1301415 九、發明說明： 【發明所屬之技術領域】 本發明係有關於一種在水 粒膠原蛋白之方法’可供應用於生 主要係在於將膠原蛋白溶人水相環境中域’其 正、負極性之電場環境中，以 ^置入於通有 原蛋白。 成奈米尺寸之微粒耀 【先前技術】 修、人=月、、且、、我中如有叉到傷害或疾病而缺損時，醫療 修補以自體移植骨組織為最佳之選擇，但骨組織之來= 限^且患者必_受二次手術之苦，但若以賤移植貝^ 排斥及併發症等缺失存在。而人卫合成的方式，提供了一 種新選擇，其中以㈣蛋白最為適合培養新的骨細胞生 長0 膠原蛋白是哺乳動物中含量最多的結構性蛋白質，廣 泛存在於皮膚、軟骨及骨骼組織中，約佔所有蛋白質含量 的20-25%，膠原蛋白約由1〇〇〇多個胺基酸組成，其中主 要是由甘胺酸〔glycine，33· 5%〕、脯胺酸〔pr〇iine， 12%〕、經基脯胺酸〔hydroxyproline，10%〕三種胺基 酸，經基脯胺酸〔hydroxyproline〕是膠原蛋白分子中特 有的胺基酸，一般可藉由羥基脯胺酸的含量來測得膠原蛋 白的濃度，這些胺基酸所組成的多生肽鏈，因甘胺酸分子 較小，可使多生肽鏈產生適度的摺疊，而使多生肽鏈以左 1301415 旋的α _ chain螺旋形式存在。三條α _ chain利用氫鍵 及i|IL水! 生基團^/成右旋的三股螺旋，此稱為膠原蛋白原 〔tropocollagen〕，而羥基脯胺酸的功能是用來形成分子 内氳鍵’以穩^三股螺旋結構。不同的胺基酸組成，胺基 與排歹J順序可組成不同的α _ chain，膠原蛋白是由三 條互相纏繞的α - 形成三股職結構且三股不同的 a - chain可構成不同型式的膠原蛋白，例如：膠原蛋白 第-^的結，為〔αι(Ι )〕_，而在透明軟骨中的膠原 蛋白第一型疋由〔ai(jj)〕3所組成的。至目前為止分佈 於動物體内的各種組織中約有”種膠原蛋白已明確被定 義，其中第二型膠原蛋白即為軟骨基質中最主要的膠原蛋 白成份，約佔透明軟骨乾重的4〇%。 在膠原蛋白原彼此會藉由分子氳鍵、疏水基團的作用 與共價鍵結形«顧維原〔eQUagen fibH1〕之後膠 原纖維原會彼此聚集形成直徑更粗的膠原纖維〔灿_ 他30’卿成的膠原纖維與其他成份〔例如：葡萄㈣ ^酶、聚玻璃醣_等〕組合而成基質〔matrix〕，膠 成一網狀結構’其功能除可避免組織被拉伸 ，，也具有分散力量的作用，提供組織所需的機械強 度0 ο「„ 膠原蛋白分子⑺表面包含非極性 極性的帶電區(Τ2)，其在疏水區_ 夕刀田Α度達到3代時，這些水分子會相互聚集 1301415 脫離膠原蛋白，使得疏水區（τι)暴露出而能相互作用；再 加上帶電區（Τ2)的靜電作用力，使得膠原蛋白分子彼此結 合，自組成纖維結構。 又因蛋白質分子，有酸性基團〔-C00H〕與鹼性基團 〔-ΝΗ2〕的兩性電解質，溶液的ΡΗ決定被分離物質的解 離程度和質點的帶電性質及所帶淨電荷量，在某一溶液中 若所帶正負電荷相等時，即是分子的淨電荷等於零，此時 溶液的這一 ΡΗ值爲該蛋白質的等電點〔isoelctric point，pi〕。若溶液PH處於等電點酸侧〔PH< pi〕，則蛋 白質帶正電荷，在電場環境中會向負極移動。若溶液PH 處於等電點鹼侧〔PH > pi〕，則蛋白質帶負電荷，會向正 極移動。在生理條件〔PH 7. 2〕下，膠原蛋白呈現電中性， 因此可以藉由調整磷酸緩衝溶液之PH值來改變膠原蛋白 的帶電性。 故膠原蛋白係為一生物可分解性天然高分子材料，因 其生物相容性高、毒性低、並可促進細胞的吸附與生長， 所以已成為醫學工程領域被廣泛應用的生醫材料，在人造 血管、人造皮膚、細胞培養基材與軟骨修復上都有不錯的 成效，但由於其顆粒之大小係會影響到組織與材料之間的 反應，同時顆粒太大時，也無法被生物體所吸收，並且造 成其周圍有纖維組織產生，所以為能克服膠原蛋白之顆粒 過大缺點，因此有如發明專利第420604號「可吸收式磷 酸鈣生醫複合材料顆粒及其製程」及第524699號「製備 1301415 膝原蛋白和生醫陶穿冷、古 為一種將膠原蛋微粒的方法」，其均係 尺寸仍相當有限，龙法，但其所能製成之微粒 吸收性上仍未達到最佳化。 之尺才戶斤以在生物 【發明内容】 由於上述習知之膠原蛋白 、 當::之顆粒，在著上述之：故:::= :水相環境中利用電場製作微教膠原蛋白之方法，其 A·使膠原蛋白存於水相環境中 膠原蛋白之溶劑，利用磷酸緩衝溶液對此 了二跋斤接近至中性，再以高速離心後，獲得益 菌的膠原蛋白； 于…、 相環境之膠錢白置於電場環境中 環境+之膠原蛋白，置入於通有 負極之間的電場環境内； C. 在受控制的卫作溫度下以 於上述之膠原蛋白 4制之_強度作用 其係利用一# $ t ^ 度下進行反應； 度’而在—特㈣工作溫 D. 維射步驟達—設定時間 其係持續—特定的反應時間； E. 獲得微粒之膠原蛋白 1301415 在電場環境下重組形成奈米微粒之膠原蛋白。 本發明具有下列之優點： 1·本發明可獲得奈米微粗之膠原蛋白，以供廣泛應用 於人造血管、人造皮膚、細胞培養基材與軟骨修復領域等 生醫材料，尤適合被生物體所迅速吸收，以提高組織之再 生能力。 2·本發明之製造步驟相當簡單，可有效降低製造之成 本，同時可縮短製造之時間，可以獲得高品質之奈米級膠 原蛋白。 【實施方法】 首先，請參閱第一圖所示，本發明之製造方法，係包 括有下列步驟：1301415 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method for applying collagen in water particles, which can be applied to the main line of the collagen in the aqueous environment of the human body. In the electric field environment, it is placed in the original protein. Particles of nanometer size [prior art] When repairing, person=month, and, if there is a defect in the injury or disease, medical repair is the best choice for autologous bone graft, but bone The organization comes = limit ^ and the patient must suffer from the second operation, but if there is a loss of transplant rejection and complications. The human-synthesis approach provides a new option, in which (4) protein is most suitable for cultivating new bone cell growth. 0 Collagen is the most abundant structural protein in mammals and is widely found in skin, cartilage and bone tissue. About 20-25% of all protein content, collagen consists of about 1 amino acid, mainly by glycine (33.5%), prionine (pr〇iine, 12%], hydroxyproline (10%) three amino acids, hydroxyproline is a unique amino acid in collagen molecules, generally by the content of hydroxyproline The concentration of collagen is measured. The polypeptide chain composed of these amino acids, due to the small molecule of glycine, can cause moderate folding of the multi-peptide chain, and the poly-peptide chain is rotated by the left 1301415. _ chain spiral exists. The three α _ chains utilize hydrogen bonds and i|IL water! The bio-group ^/ into a right-handed triple helix, which is called tropocollagen, and the function of hydroxyproline is used to form intramolecular quinone bonds. 'To stabilize the three-strand spiral structure. Different amino acid composition, the amine group and the sputum J sequence can form different α _ chain, the collagen is composed of three intertwined α - forming a three-member structure and three different a - chains can constitute different types of collagen For example, the knot of collagen -^ is [αι(Ι)]_, and the first type of collagen in hyaline cartilage is composed of [ai(jj)]3. A variety of collagens have been clearly defined in various tissues distributed so far in animals. The second type of collagen is the most important collagen component in the cartilage matrix, accounting for about 4% of the dry weight of hyaline cartilage. In the collagenogens, the collagen fibers will aggregate with each other to form a collagen fiber with a larger diameter by the action of a molecular hydrazone bond, a hydrophobic group, and a covalent bond [eQUagen fibH1]. 'Qingcheng's collagen fiber is combined with other ingredients (eg, grape (4) enzyme, poly-glass sugar, etc.) to form a matrix, which is a network structure. Its function is to prevent the tissue from being stretched. Dispersing the power to provide the mechanical strength required by the tissue 0 ο" „ Collagen molecules (7) surface contains a non-polar polarity of the charged area (Τ2), which in the hydrophobic area _ 刀 Α Α 达到 达到 达到 达到 , , , Will accumulate 1301415 from each other and leave the collagen, so that the hydrophobic region (τι) is exposed and can interact; together with the electrostatic force of the charged region (Τ2), the collagen molecules are combined with each other. Since the composition of the fiber structure. Also, due to the protein molecule, there is an ampholyte of the acidic group [-C00H] and the basic group [-ΝΗ2]. The enthalpy of the solution determines the degree of dissociation of the separated substance and the charged nature of the particle and the net charge amount. If the positive and negative charges in a solution are equal, the net charge of the molecule is equal to zero, and the enthalpy of the solution is the isoelctric point (pi) of the protein. If the pH of the solution is on the acid side of the isoelectric point [PH< pi], the protein is positively charged and moves to the negative electrode in an electric field. If the pH of the solution is at the isoelectric side of the isoelectric point [PH > pi], the protein is negatively charged and will move toward the positive electrode. Under physiological conditions [pH 7.2], collagen is electrically neutral, so the chargeability of collagen can be changed by adjusting the pH of the phosphate buffer solution. Therefore, collagen is a biodegradable natural polymer material. Because of its high biocompatibility, low toxicity, and promotion of cell adsorption and growth, it has become a widely used biomedical material in medical engineering. Artificial blood vessels, artificial skin, cell culture substrate and cartilage repair have good results, but because the size of the particles will affect the reaction between the tissue and the material, and when the particles are too large, they cannot be used by the organism. Absorbing, and causing the formation of fibrous tissue around it, so in order to overcome the disadvantage of excessive granules of collagen, it is, for example, Patent No. 420604 "Absorbable calcium phosphate biomedical composite particles and processes thereof" and No. 524699 "Preparation 1301415 Knee protoprotein and biomedical pottery wear cold, ancient is a method of collagen egg microparticles, and its average size is still quite limited, Long method, but its particle can not be optimized for absorption. . Because of the above-mentioned conventional collagen, when: granules, in the above: Therefore:::=: the method of making the micro-teaching collagen using the electric field in the aqueous environment, A. The collagen in which the collagen is stored in the aqueous environment, and the phosphate buffer solution is used to approach the neutral, and then centrifuged at a high speed to obtain the collagen of the beneficial bacteria; The gelatin white is placed in the electric field environment and the collagen is placed in the electric field environment between the negative electrodes; C. The strength of the above-mentioned collagen 4 is controlled at the controlled temperature of the temperate The system uses a #: t ^ degree to carry out the reaction; degree 'at the special (four) working temperature D. The dimensioning step reaches - set the time to continue - specific reaction time; E. obtain the particulate collagen 1301415 in the electric field The collagen is recombined to form nanoparticles in the environment. The invention has the following advantages: 1. The invention can obtain collagen of nanometer coarseness, which is widely used in biomedical materials such as artificial blood vessels, artificial skin, cell culture substrate and cartilage repairing, and is particularly suitable for being used by living organisms. It is quickly absorbed to improve the regenerative capacity of the tissue. 2. The manufacturing process of the present invention is relatively simple, and the manufacturing cost can be effectively reduced, and the manufacturing time can be shortened, and a high-quality nano-sized collagen protein can be obtained. [Method of Implementation] First, referring to the first figure, the manufacturing method of the present invention includes the following steps:
A·使膠原蛋白存於水相環境中：係先取下牛隻之膝關 節軟骨〔保存於-20°C環境中〕，去除牛膝關、節之周圍軟組 織，然後再利用4°C，0· 15M，PH7.2之磷酸缓衝溶液〔PBS〕 沖洗數次後，再將軟骨切成約為lmm3的大小，泡製15倍軟骨 體積的 4· 0M鹽酸胍〔GuCl，Guanidine hydrochloride〕 /0.05M三羥甲基氨基甲烷〔Tris base，PH7.2〕，並將軟骨 浸泡其中，用均質機攪拌數次，使軟骨中非第二型膠原蛋 白的物質溶出，48小時後再取出軟骨置於〇·5Μ醋酸溶液中 並加入0.5mg/ml的胃蛋今腌，用均質機均勻攪拌數次，使 軟骨中第二型膠原蛋白溶解出來，48小時後，利用高速離 心機12000rpm於0°C離心1小時後，取其上清液並加入〇. 86M 1301415 之氯化納進行鹽析，則可將第二型膠原蛋白析出，24小時 後再於0°C中進行12000rpm高速離心，將沈澱物及上清液分 離’所得之沈澱物即為第二型（Type II)膠原蛋白，將離 心後所得之沈澱物溶至0· 05M Tris base/O· 5M氯化鈉 〔PH7· 2〕中。先對〇· 〇5M Tris base/0· 5M氯化鈉〔ΡΗ7· 2〕 透析’再對〇· 05Μ Tris base/O· 2Μ氯化鈉〔ΡΗ7· 2〕透析數 天’最後再利用ΡΗ7. 2之磷酸缓衝溶液對此溶劑作透析至 ΡΗ7·2。最後將已透析成中性之第二型膠原蛋白以32〇〇〇rpm 於〇°C進行超高速離心3小時，取其三分之二之上清液，即 為無囷的第二型膠原蛋白。 B·將上述存於水相環境之膠原蛋白置於電場環境中： 如第一圖所示，係取3〇〇mmXi〇〇mmX3mm之兩玻璃片（！）分別 貼上導電膠帶後，並於周圍貼上絕緣膠帶，於頂部及底部 分兩邊使用壓克力塊墊高，使其上下總高度為1〇mm，最後 接上電線（2)，並將電線（2)接上高壓電源供應器（3)，頂部 接正極，底部接負極，即完成系統的架設，然後取存在於 容器（4)内水相環境中之〇.2mg/ml第二型膠原蛋白lml 〔PH7.2〕’置入於該兩玻璃片（1)之間的電場環境内。 C·在文控制的工作溫度下以一受控制之電場強度作用 於上述之膠原蛋白··其係使則· 5kv/cn^7kV/cm之電場強 度在低於3G。⑶卫作溫度下進行反應；本實施例之電場強 度為3kV/cm，工作溫度則為25。〇。 D·維持C步驟達-設定時間··其係持續分別反應一段時 1301415 間，例如1小時以上。 F·獲得微粒之膜居疋 為均將其纽料有直裡較 幻不水微粒之膠原蛋白。 執行上述步驟所獲得 ^ rr/α ^ 夕"由 ㈣仵之結果，顯不於附件-至附件五 之』此，、、片中。附件一至附件三顯示在饥環境下 Ο.2—1的第二型膠原蛋白在W、時、3小時、5小時= 日.二.，至277.7 土 4.7nm之間，且在3小時和5小時電 場反應後微粒表面變的較平滑緊實。此外，附 书 mg/ml第二型膠原蛋白在阶下錢ν/_ 作用3 4.7nm。附件五係0·5 nig/ml第二型膠原蛋白在25。〇下以 3kV/Cm電場強度作用3小時所產生微粒膠原蛋白之顯微照 片，粒徑為 1361· 1 土 292 nm。 經由上述步驟所製成之第二型膠原蛋白，係可將其滴 至銅網上，待乾燥後再以PH 7· 2，2%磷鎢酸 〔Ph〇s_ungstic Acid，PTA〕進行負染色〔聯的-staining〕，待乾燥後再將銅網置於穿透式顯微鏡下觀 察，則可觀察到電場反應後的膠原蛋白有微粒的產生。 >上述實施例中，有關電場強度、工作溫度、反應時間 等參數，僅為本發明可實施之參數條件之―，並非用以侷 限本發明之範疇；此外’本發明之實施例以第二型膠原蛋 11 1301415 白為實施標的，但其他類型之膠原蛋白亦能適用於本發 明0 12 1301415 【圖式簡單說明】 第一圖係為本發明之步驟流程圖。 第二圖係為本發明實施例之使用裝置示意圖。 第三圖係為膠原蛋白分子之組合排列示意圖。 附件一係0. 2 mg/ml第二型膠原蛋白在25°C下以3kV/cm電場 強度作用1小時所產生微粒膠原蛋白之顯微照片。 附件二係〇. 2 mg/ml第二型膠原蛋白在25°C下以3kV/cm電場 強度作用3小時所產生微粒膠原蛋白之顯微照片。 附件三係0. 2 mg/ml第二型膠原蛋白在25°C下以3kV/cm電場 強度作用5小時所產生微粒膠原蛋白之顯微照片。 附件四係0. 2 mg/ml第二型膠原蛋白在25°C下以3kV/cm電場 強度作用3小時所產生微粒膠原蛋白之顯微照片。 附件五係0. 5 mg/ml第二型膠原蛋白在25°C下以3kV/cm電場 強度作用3小時所產生微粒膠原蛋白之顯微照片。 【主要元件符號說明】 (1) 玻璃片 (2) 電線壓克力塊 (3) 南壓電源供應器 (4) 容器 (T) 膠原蛋白分子 (T1) 疏水區 (T2) 帶電區 13A· Let collagen be stored in the aqueous environment: first remove the knee joint cartilage of the cow [preserved in -20 ° C environment], remove the soft tissue surrounding the achyranthes, and then use 4 ° C, 0 · 15M, pH 7.2 phosphate buffer solution [PBS] After several times of washing, the cartilage is cut into a size of about 1mm3, and the cartilage volume of 4·0M guanidine hydrochloride [GuCl, Guanidine hydrochloride] /0.05 M Tris base (pH 7.2), and the cartilage was soaked therein, and stirred with a homogenizer several times to dissolve the non-type 2 collagen substance in the cartilage, and then the cartilage was taken out after 48 hours. 〇·5Μ acetic acid solution and add 0.5mg/ml of stomach egg, marinate, stir evenly with homogenizer several times, so that the second type collagen in cartilage dissolves. After 48 hours, use high speed centrifuge 12000rpm at 0°C After centrifugation for 1 hour, the supernatant was taken and salted out by adding sodium chloride of 86 M 1301415 to precipitate the second type of collagen. After 24 hours, high-speed centrifugation at 12,000 rpm was carried out at 0 ° C to precipitate. Separation of the substance and the supernatant 'the resulting precipitate is the second type Type II) collagen, obtained from the heart after the precipitate was dissolved to 0 · 05M Tris base / O · 5M sodium chloride in the [PH7 · 2]. First, 〇· 〇 5M Tris base/0· 5M sodium chloride [ΡΗ7· 2] dialysis 'repeated 〇 · 05 Μ Tris base/O· 2 Μ sodium chloride [ΡΗ7· 2] dialysis for several days 'final reuse ΡΗ 7. The phosphate buffer solution of 2 was dialyzed against this solvent to ΡΗ7.2. Finally, the second type collagen which has been dialyzed into neutral is ultracentrifuged at 32 ° rpm for 3 hours at 〇 ° C, and two-thirds of the supernatant is taken, which is the second type collagen without flaws. protein. B. Place the above-mentioned collagen stored in the aqueous environment in an electric field environment: As shown in the first figure, attach two pieces of glass (!) of 3〇〇mmXi〇〇mmX3mm to the conductive tape, respectively. Apply insulation tape around the top, and use the acrylic block height on both sides of the top and bottom parts to make the total height of the upper and lower parts 1〇mm. Finally, connect the wires (2) and connect the wires (2) to the high-voltage power supply. (3), the top is connected to the positive electrode, and the bottom is connected to the negative electrode, that is, the system is set up, and then the 2mg/ml type II collagen 1ml [PH7.2] is placed in the aqueous environment in the container (4). Entered in the electric field environment between the two glass sheets (1). C. At the operating temperature controlled by the text, a controlled electric field strength acts on the above-mentioned collagen. The electric field strength of 5kv/cn^7kV/cm is lower than 3G. (3) The reaction was carried out at the temperature of the temper; the electric field intensity of this example was 3 kV/cm, and the operating temperature was 25. Hey. D. Maintain C step up - set time · The system continues to react for a period of time between 1301415, for example, 1 hour or more. F·The film of the obtained microparticles is a collagen which has a straight-lined and opaque particle. The results obtained by performing the above steps ^ rr / α ^ 夕 " by (4) 仵 results, not shown in the attachment - to the fifth, this, in the film. Annexes I to III show that type II collagen in hunger environment 2-1. at W, hour, 3 hours, 5 hours = day. II., to 277.7 soil between 4.7 nm, and at 3 hours and 5 After the hourly electric field reaction, the surface of the particles becomes smoother and firmer. In addition, the attached book mg/ml type II collagen in the order of money ν / _ action 3 4.7nm. Annex 5 is 0. 5 nig / ml type II collagen at 25. A microphotograph of the microparticle collagen produced by the electric field strength of 3 kV/cm for 3 hours was used, and the particle size was 1361·1 soil 292 nm. The second type of collagen produced through the above steps can be dripped onto the copper mesh, and then dried and then negatively stained with PH 7.2, 2% phosphotungstic acid (Ph〇s_ungstic Acid, PTA). The -staining of the joint, after drying the copper mesh under a transmission microscope, it can be observed that the collagen after the electric field reaction has the generation of particles. > In the above embodiments, the parameters such as the electric field strength, the operating temperature, the reaction time, and the like are only the parameter conditions that can be implemented by the present invention, and are not intended to limit the scope of the present invention; further, the embodiment of the present invention is second. Type collagen egg 11 1301415 white is the target, but other types of collagen can also be applied to the present invention. 0 12 1301415 [Simplified description of the drawings] The first figure is a flow chart of the steps of the present invention. The second figure is a schematic diagram of a device for use in an embodiment of the present invention. The third figure is a schematic diagram of the arrangement of collagen molecules. Annex I is a photomicrograph of microparticle collagen produced by 0.2 mg/ml type 2 collagen at 25 ° C for 1 hour at an electric field strength of 3 kV/cm. Annex II: 显微. Photomicrograph of microparticle collagen produced by 2 mg/ml type 2 collagen at 25 ° C for 3 hours at an electric field strength of 3 kV/cm. Annex III is a photomicrograph of microparticle collagen produced by 0.2 mg/ml type II collagen at 25 ° C for 5 hours at an electric field strength of 3 kV/cm. Annex IV is a photomicrograph of microparticle collagen produced by 0.2 mg/ml type II collagen at 25 ° C for 3 hours at an electric field strength of 3 kV/cm. Annex 5 is a photomicrograph of microparticle collagen produced by 0.5 mg/ml type II collagen at 25 ° C for 3 hours at an electric field strength of 3 kV/cm. [Explanation of main component symbols] (1) Glass piece (2) Wire acrylic block (3) South voltage power supply (4) Container (T) Collagen molecule (T1) Hydrophobic zone (T2) Charged area 13