TW201300371A - 細胞組織膠黏劑 - Google Patents

細胞組織膠黏劑 Download PDF

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TW201300371A
TW201300371A TW101122180A TW101122180A TW201300371A TW 201300371 A TW201300371 A TW 201300371A TW 101122180 A TW101122180 A TW 101122180A TW 101122180 A TW101122180 A TW 101122180A TW 201300371 A TW201300371 A TW 201300371A
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Taiwan
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acid
group
tissue adhesive
cell tissue
cell
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TW101122180A
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English (en)
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TWI461416B (zh
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Ling-Hui Huang
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Univ Nat Cheng Kung
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Abstract

本發明提供一種細胞組織膠黏劑，其包含一交聯劑(cross-linking agent)，其具有官能基；一個以上的基質分子(matrix molecule)與前述交聯劑產生交聯，以及一與前述交聯劑之官能基反應的阻停劑(quenching agent)。本發明更提供一種細胞植入物(cell implant)，其包含前述之細胞組織膠黏劑以及一細胞源，該細胞植入物可用於導入人體。本發明藉由添加可與交聯劑之官能基反應之阻停劑，可增加細胞組織膠黏劑之鍵結強度以及降低細胞毒性；並藉由前述優點使該細胞組織膠黏劑與細胞源結合所形成之細胞植入物可被輸入至人體之位置。

Description

細胞組織膠黏劑
一種細胞組織膠黏劑，尤指一種具有較佳鍵結強度以及低細胞毒性之細胞組織膠黏劑。
生醫材料之特性在於基材的生物功能性(biological function)、生物可降解性(biodegradability)、生物相容性(compatibility)、生物可吸收性(bio-absorption)以及無細胞毒性(cytotoxicity)，尤其係應用於臨床醫學及組織工程之細胞組織膠黏劑，可藉由具有黏著性之特性，達到控制藥物釋放、調控生物相容性材料分解或促進接合血管等功效。然而，現有技術之細胞組織膠黏劑係由諸如戊二醛(glutaraldehyde,GA)、乙二醛(glyoxal)、甲醛(formaldehyde)或三聚磷酸鈉(sodium tripolyphosphate,TPP)等交聯劑(cross-linking agent)與諸如明膠(gelatin)、幾丁聚醣(chitosan)或膠原蛋白(collagen)等載體材料交聯所形成，其中交聯劑具有細胞毒性，且容易導致強烈且持續性的免疫反應(immune response)，使得細胞組織膠黏劑具有毒性。
鑒於現有技術之細胞組織膠黏劑具有毒性，故本發明之目的在於提供一種細胞組織膠黏劑，其中所包含之交聯劑與阻停劑可增強細胞組織膠黏劑的鍵結強度以及降低其細胞毒性。
為達上述目的，本發明提供一種細胞組織膠黏劑，其 包含一交聯劑(cross-linking agent)，其具有官能基；一個或以上的基質分子(matrix molecule)，其與前述交聯劑產生交聯，以及一與前述交聯劑之官能基反應的阻停劑(quenching agent)。
依據本發明，「基質分子」於此所指涉者係可於細胞外基質被發現，或可以與細胞共存之高分子量物質，並有助於保持細胞於植入位置之分子。本發明之細胞組織膠黏劑中的基質分子，偏好選用可提高膠黏劑黏度的高分子量物質。
較佳的，所述之基質分子係選自於由膠原蛋白(collagen)、透明質酸(hyaluronan)、明膠(gelatin)、纖維連蛋白(fibronectin)、彈性蛋白(elastin)、細胞粘合素(tenascin)、基膜素(laminin)、玻連蛋白(vitronectin)、硫酸乙醯肝素(heparan sulfate)、軟骨素(chondroitin)、硫酸軟骨素(chondroitin sulfate)、角質素(keratan)、硫酸角質素(keratan sulfate)、硫酸皮膚素(dermatan sulfate)、卡拉膠(carrageenan)、肝素(heparin)、甲殼素(chitin)、幾丁聚醣(chitosan)、海藻酸鹽(alginate)、瓊膠(agarose)、洋菜(agar)、纖維素(cellulose)、肝醣(glycogen)、纖維蛋白、纖維蛋白原、凝血酶、聚麩胺酸、合成聚合物(例如，丙烯酸酯、聚乳酸、聚乙醇酸、聚乳酸-甘醇酸)及其上述基質分子衍生物所組成的群組。舉例而言，纖維素的衍生物係甲基纖維素(methyl cellulose)、羧基甲基纖維素(carboxyl methyl cellulose)等。
依據本發明，「膠原蛋白」於此所指涉者包括任何自然產生的膠原蛋白或其官能變體，諸如其膠原蛋白衍生物 或降解物。
依據本發明，「透明質酸」於此所指涉者係具有陰離子性且非硫化的糖胺聚多醣(non-sulfated glycosaminoglycan)，其包括D-葡萄糖醛酸(D-glucuronic acid)和N-乙醯麩胺酸(N-acetylglucosamine)所構成的雙醣單元組合及其衍生物。
在較佳的實施例中，基質分子係選自於膠原蛋白、透明質酸及明膠所組成的群組。
在另一較佳的實施例中，基質分子係選自於膠原蛋白及透明質酸所組成的群組，且該膠原蛋白與該透明質酸的重量比係0.01至100：1。
依據本發明，「交聯劑」於此所指涉者係該交聯劑能與目標分子反應並與目標分子連結在一起，且該交聯劑通常有兩個或以上之官能反應基，該等官能反應基可相同或不同。由表1所示，交聯劑可以係含有：胺基(amine)、氫硫基(sulfhydryl)、羰基(carbonyl)、乙二醇(glycol)、羧基(carboxyl)、疊氮(azide)、亞胺酯(imidoester)、環氧化物(epoxide)、醛基(aldehyde)、haloacetyl、吡啶二硫化物(pyridyl disulfide)、醯胼(hydrazide)、光交聯基團(photo-crosslinking group)、碳二醯亞胺(carbodiimide)、雙吖丙啶(diazirine)、梔子素(genipin)、核黃素(riboflavin)、類黃酮(flavonoid)及其衍生物、羥甲基膦(hydroxymethyl phosphine)、異氰酸酯類(isocyanate)、馬來亞醯胺(maleimide)、N-羥基琥珀酰亞胺(N-hydroxy-succinimide,NHS-ester)、五氟苯酯(pentafluorophenyl ester,PFP-ester)、 補骨脂素(psoralen)及乙烯基碸(vinyl sulfone)的官能反應基化合物。
較佳的，所述之交聯劑包括，但不限於亞胺酯(imidoester)、環氧化物(epoxide)、乙二醇二環氧丙基醚(ethylene glycol diglycidyl ether)、戊二醛(glutaraldehyde)、2,3-二溴丙酰N-羥基丁二醯亞胺酯(2,3-dibromopropionyl-N-hydroxysuccinimide ester)、磺酸基 -N-羥基丁二醯亞胺酯(sulfo-N-hydroxysuccinimide ester)、苯丁酸氮芥N-羥基丁二醯亞胺酯(chlorambucil-N-hydroxysuccinimide ester)、1-乙基-3-[3-二甲氨基]碳二醯亞胺(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)、疊氮(azide)、雙吖丙啶(diazirine)、梔子素(genipin)、核黃素(riboflavin)、類黃酮(flavonoid)及其衍生物、haloacetyl、吡啶二硫化物(pyridyl disulfide)、醯肼(hydrazide)、6-(馬來醯亞胺基)己酸活性酯(6-maleimidohexanoic acid active ester)、雙琥珀亞胺醯辛二酸酯或根(disuccinimidyl suberate)、4-(N-馬來醯亞胺甲基)環己烷-1-羧酸磺酸基琥珀醯亞胺酯[sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate]及雙琥珀醯亞胺辛二酸酯[bis(sulfosuccinimidyl)suberate]。
更佳的，所述之類黃酮包括，但不限於原花青素(proanthocyanidin)、兒茶素(catechin)、表兒茶素(epicatechin)、沒食子兒茶素(epigallocatechin)、培酸表兒茶酯(epicatechingallate)、沒食子兒茶素沒食子酸酯(epigallocatechingallate)、槲黃素(quercetin)、查耳酮(chalcones)、芹菜素(apigenin)、木犀草素(luteolin)、多甲氧基黃酮(polymethoxylated flavone)、槲皮醇(quercitoi)、山柰酚(kaempferol)、楊梅素(myricetin)、花青素(anthocyanin)、白藜蘆醇(resveritrol)、異黃酮(isoflavanoid)、大豆黃酮(daidzein)、染料木黃酮(genistein)、川陳皮素(nobiletin)、桔皮素(tangeretin)及單寧酸(tannic acid)。
依據本發明，「阻停劑」於此所指涉者係與交聯劑反應基團反應的試劑，當目標分子與交聯劑交聯後，阻停劑可與未和目標官能反應基團反應的交聯劑反應基團發生反應。藉著「耗盡」未反應的反應基團，阻停劑可以完全或部分減少交聯劑的毒性。
較佳的，所述之阻停劑包括，但不限於胺基酸、寡胜肽、多胜肽、蛋白質、二胺(diamine)、寡胺(oligoamine)、多胺(polyamine)、二羧酸(dicarboxylic acid)、寡羧酸(oligo-carboxylic acid)、聚羧酸(polycarboxylic acid)、多硫醇化合物(polysulfhydryl compound)、多羥基化合物(polyhydroxy compound)、寡醣、多醣、核醣核酸(ribonucleic Acid,RNA)、去氧核醣核酸(deoxyribonucleic acid,DNA)及含單官能基或異雙官能基(heterobifunctional)之化合物。
在較佳的實施例中，當交聯劑係含有與胺基反應之基團，阻停劑包含但不限於胺基酸、多胜肽、蛋白質、雙胺、寡胺、聚胺如聚離胺酸以及聚麩醯胺酸(polyglutamine)。
在另一較佳的實施例中，當交聯劑係含有與羧基反應之基團，阻停劑包含但不限於胺基酸、多胜肽、蛋白質、二元羧酸、寡羧酸、聚羧酸或聚麩胺酸鹽(polyglutamate)。
在另一較佳的實施例中，當交聯劑係含有與硫醇基反應之基團，阻停劑包含但不限於硫醇化合物或聚硫醇化合物。
在另一較佳的實施例中，當交聯劑係含有與羥基反應之基團，阻停劑包含但不限於聚羥類的化合物。
在另一較佳的實施例中，交聯劑為梔子素，阻停劑包 含但不限於聚離胺酸(polylysine)，且聚離胺酸的平均分子量通常大於兩萬道耳吞(20 kDa)，例如，大於99 kDa，或大於212 kDa。
在另一較佳的實施例中，交聯劑為環氧化物，諸如乙二醇二環氧丙基醚(ethylene glycol diglycidyl ether)，阻停劑包含但不限於係水。
在另一較佳的實施例中，交聯劑包含但不限於係乙二醇二環氧丙基醚，阻停劑係聚離胺酸或聚麩胺酸(poly glutamic acid)。
較佳的，所述之細胞組織膠黏劑更包含一營養物質(nutrient)、一生化活性分子(bioactive agent)或細胞。
依據本發明，「營養物質」於此所指涉者係細胞生長必需的營養來源，其係胺基酸、維生素、礦物質、碳源(如葡萄糖)、脂肪酸或上述的混合物。較佳的，所述之營養物質係細胞培養基，其包括但不限於最小必須培養基(Minimum Essential Medium,MEM)、杜氏改良英格爾培養基(Dulbecco's Modified Eagle's medium,DMEM)、基礎依格培養基(Basal Medium Eagle,BME)、營養混合培養基(Ham's Nutrient Mixtures F-10或F-12)、培養基199(Medium 199)、羅斯微公園研究所培養基(Roswell Park Memorial Institute medium,RPMI medium)、安姆氏培養基(Ames' Medium)、BGJb Medium(Fitton-Jackson Modification)、克利克培養基(Click's Medium)、CMRL-1066 Medium、費雪培養基(Fischer's Medium)、格拉斯哥最小必需培養基(Glasgow Minimum Essential Medium)、Iscove's Modified Dulbecco's Medium、L-15 Medium、McCoy's 5A Modified Medium、NCTC Medium、Swim's S-77 Medium、Waymouth Medium或William's Medium E。
依據本發明，「生化活性分子」於此所指涉者係任何可增進細胞存活能力、提昇細胞增生能力或誘導細胞分化之試劑。
較佳的，所述之生化活性分子包括，但不限於胜肽、多胜肽、寡醣、多醣、小分子、生長因子、細胞素、化學趨化激素、細胞分化因子、中草藥、中草藥之有效成分，或上述物質之組合物。
較佳的，所述之生化活性分子係生長因子，其包括但不限於表皮生長因子(epidermal growth factor,EGF)、纖維母細胞生長因子(fibroblast growth factor,FGF)、血管內皮生長因子(vascular endothelial growth factor,VEGF)、結締組織生長因子(connective tissue growth factor,CTGF)、血小板衍生生長因子(platelet-derived growth factor,PDGF)、類胰島素生長因子(insulin-like growth factor,IGF)、神經生長因子(nerve growth factor,NGF)、肝細胞生長因子(hepatocyte growth factor,HGF)、群落刺激因子(colony-stimulating factor,CSF)、幹細胞因子(stem cell factor,SCF)、角質細胞生長因子(keratinocyte growth factor,KGF)、白血球生長因子(granulocyte colony-stimulating factor,GCSF)、巨噬細胞群落刺激因子(granulocyte macrophage colony-stimulating factor)、神經膠衍生神經滋養因子(glial derived neurotrophic factor,GDNF)、毛狀神經 營養因子(ciliary neurotrophic factor)、內皮細胞單核細胞活性多肽(endothelial-monocyte activating polypeptide)、表皮中性球活性多肽(epithelial neutrophil activating peptide)、紅血球生成素(erythropoietin)、骨塑型蛋白(bone morphogenetic protein)、腦衍生神經滋養因子(brain-derived neurotrophic factor)、血清素(serotonin)、溫韋伯氏因子(von Willebrand factor)、轉換生長因子-β(transforming growth factor-β,TGF-β)、腫瘤壞死因子(tumor necrosis factor,TNF)或上述物質之組合物。
在較佳的實施例中，該生化活性分子係一細胞素/化學趨化激素(cytokines/chemokine)，其包括但不限於胸腺表現化學趨素(breast-expressed chemokine,BRAK)、腎表現化學趨化激素(kidney-expressed chemokine,CXCL14)或上述物質之組合物。
在另一較佳的實施例中，該生化活性分子係一細胞分化因子，其包括但不限於人工合成皮質類固醇(dexamethasone)、丙酮酸鈉(sodium pyruvate)、維生素C磷酸鹽(ascorbic acid-2-phosphate)、維生素C(ascorbic acid)、維生素A酸(retinoic acid)、脯胺酸(proline)、胰島素(insulin)、運鐵蛋白(transferrin)、亞硒酸(selenous acid)、亞油酸(linoleic acid)、血清白蛋白、轉換生長因子-β3(transforming growth factor beta 3,TGF-ß3)或上述物質之組合物。
在另一較佳的實施例中，該分化因子為可促進間葉幹細胞的軟骨生成、骨新生[如地塞米松(dexamethasone)、維 生素C(ascorbic acid)、β-磷酸甘油(β-glycerol phosphate)]、脂肪生成[如胰島素、異丁基甲基黃嘌呤(isobutylmethylxanthine)、地塞米松、引朵美洒辛(indomethacin)]、心肌分化[如激活素A(activin A)、骨形態生成蛋白4(bone morphogenetic protein-4,BMP-4)]、內皮細胞分化[如內皮細胞(EBM-2)、地塞米松以及血管內皮生長因子(VEGF)]、平滑肌細胞分化[如血小板衍生生長因子(PDGF)]、神經誘導[如鹼性纖維母細胞生長因子(basic fibroblast growth factor,bFGF)、表皮生長因子(EGF)、B27 supplement、二甲基亞碸(DMSO)、丁基羥基甲氧苯(butylated hydroxyanisole)、錦紫蘇素(forskolin)、丙戊酸(valproic acid)、氯化鉀、K252a以及N2 supplement]或內胚層譜系分化(endodermal lineage differentiation)[如地塞米松、肝細胞生長因子(HGF)和纖維母細胞生長因子-4(FGF-4)]。
在更佳的實施例中，生化活性分子係中草藥或是中草藥之有效成分。
較佳的，所述之細胞係由哺乳類動物所獲得的細胞源。
更佳的，所述之細胞源係幹細胞、組織前驅細胞(tissue progenitor cells)、芽細胞、成纖維母細胞、組織細胞、基質細胞、基因工程細胞或上述細胞之組合物。
較佳的，所述之幹細胞，其包括但不限於胚胎幹細胞、胎盤幹細胞、骨髓幹細胞、基質細胞(如脂肪基質細胞)、間葉幹細胞、人工多能幹細胞(induced pluripotent stem cells,iPSC)，進而使含有細胞源以及細胞組織膠黏劑之細胞植入物，能被輸送到人體之目標位置。
本發明更提供一種細胞植入物(cell implant)，其包含前述之細胞組織膠黏劑以及一細胞源，該細胞植入物可用於導入人體。依據本發明，「導入人體」於此所指涉者係將細胞植入物以任一種方式由人體外移至人體內，其包括任何可將細胞植入物置於人體之方法。
本發明藉由添加可與交聯劑之官能基反應之阻停劑，該阻停劑可與未和目標官能反應基團反應的交聯劑反應基團發生反應，進而完全或部分減少交聯劑的毒性，亦可增加細胞組織膠黏劑之鍵結強度；並藉由前述優點使該細胞組織膠黏劑與細胞源結合所形成之細胞植入物可被植入至人體之位置，以進行組織修復和其它的治療目的。
本發明將由下列的實施例做為進一步說明，這些實施例並不限制本發明前面所揭示的內容。熟習本發明之技藝者，可以做些許之改良與修飾，但不脫離本發明之範疇。
製備例 材料與方法
1.基質分子
基質分子可由任何自然產生的膠原蛋白或其官能變體以及透明質酸製備而得。
(1)膠原蛋白
目前，至少已發現28種不同基因物種之膠原蛋白。膠原蛋白可以簡單的從人類或動物的富含膠原蛋白組織中分離純化出來，如皮膚、肌腱、韌帶和骨組織。分離純化膠原蛋白方法於此領域中已是眾所周知。(可參照例如：美國專 利5,512,291、美國專利申請公開20040138695號公報、Methods in Enzymology,vol.82,pp.33-64,1982、J.Am.Leather Chemists Assoc.,Vol.LXV,pp.440-450,1970以及美國專利6,090,996)
膠原蛋白亦能使用重組技術製備，如在Advanced Tissue Sciences(La Jolla,Calif.)所描述的，或從不同的供應商購得(如Fibrogen；South San Francisco,Calif.)。以下是一實施例：牛深屈肌腱，除去脂肪及筋膜後用水洗滌，凍結，並以切片器切成0.5毫米(mm)薄片。先將適量切片肌腱用50毫升(mL)的水在室溫下萃取24小時。丟棄水溶性部分，然後切片過的肌腱與酸性溶液(如0.2 N鹽酸)在適當的溫度(如室溫)下萃取一段適當的時間(如12小時至24小時)。
接著，使用水來洗去殘留在肌腱上的酸液。沖洗過的肌腱使用鹼性溶液(如0.75 M氫氧化鈉)在適當的溫度(如室溫)進行萃取一段適當的時間(如12小時至24小時)。之後移除鹼性溶液，使用酸性溶液(如0.1 N鹽酸)來中和切片肌腱直到pH值為4至7(如pH 5)，接著重複使用水來洗去殘餘在肌腱的鹼液。之後將肌腱使用醇類(如異丙醇)來於室溫中脫脂一段足夠的時間(如16小時)。將萃取物倒出並且使用醇類(如異丙醇)於室溫中對肌腱作進一步萃取一段時間(如12小時至24小時)，形成含膠原蛋白的溶液，可以在乾淨的抽風櫃中乾燥。因此形成的膠原蛋白粉可以溶散於酸性溶液(如0.5 M或0.25 M醋酸)中，在含有蛋白水解酶(如胰蛋白酶或胃蛋白酶)的存在下於4℃中反應一段適當的時間。使用100目之不銹鋼網篩來過濾混合物，並可以使用 5%食鹽水使可溶性膠原蛋白沈澱，沈澱出的膠原蛋白可用上述的酸性溶液再溶解，因此形成的溶液可以使用100目不銹鋼網篩來過濾，以移除不溶微粒。然後此膠蛋白溶液於純水中透析來除去溶液中的酸。
(2)透明質酸
透明質酸可以從天然物中分離出來，如囊狀鏈球菌、雞冠、軟骨、滑膜關節液、臍帶、皮膚組織和眼睛的玻璃體，藉由諸如Guillermo Lago et al.Carbohydrate Polymers 62(4)：321-326,2005及Ichika Amagai et al.Fisheries Science 75(3)：805-810,2009所述之現有技術方法。或者，可向商業的供應商，例如Genzyme Corporation、Lifecore Biomedical,LLC以及Hyaluron Contract Manufacturing購買。
天然的透明質酸的衍生品包括但不限於下列透明質酸酯(hyaluronan esters)、己二酸二醯肼-修飾透明質酸(adipic dihydrazide-modified hyaluronan)、透明質酸醯胺產品(hyaluronan amide products)、交聯透明質酸(crosslinked hyaluronic acid)、半脂丁二酸(hemiesters of succinic acid)或透明質酸之重金屬鹽類、部分或全脂化透明質酸、硫化透明質酸(sulphated hyaluronic acid)、N-硫化透明質酸(N-sulphated hyaluronic acid)及胺或二胺修飾透明質酸。
透明質酸衍生物亦可藉由一個以上之官能基化學修飾而獲得，其中該官能基包括，但不限於羧酸基(carboxylic acid group)、羥基(hydroxyl group)、還原性端基(reducing end group)及N-乙醯基(N-acetyl group)；其中羧酸基的修飾反 應可藉由碳二醯亞胺(carbodiimide)及雙醯肼(bishydrazide)之催化或酯化反應(esterification)來修飾；其中羥基的修飾反應，包括但不限於硫酸化(sulfation)、酯化、異脲耦合(isourea coupling)，溴化氰活化(cyanogen bromide activation)及高碘酸氧化(periodate oxidation)；還原性端基可藉由還原胺化(reductive amination)以修飾還原性端基。
透明質酸也可連結於磷脂質、染料(如螢光基團或發色團)以及用以製備親核性基質的試劑。
天然的透明質酸之衍生物也可以藉著使用交聯劑、分子內酯化(internal esterification)、光交聯反應(photocross-linking)或表面電漿處理(surface plasma treatment)而獲得，其中使用之交聯劑包括，但不限於雙環氧化物(bisepoxide)、二乙烯碸(divinylsulfone)、雙碳二亞胺(biscarbodiimide)、分子較小的雙官能基架橋劑、甲醛(formaldehyde)、異氰酸環己酯(cyclohexyl isocyanide)、離胺酸乙酯(lysine ethyl ester)、金屬陰離子、醯肼(hydrazide)或上述之組合。
目前已顯示透明質酸，特別是高分子量的透明質酸(即大於5 kDa)，可有效地促進血管新成，進而促進傷口癒合。例如美國暫時申請案61/390,789，透明質酸之分子量係50 kDa至5,000 kDa、70kDa至1,500 kDa、200 kDa至1,500 kDa、500 kDa至1,500 kDa或700 kDa至1,500 kDa。
當細胞生長於膠原蛋白或透明質酸之基質中，具有良好存活能力。本發明之細胞組織膠黏劑之基質分子，其中透明質酸之濃度係每毫升0.001毫克(mg/mL)至100 mg/mL；膠原蛋白之濃度係0.001 mg/mL至100 mg/mL。
較佳的，膠原蛋白之濃度為0.1 mg/mL至100 mg/mL，透明質酸之濃度係0.01 mg/mL至35 mg/mL。
更佳的，膠原蛋白之濃度係3 mg/mL至75 mg/mL(如6 mg/mL或9 mg/mL)，透明質酸之濃度係0.2 mg/mL至20 mg/mL。
2.交聯劑
此處所使用之交聯劑係梔子素。
3.阻停劑
此處所使用之阻停劑包括精胺(spermine)、魚胺類(protamine)、1-6己二胺(1,6-hexanediamine)，聚離胺酸則係不同組別的分子量。聚離胺酸的平均分子量分別為3.4 kDa、20 kDa、99 kDa、212 kDa以及225 kDa。
實施例1：細胞組織膠黏劑之連結強度測試
將等當量的聚離胺酸與300 mg/mL之明膠在磷酸鹽緩衝液中混合，以形成聚離胺酸-明膠溶液，再將等體積且濃度為20 mg/mL的梔子素溶液與上述的聚胺-明膠溶液混合以形成細胞組織膠黏劑，再加到豬皮樣品[其尺寸大小係10x30x0.7至0.9立方公釐(mm3)]的真皮側。兩片豬皮樣品以真皮面相對，中間為細胞組織膠黏劑(10x10 mm2)，將50克重的砝碼放在兩片豬皮樣品的上方30分鐘，並以LRX材料測試系統(Lloyd Instruments Ltd.,England)以每分鐘10釐米(mm/min)速率的拉力下，分離兩片豬皮樣品以測試細胞組織膠黏劑的連結強度。
結果顯示當聚離胺酸的分子量高於20 kDa時可增進細 胞組織膠黏劑的連結強度，且含有聚離胺酸的細胞組織膠黏劑的連結強度會隨著聚離胺酸濃度的上升而增強，直到聚離胺酸濃度高達46.8 mN。對於使用17.5 mN聚離胺酸製成的細胞組織膠黏劑以及未使用聚離胺酸的膠黏劑進行的研究顯示，含有聚離胺酸的細胞組織膠黏劑之連結強度的最大值大於未使用聚離胺酸的細胞組織膠黏劑。
無論梔子素的濃度，隨著聚離胺酸含量的升高，細胞組織膠黏劑的連結強度將上升；此外，連結強度也隨著梔子素濃度的提高而增強。
實施例2：細胞組織膠黏劑之毒性評估
將10 μL之細胞組織膠黏劑加入24孔盤之培養皿中央，再種入2x104個細胞/孔(cell/well)之纖維母細胞，並培養於5%二氧化碳(CO2)、37℃環境下歷經38小時。存活的細胞數量由血球計數板來定量。
無聚離胺酸之細胞組織膠黏劑的毒性隨著梔子素濃度的上升而提高。加入聚離胺酸可顯著的減少梔子素的毒化效應。當使用7.5 mg/mL的梔子素與46.8 mN的聚離胺酸時，觀察不到顯著的細胞毒性效應(cytotoxic effect)。
實施例3：細胞組織膠黏劑之流變性質(rheological properties)
本實施例之目的係探討聚離胺酸對於細胞組織膠黏劑的物理及化學特性的影響。藉由RheoStress RS 150(Haake,Germany)可測量含有及未含聚離胺酸的細胞組織膠黏劑的流變性質，並即時記錄彈性儲存模數(elastic storage modulus,G')及反應溫度於固定剪應力(fixed shear stress)為 1帕(Pa)以及頻率為1赫茲(Hz)之震盪測試條件下之數據。
結果顯示，彈性儲存模數之數值隨著細胞組織膠黏劑從液態的形成而增加。彈性儲存模數之數值在加入聚離胺酸時有顯著的提高。彈性儲存模數之數值在不同反應溫度的變化中偵測，並且當反應在50℃下進行，反應時間為90分鐘時顯示彈性儲存模數之數值有急劇的上升。此相轉移現象顯示聚離胺酸參與了細胞組織膠黏劑的交聯反應，並且增加了細胞組織膠黏劑的彈性儲存模數。無添加聚離胺酸時則沒有觀察到相轉移現象。

Claims (23)

  1. 一種細胞組織膠黏劑，包括：一交聯劑(cross-linking agent)，其具有官能基；一個以上基質分子(matrix molecule)，其與前述交聯劑產生交聯；以及一與前述交聯劑之官能基反應的阻停劑(quenching agent)。
  2. 如請求項1所述之細胞組織膠黏劑，其中該交聯劑係選自於含有胺基(amine)、氫硫基(sulfhydryl)、羰基(carbonyl)、乙二醇(glycol)、羧基(carboxyl)、疊氮(azide)、亞胺酯(imidoester)、環氧化物(epoxide)、醛基(aldehyde)、haloacetyl、吡啶二硫化物(pyridyl disulfide)、醯胼(hydrazide)、光交聯基團(photo-crosslinking group)、碳二醯亞胺(carbodiimide)、雙吖丙啶(diazirine)、梔子素(genipin)、核黃素(riboflavin)、類黃酮(flavonoid)及其衍生物、羥甲基膦(hydroxymethyl phosphine)、異氰酸酯類(isocyanate)、馬來亞醯胺(maleimide)、N-羥基琥珀酰亞胺(N-hydroxy-succinimide,NHS-ester)、五氟苯酯(pentafluorophenyl ester,PFP-ester)、補骨脂素(psoralen)、乙烯基碸(vinyl sulfone)的官能反應基化合物所組成的群組。
  3. 如請求項1所述之細胞組織膠黏劑，其中該基質分子係選自於由膠原蛋白(collagen)、透明質酸(hyaluronan)、明膠(gelatin)、纖維連蛋白(fibronectin)、彈性蛋白(elastin)、細胞粘合素(tenascin)、基膜素(laminin)、玻連蛋白 (vitronectin)、硫酸乙醯肝素(heparan sulfate)、軟骨素(chondroitin)、硫酸軟骨素(chondroitin sulfate)、角質素(keratan)、硫酸角質素(keratan sulfate)、硫酸皮膚素(dermatan sulfate)、卡拉膠(carrageenan)、肝素(heparin)、甲殼素(chitin)、幾丁聚醣(chitosan)、海藻酸鹽(alginate)、瓊膠(agarose)、洋菜(agar)、纖維素(cellulose)、肝醣(glycogen)、纖維蛋白、纖維蛋白原、凝血酶、聚麩胺酸、丙烯酸酯、聚乳酸、聚乙醇酸、聚乳酸-甘醇酸及上述基質分子之衍生物所組成的群組。
  4. 如請求項1所述之細胞組織膠黏劑，其中該阻停劑係選自於由胺基酸、寡胜肽、多胜肽、蛋白質、二胺(diamine)、寡胺(oligoamine)、多胺(polyamine)、二羧酸(dicarboxylic acid)、寡羧酸(oligo-carboxylic acid)、聚羧酸(polycarboxylic acid)、多硫醇化合物(polysulfhydryl compound)、多羥基化合物(polyhydroxy compound)、寡醣、多醣、核醣核酸(ribonucleic Acid,RNA)、去氧核醣核酸(deoxyribonucleic acid,DNA)及含單官能基或異雙官能基(heterobifunctional)之化合物所組成的群組。
  5. 如請求項1所述之細胞組織膠黏劑，其中該交聯劑係選自於由亞胺酯(imidoester)、環氧化物(epoxide)、乙二醇二環氧丙基醚(ethylene glycol diglycidyl ether)、戊二醛(glutaraldehyde)、2,3-二溴丙酰N-羥基丁二醯亞胺酯(2,3-dibromopropionyl-N-hydroxysuccinimide ester)、磺酸基-N-羥基丁二醯亞胺酯(sulfo-N-hydroxysuccinimide ester)、苯丁酸氮芥N-羥基丁二醯亞胺酯(chlorambucil-N- hydroxysuccinimide ester)、1-乙基-3-[3-二甲氨基]碳二醯亞胺(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)、疊氮(azide)、雙吖丙啶(diazirine)、梔子素(genipin)、核黃素(riboflavin)、類黃酮(flavonoid)及其衍生物、haloacetyl、吡啶二硫化物(pyridyl disulfide)、醯肼(hydrazide)、6-(馬來醯亞胺基)己酸活性酯(6-maleimidohexanoic acid active ester)、雙琥珀亞胺醯辛二酸酯或根(disuccinimidyl suberate)、4-(N-馬來醯亞胺甲基)環己烷-1-羧酸磺酸基琥珀醯亞胺酯[sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate]及雙琥珀醯亞胺辛二酸酯[bis(sulfosuccinimidyl)suberate]所組成的群組。
  6. 如請求項1所述之細胞組織膠黏劑，其中該交聯劑係梔子素，該阻停劑係聚離胺酸(polylysine)。
  7. 如請求項6所述之細胞組織膠黏劑，其中該聚離胺酸的平均分子量大於20 kDa。
  8. 如請求項7所述之細胞組織膠黏劑，其中該聚離胺酸的平均分子量大於99 kDa。
  9. 如請求項8所述之細胞組織膠黏劑，其中該聚離胺酸的平均分子量大於212 kDa。
  10. 如請求項1所述之細胞組織膠黏劑，其中該交聯劑係乙二醇二環氧丙基醚(ethylene glycol diglycidyl ether)，該阻停劑係水。
  11. 如請求項1所述之細胞組織膠黏劑，其中該交聯劑係乙二醇二環氧丙基醚，該阻停劑係聚離胺酸或聚麩胺酸(poly glutamic acid)。
  12. 如請求項3所述之細胞組織膠黏劑，其中該基質分子係選自於由膠原蛋白、透明質酸及明膠所組成的群組。
  13. 如請求項12所述之細胞組織膠黏劑，其中該基質分子係選自於由膠原蛋白及透明質酸所組成的群組。
  14. 如請求項3所述之細胞組織膠黏劑，其中該基質分子係明膠。
  15. 如請求項10或11任一項所述之細胞組織膠黏劑，其中該基質分子係膠原蛋白。
  16. 如請求項10或11任一項所述之細胞組織膠黏劑，其中該基質分子係明膠。
  17. 如請求項1所述之細胞組織膠黏劑，其更包括一營養物質及一生化活性分子。
  18. 如請求項17所述之細胞組織膠黏劑，其中該營養物質係細胞培養基。
  19. 如請求項17所述之細胞組織膠黏劑，其中生化活性分子係選自於生長因子、細胞素、化學趨化激素、細胞分化因子、中草藥、中草藥之有效成分，或上述物質之組合物所組成的群組。
  20. 如請求項19所述之生長因子係選自於由表皮生長因子(epidermal growth factor,EGF)、纖維母細胞生長因子(fibroblast growth factor,FGF)、血管內皮生長因子(vascular endothelial growth factor,VEGF)、結締組織生長因子(connective tissue growth factor,CTGF)、血小板衍生生長因子(platelet-derived growth factor,PDGF)、類胰島素生長因子(insulin-like growth factor,IGF)、神經生長因子(nerve growth factor,NGF)、肝細胞生長因子(hepatocyte growth factor,HGF)、群落刺激因子(colony-stimulating factor,CSF)、幹細胞因子(stem cell factor,SCF)、角質細胞生長因子(keratinocyte growth factor,KGF)、白血球生長因子(granulocyte colony-stimulating factor,GCSF)、巨噬細胞群落刺激因子(granulocyte macrophage colony-stimulating factor)、神經膠衍生神經滋養因子(glial derived neurotrophic factor,GDNF)、毛狀神經營養因子(ciliary neurotrophic factor)、內皮細胞單核細胞活性多肽(endothelial-monocyte activating polypeptide)、表皮中性球活性多肽(epithelial neutrophil activating peptide)、紅血球生成素(erythropoietin)、骨塑型蛋白(bone morphogenetic protein)、腦衍生神經滋養因子(brain-derived neurotrophic factor)、血清素(serotonin)、溫韋伯氏因子(von Willebrand factor)、轉換生長因子-β(transforming growth factor-β,TGF-β)、腫瘤壞死因子(tumor necrosis factor,TNF)，或上述物質之組合物所組成的群組。
  21. 如請求項1或17任一項所述之細胞組織膠黏劑，其進一步包括細胞源。
  22. 如請求項13所述之細胞組織膠黏劑，其中膠原蛋白和透明質酸的重量比係0.01至100：1。
  23. 一種細胞植入物(cell implant)，其中該細胞植入物(cell implant)包含有如請求項1或17任一項所述之細胞組織膠黏劑及一細胞源。
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