Name: doxycycline loaded collagen-chitosan composite scaffold for the accelerated healing of diabetic wounds
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作者感谢阿希什·瓦德瓦尼博士。（印度奥蒂JSS药学院药物生物技术系助理教授兼系主任）协助 体外 细胞生存能力研究。
作者要感谢新德里大学和高等教育机构科技基础设施改善基金（DST-FIST）支持我们系。
作者也想感谢三菊先生。S 和斯里拉姆先生纳鲁库拉M.制药学生的支持，在视频拍摄。
这项研究得到了JSS高等教育与研究学院（JSSAHER）的支持。

所有动物程序都得到了印度奥蒂JSS药学院动物伦理委员会的批准。
1. 通过冷冻干燥方法制备 DOX 装载多孔脚手架
<ol><li>将 1.2 克 COL 添加到 100 毫升水（例如米利波尔），并留出以防肿胀。</li>
	<li>在 2000 rpm 时搅拌肿胀的 COL 分散，以确保 COL 完全解散。</li>
	<li>通过溶解约 0.8 克 CS 在 100 mL 的 1% 醋酸中准备 CS 解决方案。</li>
	<li>在 2000 rpm 时将 CS 解决方案搅拌过夜，以确保均?...

<ol>
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本研究的主要目的是确定 DOX 装载的 COL-CS 脚手架对大鼠 DW 愈合的影响。CL 和 NCL 在形态、肿胀指数、体外释放动力学和生物相容性方面进行了准备和评估。
DOX 加载 NCL 和 CL 脚手架的特征 已准备好的脚手架被发现多孔，毛孔相互连接。这些相互关联的毛孔确保多孔、海绵状的性质，有助于将氧气和营养物质适当扩散到细胞中，从而促进增殖和迁移，从而加速愈...

糖尿病（DM）是一种身体未能提供胰岛素或对直接糖的异常消化结果作出反应的情况，导致血糖 1的激增。DM 最连续和最破碎的纠缠是糖尿病伤口 （DW）。大约25%的DM患者有机会在有生之年建立 DW。DW 的阻碍愈合被认可为 DM 的三种病变：免疫病变、血管病和神经病变。每当DW得不到治疗，它可能导致坏死的发展，因此促使有关器官 2的删除。
大量的治疗方法，如指导病人（每天检查伤口，清洁伤口，避免对伤口造成压力的活动，定期葡萄糖监测等），控制他们的血糖，伤口脱脂，压力卸载，医疗程序，高压氧疗法，和先进的治疗是在实践3，4。鉴于多因素病理生理条件和与这些药物相关的意外费用，这些药物大多未能解决DW护理至关重要的所有先决条件。即使DW发病机制是多因素的，但组织管理不当的持续炎症是DW 5、6中延迟愈合的实际原因。
DW中炎症和亲炎症调解人的增加水平导致导致伤口愈合延迟的生长因子减少2，6。DW 中细胞外矩阵 （ECM） 的形成被认可为矩阵金属蛋白酶 （MMP） 水平增加，导致已形成的 ECM 的快速降解。在MMP中，MMP-9被报告为主要中间人，负责长期炎症和快速ECM降解7。据说，局部治疗与抗炎药物，降低MMP-9的升高水平，重新建立皮肤平衡，框架安排，并提示更好的愈合DWs8，9。 
多西环素（DOX），一种MMP-9抑制剂，被选择来抑制MMP-9的升高水平，MMP-9是负责10、11、12号DWs持续炎症的主要炎症调解人。此外，DOX 拥有抗氧化剂（产生能够与活性氧物种结合的自由羟基和苯氧基）13和抗凋零（抑制卡斯帕塞表达和线粒体稳定）14项对治疗 DW 至关重要的活动。选择了包含 DOX、胶原蛋白 （COL） 和奇托桑 （CS） 的框架安排。COL的选择取决于它如何帮助提供必要的框架负责机械强度和组织再生15。另一方面，CS在结构上与糖糖糖素同源，与几个伤口愈合阶段相关。另据报道，CS持有显著的抗菌特性15。因此，DOX 的 COL/CS 脚手架被配制以抑制长期炎症，然后支持矩阵形成，以便在 DM 条件下成功愈合伤口。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-ethyl-(3-3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>E7750</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(N-morpholino) ethane sulfonic acid (MES)</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>137074</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(4, 5 dimethyl thiazole-2 yl) -2, 5-diphenyl tetrazolium bromide (MTT)</td>
			<td>Thermo Fisher, Mumbai, India</td>
			<td>M6494</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep freezer verticle</td>
			<td>Labline Instruments, Kochi, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis sack</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>D6191-Avg. flat width 25 mm (1.0 in.), MWCO 12,000 Da</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma chemicals Co. Ltd, Mumbai, India</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Elisa kit</td>
			<td>R&#38;D Systems</td>
			<td>RMP900</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli (E. coli)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 2567</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer-SZ042</td>
			<td>Sub-Zero lab instruments, Chennai, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical Stirrer-RQ-122/D</td>
			<td>Remi laboratory instruments, Mumbai, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium molecular weight Chitosan</td>
			<td>Sisco Research Laboratories Pvt. Ltd., Mumbai, India</td>
			<td>18824</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome-RM2135</td>
			<td>Leica, U.K</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblast cells (3T3-L1)</td>
			<td>National Centre for Cell Sciences, Pune, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiple plate reader -Inifinte M200 Pro</td>
			<td>Tecan Instruments, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxy succinimide (NHS)</td>
			<td>Sigma chemicals Co. Ltd, Mumbai, India</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa (P. aeruginosa)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscopy (SEM)-S-4800</td>
			<td>Hitachi, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH) pellets</td>
			<td>Qualigen fine chemicals, Mumbai, India</td>
			<td>Q27815</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus (S. aureus)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus epidermis (S. epidermis)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 5270</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptozotocin (STZ)</td>
			<td>Sisco Research Laboratories Pvt. Ltd., Mumbai, India</td>
			<td>14653</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-1 rat Collagen</td>
			<td>Sigma chemicals Co. Ltd, Mumbai, India</td>
			<td>C7661</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet&#8211;visible spectroscopy-1700</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

doxycycline loaded collagen-chitosan composite scaffold for the accelerated healing of diabetic wounds

糖尿病的一个主要并发症是糖尿病伤口（DW）。糖尿病炎症的延长阶段阻碍了导致伤口愈合延迟的进一步阶段。我们选择多西环素（DOX），作为一种潜在的药物的选择，因为它的抗菌性能，以及它报告的抗炎特性。目前的研究旨在制定DOX加载胶原蛋白-奇托桑非交联（NCL）和交联（CL）脚手架，并评估其在糖尿病条件下的愈合能力。脚手架的定性结果表明，与 DOX-NCL 脚手架相比，DOX-CL 脚手架具有理想的孔隙性、低肿胀和降解率以及 DOX 的持续释放。 体外 研究表明，与处理和控制组的 CL 脚手架相比，DOX-CL 脚手架具有生物相容性和增强的细胞生长。抗菌研究表明，DOX-CL脚手架比CL脚手架对DW中最常见的细菌更有效。使用链球菌素和高脂肪饮食诱导的DW模型，观察到DIX-CL脚手架治疗组的伤口收缩速度明显（p≤0.05）。使用 DOX-CL 脚手架可以证明是治疗 DW 的有希望的方法。

DOX 加载 NCL 和 CL 脚手架的特征 在目视检查中，NCL 和 CL 脚手架被发现为彩色奶油色。此外，两个脚手架似乎都像海绵，僵硬和无弹性时，身体检查。NCL 和 CL 脚手架的 SEM 图像显示在 图 1中。从数字上看，通过形成分子间联系，在交叉链接后，孔径尺寸明显减少。此外，NCL和CL脚手架孔隙度分别为92.3±4.21和71.35±2.65。NCL和CL脚手架的吸水百分比为750±11.4%和492±8....

Watch this Scientific Journal Video about Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds at JoVE.com

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

准备好的 DOX-CL 脚手架满足了机械强度、孔隙度、吸水率、降解率、持续释放、抗菌、生物相容性和抗炎特性的理想 DW 敷料的先决条件，这些都被认为是 DW 中受损组织恢复的关键。

多西环素加载胶原蛋白-奇托桑复合支架，用于加速治疗糖尿病伤口

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an ...

目前的研究工作是多西环素加载胶原蛋白-奇托桑复合脚手架，以加速愈合糖尿病伤口。糖尿病是一种慢性代谢疾病，其特征是血糖水平升高。个人患糖尿病的时间越长，患心血管疾病、肾病、神经病变、视网膜病变和足部损伤等并发症的风险就越高。脚部神经损伤和脚部血流不良会增加各种足部并发症的风险。如果不加以治疗，这些伤口最终可能需要脚趾、脚或腿部截肢。大约，25%的糖尿病患者有机会在有生之年建立糖尿病伤口。糖尿病伤口的治疗管理仍然是迄今为止需要最终跨越的一个障碍。尽管糖尿病伤口的病理生理学是多因素的，但人们相信，使用抗炎药物进行局部治疗可以降低MMP-9的升高水平，并重建皮肤平衡框架安排，并促使糖尿病伤口愈合。在这项研究中选择了多西环素，MMP-9抑制剂，以抑制MMP-9的这些高浓度，这是导致糖尿病伤口持续炎症的主要炎症分子。另一方面，生物材料几十年来一直吸引着人类，在高效药物和大分子输送以及定制生物医学设备开发方面，生物材料一直是许多改变游戏规则的医学突破的核心。因此，通过这个简短的介绍，我们准备了一个多西环素加载胶原蛋白-奇托桑脚手架来衰减，以加速糖尿病伤口的延迟愈合。这是印度奥蒂JSS药学院药剂系的研究学者巴拉特·库马尔·雷迪。今天，我们在这里研究的程序，如何准备多西环素加载胶原蛋白-奇托桑脚手架，以加速愈合糖尿病伤口。让我们来一瞥这个程序，如何准备。我们将准备1.2%的胶原蛋白溶液，为此我们服用1.2克I型胶原蛋白，这是从老鼠尾部来源。为此，我们添加了100毫升的米利波尔水，并保留在一边半小时，以便让胶原蛋白在水中膨胀。下一步，准备了0.8%的甲基托桑溶液，为此我们服用了0.8克中等分子量的甲基托桑，这与100毫升1%醋酸混合。这个解决方案也，我们已经搁置了半个小时。半小时后，两种解决方案在2000年RPM的一次机械搅拌中进行了通宵。在一夜之间搅拌胶原蛋白溶液中，我们正在慢慢添加一克多西环素。含有这种胶原蛋白和多西环素的溶液在2000年RPM上经过了半小时的搅拌。为了这个解决方案，我们正在慢慢添加隔夜搅拌的奇托桑溶液。这一点，确保它被标记为物理混合物，其中含有多西环素，胶原蛋白，甲苯丙胺。这种物理混合物，我们已经进行了半小时的搅拌在2000年RPM。半小时后，这些物理混合物被转移到各自的铝模具中，其尺寸为2到2厘米，厚度为0.5厘米。后来，这些模具被转移到一个金属容器中。这些金属容器中含有铝模具，被置于深度冻结。24小时后，我们可以清楚地看到含有多西环素、胶原蛋白和奇托桑的凝固物理混合物。这些凝固的物理混合物进一步受到冻血症。在零下85摄氏度或零下4摄氏度下进行了72小时的嗜血性手术。72小时后，从嗜血剂中取出嗜血材料。在这里，我们可以清楚地看到完全干燥的物理混合物，这只不过是被称为多西环素加载胶原蛋白-奇托桑脚手架。这些脚手架被置于交联状态，为了进行交联，最初，MES 缓冲器是通过取 0.488 克 MES 和 15 毫升水来准备的，我们从中取出 20 毫升 MES 缓冲器，并单独转移到您的烧饼中。在这20毫升烧甲机中，添加了50毫克含有胶原蛋白-奇托桑的多西环素。这个脚手架的浸泡进行了半个小时。半小时后，从20毫升MES缓冲器中取出装有胶原蛋白-奇托桑脚手架的多西环素。此外，它被浸泡在含有19.5毫升MES缓冲液、0.1264克EDC和0.014克NHS的溶液中。浸泡时间间隔为四小时，以完成交联。交联后，将装有胶原蛋白-奇托桑脚手架的交叉链接从溶液中取出并转移到容器中，该容器在之前的程序中已详细说明，并单独干燥。在这里，我们可以清楚地看到非交联和交叉链接的脚手架，以及相同的图像。从图像中可以明显看出，由于形成强大的分子内键，脚手架的交叉链接导致毛孔尺寸减少。雄性威星鼠被带走，两个成两厘米的标记被绘制在胸腔区域。切除伤口围绕标记创建，伤口追踪在 OHP 表上。伤口的痕迹是使用图形方法测量的。动物身上的伤口大约是176毫米。后来，用交叉链接的多西环素加载胶原蛋白-奇托桑脚手架与主要敷料进行治疗，并监测了21天。一旦从麻醉中恢复动物，每只动物都单独被关在笼子里。这些动物被喂食正常饮食和水。所有动物在造成伤口后，被分成三组，每组包含六只动物。在这里，我们可以清楚地看到零天和七天的区别，其中多西环素加载的交联脚手架伤口减少约48%，在这张图片中，我们可以清楚地看到第14天和第21天的区别 在HMB染色的帮助下，所有含有胶原蛋白的胶原蛋白交叉链接脚手架的组被隔离并处理为组织病理学研究。在这里，我们可以清楚地看到重新环化表皮，在第21天，在多西环素加载胶原蛋白奇托桑脚手架。而在控制和交叉链接的脚手架，只有安慰剂，仍然表皮必须形成。与我们自己的研究一样，制备的多氧环素交联脚手架也表现出许多理想的特性，如良好的机械强度、生物相容性、持续释放吸水以及抗菌和抗炎特性。这些脚手架可以是治疗糖尿病伤口的理想剂量形式。

Research

JoVE Journal

Bioengineering

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

科研

生物工程

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

制造业的鲁棒天然纤维预制体的利用细菌纤维素为粘结剂

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

糖尿病大鼠模型神经传导阻滞的超声工具

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

成年大鼠复合再生外周神经接口（C-RPNI）的制造

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...