雙相型玻尿酸膠體於牙髓組織再生之應用：樣本製備與動物實驗之研究

Abstract

玻尿酸（hyaluronic acid, HA）材料因擁有良好生物相容性而廣泛應用於醫學領域，為了增加臨床實用性，雙相型玻尿酸膠體材料（biphasic hyaluronic acid gel, biHAG）結合了交聯型玻尿酸（crosslinked hyaluronic acid gel, cHAG）可提供穩定立體結構，以及非交聯型玻尿酸（non-crosslinked hyaluronic acid, ncHA）可於生物降解後釋放小分子玻尿酸並促進血管新生的特性， 做為牙髓組織再生之支架材料極具潛力。本研究團隊先前使用1,4-丁二醇縮水甘油醚（1,4-butanediol diglycidal ether, BDDE）做為交聯劑，並針對ncHA混合比例、膠體顆粒大小、交聯程度等因素進行各種不同 biHAG材料研發， 並證實命名為L80的材料系統具有良好黏彈性、可注射性與生物相容性，並在生物降解率有最佳的表現。然而進一步進行動物實驗，則因牙根樣本處理問題，導致無法取得可進行組織學分析的樣本，以探討L80在牙髓再生的應用性。因此本研究首要目標是使用完整牙齒與牙髓再生研究常用的牙根片段實驗模型做為樣本，提出在牙根表面製備人工窩洞以增進固定效率的新做法，比較牙根窩洞製備與脫鈣液種類對於樣本組織形態的影響，以建立最佳化的牙齒樣本組織固定和脫鈣處理程序。並將其應用到無菌小鼠皮下植入牙根片段的de novo動物實驗模型之樣本製備處理，以驗證所研發之L80在牙髓組織再生應用之潛力。研究結果發現，無論是完整牙齒組或是牙根片段組，經過牙根進行窩洞製備處理並搭配莫爾氏溶液進行脫鈣，可達成良好組織固定並大幅縮短樣本脫鈣時間，且不影響樣本的組織形態特徵，是適合用於牙齒樣本組織學分析的研究方法。而將L80與人類牙根尖乳突幹細胞（stem cell from apical papilla, SCAP）置入長3 mm牙根片段，並植入無菌小鼠背部皮下培養四週的動物實驗結果顯示，根管內未放置材料與細胞的控制組之根管有大量脂肪組織伴隨疏鬆結締組織的生成，而根管中放入L80的組別，根管空間則由富含血管的纖維結締組織所構成並伴隨少量殘餘材料所遺留的空腔。將L80與SCAP合併置入根管中的L80+SCAP組，可觀察根管內有較為緻密且富含血管的纖維結締組織生成，並伴隨有大量具有較大體積與較大細胞核的細胞散佈，推測可能是由置入的人類SCAP分化而來；同時可見這些細胞沿根管壁排列形成一層特化細胞層排列，疑似為類牙本質母細胞（odontoblast-like cells），具有類牙髓組織（pulp-like tissue）結構。總結而言，L80具有促進血管新生與纖維結締組織生長的效應，同時可阻擋脂肪細胞長入根管；而L80+SCAP可促進根管內纖維結締組織的生成能力，呈現類牙髓組織結構，證實其在誘導牙髓組織再生應用的潛力。

Hyaluronic acid (HA) is widely used in the medical field due to its excellent biocompatibility. The biphasic hyaluronic acid gel (biHAG), containing cross-linked hyaluronic acid gel (cHAG) as the major component for the strength and stability and non-crosslinked HA (ncHA) as additive for releasing small HA fragments to promote angiogenesis, shows great potential as scaffold material for pulp tissue regeneration. Our research team developed biHAGs with various mixing ratio of ncHA, granule sizes and degree of crosslinking using 1,4-butanediol diglycidyl ether (BDDE) as the crosslinking agent. We demonstrated the good viscoelasticity, injectability, and biocompatibility of biHAGs and found the biHAG with low crosslinking, named as L80, presenting favorable biodegradation behavior. However, due to issues with tissue preparation, no viable root samples were available for histological analysis to demonstrate the potential of L80 in pulp regeneration in vivo. Therefore, the primary aim of this study is to develop optimized histological processing protocols for dental tissues. This involved using whole teeth and root segments, prepared with artificial root cavities and treated with various decalcification solutions. Additionally, the potential of L80 for pulp regeneration in vivo was investigated using the de novo root implantation model in SCID mice, following the newly established optimized histological processing protocols. The results demonstrated that cavity preparation on dental roots combined with the use of Morse solution for decalcification achieved effective tissue fixation, significantly reduced decalcification time, and preserved the characteristic morphological features. This preparation protocol is suitable for histological analysis of dental tissue samples. Subsequently, 3 mm thick root fragment containing L80 and stem cells from the apical papilla (SCAP) in the canal were subcutaneous implanted in SCID mice for 4 weeks and the specimens were prepared for histological analysis. Histological observations reveled that the entire canal space in L80 group was filled with fibrous connective tissue containing abundant blood vessels, with some small empty spaces due to residual materials. In contrast, the control group show half of the canal space containing adipose tissue, while the other half exhibited loosely formed connective tissue formation. In L80+SCAP group, dense and vascular-rich fibrous connective tissue, with abundant larger cells with larger nuclei, was observed within the canal. Additionally, pulp-like tissue with a layer of large cells arranged along the root canal wall, suspected to be odontoblast-like cells, was noted. In summary, L80 promotes angiogenesis and fibrous tissue formation within the canal and can prevent adipose tissue infiltration into the canal space. Co-culturing L80 with human SCAP enhanced the formation of pulp-like tissue formation within the canal, highlighting its potential in pulp tissue regeneration applications.