这项工作是由美国国立卫生研究院（HL068915到DBC），从长尾研究基金（以CAP）新研究员奖的研究经费，格兰特在急救来自美国心脏协会（12GRNT11910008到DBC）的支持从儿童心脏基金会（以DBC），并捐赠给波士顿儿童医院心脏传导基金，瑞恩家庭养老和由大卫普尔曼的研究经费。

在这里，我们将展示COL1从羔羊皮隔离。作为写入，该协议也可以用来成功地从兔和人的皮肤隔离COL1。 1。准备皮肤样本<ol><li>将所有溶液平衡至4℃后才能使用。 </li><li>冲洗皮肤样本（25-50克）在冰冷的DH 2 O和删除任何羊毛，毛皮，或头发脱毛膏。 </li><li>使用单刃刀片刮结缔组织和脂肪的样品净化。 </li><li>冲洗在冰冷的DH 2 O样品</li><li>切片皮肤样品切成1厘米×1厘米的块用单刃刀片。 </li></ol> 2。除去溶解的中非胶原材料<ol><li>称出5克每50ml样品的锥形管中，并加入30毫升冰冷的0.5M的乙酸钠。 </li><li>混合管1分钟，使用50毫升管适配器在台式均质6米/秒的设置。 </li><li>丢弃SUPernatant和重复，共7醋酸钠洗涤周期。 </li><li>冲洗样品在冰冷DH 2 O和混合一次，以除去残余的乙酸钠。 </li><li>用刮刀压靠管的一侧的样品以除去过量的液体，然后转移到一个新的50ml锥形管中。 </li></ol> 3。 I型胶原蛋白的提取<ol><li>在第2次清洗样本毫升/克，0.075M柠檬酸钠缓冲液为6米/秒的台式匀浆器压缩所述样品和每次洗涤后弃去上清1分钟。 </li><li>加的，0.075M柠檬酸钠缓冲液中的淡水2毫升/克的等分试样的样品。 </li><li>执行6个连续1分钟，6米/秒台式均质混合搅拌周期不删除每个周期之间的缓冲器。 </li><li>转移厚，上层清液的收集管。 </li><li>增加一个额外的1毫升/克，0.075M柠檬酸钠缓冲液的样品并进行最后一个台式匀浆器激越化周期。 </li><li>将此最终上清收集管。 </li><li>离心收集管在3200×g离心10分钟，在4℃下</li><li>将上清转移至100,000分子量的车厢顶部切断离心过滤装置。 </li><li>离心机在3,200 xg离心30分钟，在4°C。 </li><li>从上部隔室转移纯化COL1到一个干净的锥形管中并储存于4℃。 </li></ol>

<ol>
	<li>Nageotte, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+fibrillaire+in+vitro+du+collag&#232;ne+dissous+dans+un+acide+dilu&#233;.">Coagulation fibrillaire in vitro du collag&#232;ne dissous dans un acide dilu&#233;.</a> CR hebd. s&#233;ances Acad. Sei. 184, 115 (1927).</li><li>Schmitt, F. O., Hall, C. E., Jakus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+investigation+of+the+structure+of+collagen.">Electron microscope investigation of the structure of collagen.</a> J. Cell Comp. Physiol. 20, 11-33 (1942).</li><li>Choi, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am. J. Pathol. 169, 72-85 (2006).</li><li>Pacak, C. A., Cowan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+myogenic+engineered+tissue+constructs.">Fabrication of myogenic engineered tissue constructs.</a> J. Vis. Exp. (27), (2009).</li><li>Cliche, S., Amiot, J., Avezard, C., Gariepy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+characterization+of+collagen+with+or+without+telopeptides+from+chicken+skin.">Extraction and characterization of collagen with or without telopeptides from chicken skin.</a> Poultry Sci. 82, 503-509 (2003).</li><li>Rajan, N., Habermehl, J., Cote, M. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nat. Protoc. 1, 2753-2758 (2006).</li><li>Seifter, S., Gallop, P., Colowick, S., Kaplan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+properties+of+soluble+collagens.">Preparation and properties of soluble collagens.</a> Methods in Enzymology. , (1963).</li><li>Pacak, C. A., Powers, J. M., Cowan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrarapid+purification+of+collagen+type+I+for+tissue+engineering+applications.">Ultrarapid purification of collagen type I for tissue engineering applications.</a> Tissue Eng. Part C Methods. 17, 879-885 (2011).</li><li>Ohan, M. P., Dunn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+stabilizes+collagen+sterilized+with+gamma+irradiation.">Glucose stabilizes collagen sterilized with gamma irradiation.</a> J. Biomed. Mater. Res. A. 67, 1188-1195 (2003).</li><li>Tyan, Y. C., Liao, J. D., Lin, S. P., Chen, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+the+sterilization+effect+of+gamma+ray+irradiation+of+immobilized+collagen+polypropylene+nonwoven+fabric+surfaces.">The study of the sterilization effect of gamma ray irradiation of immobilized collagen polypropylene nonwoven fabric surfaces.</a> J. Biomed. Mater. Res. 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F., Liepmann, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vascular+smooth+muscle+cells+by+micropatterning.">Regulation of vascular smooth muscle cells by micropatterning.</a> Biochem. Biophys. Res. Comm. 307, 883-890 (2003).</li><li>Leighton, J., Justh, G., Esper, M., Kronenthal, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-coated+cellulose+sponge:+three+dimensional+matrix+for+tissue+culture+of+Walker+tumor+256.">Collagen-coated cellulose sponge: three dimensional matrix for tissue culture of Walker tumor 256.</a> Science. 155, 1259-1261 (1967).</li></ol>

作者没有竞争经济利益披露。

这种方法的一个关键组成部分是真皮样品的反复有效的压缩，去除多余的液体。关键是通过压缩样本以除去尽可能多的醋酸钠尽可能在步骤2.5。我们用一个抹刀压实对管侧的皮肤，但是，也可以采用粗棉布，虽然这将须除去样品从管中，并增加来执行这些步骤所需的时间。同样地，以压缩的样本，在步骤3.1，使柠檬酸钠的量是在随后的提取步骤准确是重要的。未能充分从在上述任何步骤除去样品?...

几十年来，研究人员和商业厂商已经分离出溶解的COL1从组织来源，包括皮肤和腱使用简单的酸提取协议接着进行中和，这导致组织COL1原纤维基质的重悬浮，可用于某些变异的分类为多种生物医学应用1-4。虽然有对COL1临床应用的例子很多，少数这些采用autologously衍生COL1因为准备这种蛋白质的需要冗长的提取需要几天或几周进行5-7。因此，研究调查员和医师通常使用COL1的正在使用非人组织如鼠，牛或猪的真皮或皮肤用从尸体取出或以下包皮环切制备昂贵，商业制剂。对于再生应用，这些产品很多变化表现出对于自己的能力，形成固体矩阵或能够支持细胞粘附和生长的组织结构。因此，存在一个明显的需要，它能够快速从方便和丰富的自体组织源中提取COL1一个新的标准化方法。 这种方法可以节省时间和金钱在研究环境，让COL1可以从感兴趣的动物模型中提取并用于组装的胶原蛋白为基础的脚手架工程化组织的临床前试验。与此同时，具有在临床使用迅速隔离，autologously衍生COL1的选项会增加，否则将接收同种异体或异种制剂的患者的整体安全性。用于接收自体制剂的患者，这个简化的方法可大大减少活检收集和胶原蛋白应用程序之间的时间间隔。基于这些原因，我们试图通过使用高速AG改善后一个历史悠久的COL1分离和提纯方法itation和尺寸排阻离心。这种方法简单执行使用标准缓冲液和设备在许多实验室中找到。通过采用高速搅拌，我们的协议减少所需的时间来分离可溶性的，真皮COL1从约10天到小于3小时8。重要的是，这种方法可以容易地执行在临床上以制备autologously衍生COL1用于在一组程序中的患者使用。

<table border="0" cellpadding="0" cellspacing="0" width="544">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
		</tr>
	</tbody>
</table>

an improved method for the preparation of type i collagen from skin

可溶性Ⅰ型胶原（COL1）被广泛地用作用于细胞培养的粘合剂基体和作为再生的应用程序的蜂窝式脚手架。临床上，这种蛋白是广泛用于美容手术，经皮注射，骨移植术，和重建手术。 COL1对这些程序的来源通常是非人，这增加了炎症和排斥反应的可能性，以及外源性疾病的传播。鉴于此，一种方法能够有效地和快速地从数量autologously衍生组织有限净化COL1会规避很多这样的问题，但是，标准隔离协议是漫长的，往往需要大量的胶原组织。这里，我们表明一个有效的COL1的提取方法，可降低从约10天分离并纯化该蛋白为小于3小时的时间。我们在许多外科手术的选择，因为它的可用性真皮作为我们的组织来源。 Ŧ他的方法是使用传统的提取缓冲液结合有力的搅拌和离心过滤从少量真皮的获得高纯度的可溶性COL1。简单地说，皮肤活检进行彻底的冰冷的DH 2 O去除脂肪，结缔组织，和头发后清洗。将皮肤样品剥离用0.5M的乙酸钠和一个高速台式匀浆非胶原蛋白质和多糖的。从残余的固体胶原随后与使用匀浆机以0.075M的柠檬酸钠缓冲液提取。这些提取物是使用100,000兆瓦截止离心过滤器能产生可比较的或优质的商业产品或那些使用传统方法得到的COL1制剂纯化。我们预计这种方法将有助于autologously衍生COL1的为众多的研究和临床应用的利用率。

这COL1隔离协议需要大约2小时，15分钟即可完成。图1示出的示意图，概述在此过程中的主要步骤。制备样品并进行高速鼓动的7个循环和漂洗用乙酸钠约需35分钟（图1A-C）。执行一个搅拌和冲洗周期与DH 2 O大约需要5分钟（图1D和1E）。添加柠檬酸钠和试样施加1搅拌和冲洗循环后搅拌6个周期需要大约45分钟（图1F和1G）。从提取液...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

对于可溶性Ⅰ型胶原（COL1）隔离的传统程序需要从开始10天左右，由于长时间的缓冲液孵育和纤丝费力再悬浮完成。在这里，我们描述了从真皮小活检净化COL1，在不到3小时的方法。

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

an-improved-method-for-the-preparation-of-type-i-collagen-from-skin

Research

JoVE Journal

Bioengineering

22.7K 次观看.  Boston Children's Hospital. 对于可溶性Ⅰ型胶原（COL1）隔离的传统程序需要从开始10天左右，由于长时间的缓冲液孵育和纤丝费力再悬浮完成。在这里，我们描述了从真皮小活检净化COL1，在不到3小时的方法。 

视频: An Improved Method for the Preparation of Type I Collagen From Skin

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三维人体皮肤重建模型:一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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

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 for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

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 ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

一种改进的机械性能测试方法来评估骨种植体支抗

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...