffold was 439±56 µm in the pore size with 81.7±1.2 % in porosity. The microspheres were 9.29±1.89 µm in diameter, with an encapsulation efficiency of 64.9%±4.59 %, and encapsulating molecules were sustained release without obvious initial burst release in 2 weeks. In the first animal study, ridge augmentation was evident in all specimens treated with composites from the micro-CT imaging in 2 and 4 weeks. Stronger bone fluorochromatic signals were noted in Groups BMPi and BMPm compared to Group HAP. In the first 2 weeks, osteogenesis was significantly greater in Group BMPm relative to Group HAP (p<0.05). Supra-alveolar osseointegration with lamellar bone formation was only evident in Group BMPm in 4 weeks. In the second animal study, although no significant difference in osteogenesis among groups was noted from the micro-CT imaging, apparently greater osteogenesis was noted in Group HAPs relative to Groups HAPr and Group DBBM. In conclusion, gelatin/HA/ß-TCP cryogel scaffold showed the potential to promote alveolar ridge augmentation, and in combination with control-released BMP-2 microspheres further promoted supra-alveolar osteogenesis as well as osseointegration. In the gelatin/HA/ß-TCP cryogel scaffold, smaller size (50-100 µm) of HA/ß-TCP appeared to showed better osteogenic potential than the regular size (250-1000 µm) of HA/ß-TCP.