Name: in vitro synthesis of native, fibrous long spacing and segmental long spacing collagen
Uploaded: 2012-09-20T12:00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen at JoVE.com

We would like to thank all the many past undergraduate, graduate and post-doctoral students who have contributed their time and effort to the study of collagen fibrogenesis in our laboratory. The Natural Sciences and Engineering Research Council of Canada has continuously provided funding for our collagen studies.

NOTE: The water used in all the protocols has a resistivity &gt;18 M&Omega;.cm.
1. Native Collagen Fibrils
<ol>
	<li>Combine 6 &mu;l of water, 20 &mu;l of 200 mM Na2HPO4 adjusted to pH 7 with HCl and 10 &mu;l of 400 mM KCl in a microfuge tube and mix.</li>
	<li>Add 4 &mu;l of collagen monomer (~3 mg/ml in 0.01 N HCl) to the microfuge tube and mix. The solution should be clear and colorless.</li>
	<li>Place the microfuge tube containing the reaction mixture in a heat block that has been pre-warmed to 37 &deg;C and leave for 3 to 4 hr. At this point, the solution should be slightly cloudy and contain mainly native collagen fibrils.</li>
</ol>
2. Fibrous Long Spacing Collagen
<ol>
	<li>Dialyze 1 ml of collagen monomer (~3 mg/ml in 0.01 N HCl) using 12-14 kDa MWCO membrane against 400 ml of water, changing the water four times over a period of 24 hr at room temperature. The dialyzed collagen monomer that is not used right away can be stored at 4 &deg;C for future use.</li>
	<li>Combine 20 &mu;l of water, 20 &mu;l of 3 mg/ml &alpha;1-acid glycoprotein in water and 20 &mu;l of dialyzed collagen monomer in a microfuge tube. The solution should be clear and colorless.</li>
	<li>Leave the microfuge tube containing the reaction mixture at room temperature for 30 min. At this point, the solution should be cloudy and contain predominantly FLS collagen fibrils.</li>
</ol>
3. Segmental Long Spacing Collagen
<ol>
	<li>Combine 67 &mu;l of water, 60 &mu;l of 100 mM glycine-HCl buffer at pH 3.3 and 40 &mu;l of 10 mg/ml ATP in water in a microfuge tube and mix.</li>
	<li>Add 33 &mu;l of collagen monomer (~3 mg/ml in 0.01 N HCl) to the microfuge tube and mix. The solution should be clear and colorless.</li>
	<li>Leave the microfuge tube containing the reaction mixture at room temperature for 2 hr. At this point, the solution will still appear clear, but should contain mostly SLS collagen.</li>
</ol>
4. AFM Characterization
<ol>
	<li>Cleave the surface of a piece of mica attached to an AFM substrate by covering the mica surface with a piece of sticky tape and then peeling it away. Check the tape afterwards to confirm that an entire layer of mica was cleaved in order to reduce irregularities on the surface and ensure that the old sample is removed if the mica is being reused.</li>
	<li>Apply 20 &mu;l of the collagen fibril solution to the freshly cleaved mica substrate. Leave for 5 min.</li>
	<li>Gently rinse off solution of collagen fibril with water. It is advisable not to apply the water directly onto the center of the mica substrate but at the edge and to allow it to flow across the sample.</li>
	<li>Dry the surface under a gentle stream of nitrogen gas. It is advisable to direct the stream at the edge and not at the center of the mica substrate.</li>
	<li>Under an optical microscope at 200&times; magnification, the native and FLS collagen samples should show clumps of fibrils. SLS collagen will not be visible.</li>
	<li>The unique characteristics of the three different collagen structures are observable by AFM. Generally, we image native collagen fibrils with an AFM in intermittent contact mode using silicon AFM probes, whereas FLS and SLS collagen fibrils we typically image with an AFM in contact mode using silicon nitride AFM probes. We do this because of time and cost considerations. Silicon AFM probes are usually sharper than silicon nitride AFM probes, which make them particularly useful for imaging the narrower banding structure of native collagen fibrils. However, silicon AFM probes are much more prone to contamination by collagen samples than silicon nitride AFM probes and need to replaced frequently. Therefore, we reserve the use of silicon AFM probes mostly for imaging native collagen fibrils where resolution is a greater necessity and we operate in intermittent contact mode to prolong their lifetime of usefulness.</li>
</ol>
We recommend an initial 100 &times; 100 &mu;m2 scan which should show at least a few collagen constructs. From there, zoom in to scan sizes of 10 &times; 10 &mu;m2 and then 2 &times; 2 &mu;m2 to observe the banding periodicity of native and FLS collagen, or the finer features of an SLS collagen. It is also a good idea to check at least two other regions on the collagen sample to verify that the initial scans are representative.

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	<li>Kadler, K. E., Baldock, C., Bella, J., Boot-Handford, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+at+a+glance.">Collagen at a glance.</a> J. Cell Sci. 120, 1955-1958 (2007).</li><li>Abraham, L. C., Zuena, E., Perez-Ramirez, B., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+Collagen+Characterization+for+Bio+Studies.">Guide to Collagen Characterization for Bio Studies.</a> J. Biomed. Mat. Res. 87B, 264-285 (2008).</li><li>Baselt, D. R., Revel, J. -. P., Baldeschwieler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subfibrillar+Structure+of+Type+I+Collagen+Observed+by+Atomic+Force+Microscopy.">Subfibrillar Structure of Type I Collagen Observed by Atomic Force Microscopy.</a> Biophys. J. 65, 2644-2655 (1993).</li><li>Petruska, J. A., Hodge, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Subunit+Model+for+Tropocollagen+Macromolecule.">A Subunit Model for Tropocollagen Macromolecule.</a> Proc. Natl. Acad. Sci. U.S.A. 51, 871-876 (1964).</li><li>Smith, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Pattern+in+Native+Collagen.">Molecular Pattern in Native Collagen.</a> Nature. 219, 157-158 (1968).</li><li>Gross, J., Highberger, J. H., Schmitt, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extract+of+collagen+from+connective+tissue+by+neutral+salt+solutions.">Extract of collagen from connective tissue by neutral salt solutions.</a> PNAS. 41, 1-7 (1955).</li><li>Gale, M., Pollanen, M. S., Markiewicz, P., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+assembly+of+collagen+revealed+by+atomic+force+microscopy.">Sequential assembly of collagen revealed by atomic force microscopy.</a> Biophys. J. 68, 2124-2128 (1995).</li><li>Wood, G. C., Keech, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+fibrils+from+collagen+solutions.">The formation of fibrils from collagen solutions.</a> Biochem. J. 75, 588-597 (1960).</li><li>Williams, B. R., Gelman, R. A., Poppke, D. C., Piez, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+Fibril+formation.">Collagen Fibril formation.</a> J. Biol. Chem. 253, 6578-6585 (1978).</li><li>Holmes, D. F., Capaldi, M. J., Chapman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+collagen+fibrils+in+vitro;+the+assembly+process+depends+on+the+initiating+procedure.">Reconstitution of collagen fibrils in vitro; the assembly process depends on the initiating procedure.</a> Int. J. Biol. Macromol. 8, 161-166 (1986).</li><li>Paige, M. F., Rainey, J. K., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrous+long+spacing+collagen+ultrastructure+elucidated+by+atomic+force+microscopy.">Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.</a> Biophysical Journal. 74, 3211-3216 (1998).</li><li>Highberger, J. H., Gross, J., Schmitt, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+mucoprotein+with+soluble+collagen;+an+electron+microscope+study.">The interaction of mucoprotein with soluble collagen; an electron microscope study.</a> PNAS. 37, 286-291 (1951).</li><li>Ghadially, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ultrastructural pathology of the cell and matrix. , (1988).</li><li>Paige, M. F., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+assembly+of+segmental+long+spacing+collagen+studied+by+atomic+force+microscopy.">Ultrastructure and assembly of segmental long spacing collagen studied by atomic force microscopy.</a> Micron. 74, 355-361 (2001).</li><li>Schmitt, F. O., Gross, J., Highberger, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+particle+type+in+certain+connective+tissue+extracts.">A new particle type in certain connective tissue extracts.</a> PNAS. 39, 459-470 (1953).</li><li>Bruns, R. R., Hulmes, D. J. S., Therrien, S. F., Gross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procollagen+segment-long-spacing+crystallites:+their+role+in+collagen+fibrillogenesis.">Procollagen segment-long-spacing crystallites: their role in collagen fibrillogenesis.</a> PNAS. 76, 313-317 (1979).</li><li>Highberger, J. H., Gross, J., Schmitt, F. O. <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+observations+of+certain+fibrous+structures+obtained+from+connective+tissue+extracts.">Electron microscope observations of certain fibrous structures obtained from connective tissue extracts.</a> JACS. 72, 3321-3322 (1950).</li><li>Orekhovich, V. N., Tustanovsky, A. A., Orekhovich, K. D., Plotnikova, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O+prokollagene+kohzi.">O prokollagene kohzi.</a> Biokhimiya. 13, 55-60 (1948).</li><li>Lin, A. C., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+ultrastructure+of+fibrous+long+spacing+collagen+by+parallel+atomic+force+and+transmission+electron+microscopy.">Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy.</a> Proteins. 49, 378-384 (2002).</li></ol>

No conflicts of interest declared.

Purified collagen monomer is stable at low pH and temperature and the formation of native collagen fibrils essentially involves raising the pH and temperature of the collagen monomer solution. Procedures for the reconstitution of banded collagen fibrils from monomers have existed for more than 50 years. The procedure that our laboratory used in our first studies with collagen 15 years ago 7 was based on procedures summarized by Chapman and co-workers in 1986 10. The conditions for collagen fibril formation in that earlier work were 0.18 mg/ml of collagen monomer in Na2HPO4/KH2PO4 (I = 0.2, pH 7.4) buffer at 34 &deg;C. The drawback of this and other procedures that we have tried is that there are always unbanded fibrils formed alongside the desired banded fibrils. Our new conditions are ~0.3 mg/ml of collagen monomer in 100 mM phosphate and 100 mM KCl at pH 7 left at 37 &deg;C for 3-4 hr. The procedure described herein differs in every aspect from the earlier procedure for making native collagen fibrils. But most noticeably, we have doubled the ionic strength by raising the buffer concentration and adding 100 mM KCl. The increase in ionic strength results in more consistent formation of exclusively banded collagen fibrils.
In our experience, the only times that this procedure has not produced native type collagen was when the collagen monomer solution was past the manufacturer's recommended date of use. When this occurs, what is observed ranges from fewer fibrils that are all unbanded to many fibrils still observed but predominately unbanded. Note that expired collagen monomer can often still be successfully used to make native collagen, but when it fails, new collagen monomer should definitely be purchased.
FLS was first identified by Highberger et al. 17 in collagen preparations credited to Orekhovich et al. 18. Further studies led Highberger and Schmitt to conclude that it was &alpha;1-acid glycoprotein that promotes the formation of FLS 12. We were later able to replicate the procedure for making FLS collagen by combining commercially available collagen monomer and &alpha;1-acid glycoprotein at low pH and slowly allowing the pH to rise by dialyzing the mixture against water for 24 hr 11. We now present a modification of that procedure by dialyzing the collagen monomer against water first and simply combining it with &alpha;1-acid glycoprotein in water to form FLS collagen. The conditions for FLS collagen assembly described here are much less time consuming. The collagen monomer still needs to be pre-dialyzed against water, but it can be done in bulk and then stored at 4 &deg;C stably until it is used. This new procedure yields predominately FLS banded collagen fibrils.
From our experience, &alpha;1-acid glycoprotein with a lower combined protein and water content, and by inference higher sugar content, is critical for successfully making FLS fibrils. We typically check with the manufacturer that the &alpha;1-acid glycoprotein lot we use has a combined % protein and water content of 82 or less. And as with the procedure for native collagen synthesis, collagen monomer that is too old can also result in the failure to produce FLS collagen. In addition, the purity of the water used for the dialysis is critical in this procedure. For example, dialysis of collagen monomer against even ~8 M&Omega;.cm rather than &gt;18 M&Omega;.cm water results in a collagen solution that will not form FLS collagen.
Of the three collagen fibril types, the easiest to make is SLS collagen. The earliest preparation of SLS was described by Schmitt and co-workers 15. We published an adaption of that procedure 12 years ago for making SLS collagen using commercially available collagen 14. The procedure simply entailed combining 2 mg/ml ATP and 0.5 mg/ml of collagen monomer in 0.05% (v/v) acetic acid at pH 3.5, and leaving the mixture at room temperature overnight.
In our experience, the critical aspect of SLS assembly that must be controlled is the pH of the reaction solution. SLS assembled in more basic conditions (~pH 3.6-3.9) results in aggregated clumps of crystallites. More acidic conditions (~pH 2.9-3.2) results in thinner, more separated crystallites than ones assembled under the ideal conditions of pH 3.3-3.5. Reaction pHs outside this range (&lt; 2.8 and &gt; 4) do not yield any SLS collagen. In the past, we adjusted the pH of the reaction mixture by the addition of acetic acid. To simplify the procedure even further, in this manuscript we introduced a glycine-HCl buffer to control the pH.
The three different collagen structures formed from the protocols described above have characteristic features that can only be discerned by instruments capable of nanometer resolution. Native and FLS collagen are characterized by banding periodicities of ~67 nm and ~270 nm. The longest dimension of SLS collagen is just ~360 nm. We have described specifically how we use an AFM to characterize the three different collagen structures. However, any AFM operating in contact or intermittent contact mode with any AFM probe having &lt;10 nm radius should also be able to image these collagen structures. In addition, electron microscopy can be and has been used to characterize these collagen structures 19 .
The major advantage of these procedures is that they produce predominantly the desired collagen construct. The only other form of collagen that is found in these reactions is the starting monomer, which is often visible in the background of the AFM images. In the case of native and FLS collagen, they can be easily separated from most of the unreacted monomer by repeated centrifugation and washing of the resulting native or FLS collagen pellet with water.
We have presented simple and reliable procedures for making native, FLS and SLS collagen using advanced, commercially available starting materials. The collagen monomer used in all these procedures is commercially available in a purified form. &alpha;1-Acid glycoprotein and ATP are also both available commercially.

Collagen is a class of structural proteins that have a triple helical structure formed from three polypeptide chains. These triple helical monomers further assemble in a hierarchal manner to form progressively larger structures. There are at least 28 genetically different collagen types that have been identified 1. Combined, collagens make up 30% of the total protein found in animals, with Type I the predominant form accounting for up to 90% of all the collagen proteins 2. Type I collagen is found as fibrillous structures in skin, ligaments, tendons and bones. Atomic force microscope (AFM) and electron microscope (EM) images of Type I native collagen fibers typically show banding with a characteristic period of ~67 nm (often referred to as D-banding) 3-5.
Type I collagen monomer is a triple helical heterotrimer consisting of two identical &alpha;1(I) chains and one &alpha;2(I) chain, each more than a thousand amino acids long. It is long and thin with dimensions of 300 nm in length and 1.4 nm in diameter. Type I collagen monomer can be readily obtained by breaking down naturally formed higher order structures 6. We 7 and many others 8-10 have shown that these soluble monomer building blocks can then be used to reconstruct D-banded fibrils in vitro (Figure 1).
In addition to native collagen fibrils, two other higher order collagen structures have been found to form in vitro using the same monomer building block. The two structures are fibrous long spacing (FLS) and segmental long spacing (SLS) collagen (Figure 1). FLS collagen is a fibrillous construct like native collagen, but is characterized by a larger banding periodicity than native collagen 11. In vitro, FLS collagen forms when the collagen monomer is combined with &alpha;1-acid glycoprotein 12. FLS collagen has been observed in vivo as well, although rarely 13. SLS collagen is described as crystallites because the monomers are aligned in register like in a crystal lattice 14. SLS collagen forms when the collagen monomer is combined with adenosine triphosphate (ATP) 15. Naturally occurring SLS collagen has been observed in the culture medium of fibroblasts but not in extracted tissue samples, likely due to its small size and lack of distinguishable topographical features 16.
The goal of this manuscript is to present detailed procedures for making native collagen fibrils as well FLS and SLS collagen that even the most inexperienced researcher can reproduce. Using commercially available advanced starting materials, we have tried to make the protocols as simple as possible and still have them work reliably. We also detail how to use an AFM to characterize the different collagen structures that are made. This work will be beneficial to researchers who want to study one or more of these higher order collagen structures and/or use them as precursors in other applications, but currently lack of the expertise or simply do not wish to work with tissue samples.

<table border="1">
 <tbody>
 <tr>
 <td>Material/reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Type I collagen monomer (~3 mg/ml in 0.01 N HCl)</td>
 <td>Inamed Advanced Biomatrix</td>
 <td>5409 5005-B</td>
 <td>Lot 1261443 (2.9 mg/ml, pH 2.1) Lot 1629346 (3.2 mg/ml, pH 2.1) Lot 6006 (3.2 mg/ml, pH 2.1)</td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Research Biochemicals International</td>
 <td>A-141</td>
 <td>Lot FBJ-1194A</td>
 </tr>
 <tr>
 <td>&alpha;1-acid glycoprotein from bovine plasma </td>
 <td>Sigma-Aldrich</td>
 <td>G3643</td>
 <td>Lot 051K7440 (89.8%) Lot 011K7410 (82%) Lot 065K7405 (71.2%) Lot 063K7495 (71.9%) Lot 117K7535 (75.9%) 
 (protein + water content)</td>
 </tr>
 <tr>
 <td>a1-acid glycoprotein from human plasma </td>
 <td>Sigma-Aldrich</td>
 <td>G9885</td>
 <td>Lot 128H7606 (70.9%) Lot 049K7565V (68%) (protein + water content)</td>
 </tr>
 <tr>
 <td>mica</td>
 <td>Ted Pella</td>
 <td>53</td>
 <td></td>
 </tr>
 <tr>
 <td>Pointprobe - Silicon SPM-Sensor</td>
 <td>Nanoworld</td>
 <td>NHC</td>
 <td>Silicon AFM probe that we use for intermittent contact mode</td>
 </tr>
 <tr>
 <td>Veeco Nanoprobe Tips</td>
 <td>Veeco (now Bruker AFM probes)</td>
 <td>NP-S</td>
 <td>Silicon nitride AFM probe that we use for contact mode</td>
 </tr>
 </tbody>
</table>

in vitro synthesis of native, fibrous long spacing and segmental long spacing collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

Native collagen
The reaction mixture of ~0.3 mg/ml collagen monomer in 100 mM phosphate and 100 mM KCl at pH 7 left at 37 &deg;C for 3-4 hr will yield a solution containing native type collagen fibrils with clear ~67 nm D-banding, without unbanded fibrils.
Under an optical microscope at 200&times; magnification, clumps of several fibrils can be normally seen on the mica substrate particularly when viewed by differential interference contrast (DIC) microscopy (Figure 2). In a typical sample, a random 100 &times; 100 &mu;m2 scan by AFM will usually show at least a few fibrils that are 5-50 microns long (Figures 3a-c). Individual, separated collagen fibrils can be easily identified at this stage. With a 512 &times; 512 pixel2 image scan, zooming into a 10 &times; 10 &mu;m2 scan size shows that all the fibrils are banded (Figures 3d-f). In order to accurately measure the banding periodicity, it is best to zoom into a 2 &times; 2 &mu;m2 scan size (Figures 3g-i).
Banding periodicity can be determined by simply measuring and averaging the longitudinal distance between several peaks or valleys. Alternatively, if the AFM software allows, a one dimensional Fourier transform along a longitudinal cross-section can also be used. Figure 4 shows an AFM height image of a fibril and a corresponding longitudinal cross-section.
FLS collagen
The reaction mixture of 1 mg/ml &mu;1-acid glycoprotein and ~1 mg/ml collagen monomer in water left at room temperature for 30 min will yield a solution of FLS collagen fibrils.
Clumps of fibrils, similar to what is observed in the case of native collagen, can be easily seen on the mica substrate under an optical microscope at 200&times; magnification. In a typical sample, a random 100 &times; 100 &mu;m2 scan by AFM will usually show at least a few fibrils. With a 512 &times; 512 pixel2 image scan size, the banding periodicity can be measured by zooming into a 10 &times; 10 &mu;m2 scan (Figure 5a) or more accurately with a 2 &times; 2 &mu;m2 scan (Figure 5b). Figure 5c shows a longitudinal cross-section of a FLS fibril.
SLS collagen
The reaction mixture of 2 mg/ml ATP and ~0.5 mg/ml of collagen monomer in 100 mM glycine-HCl buffer at pH 3.3 left at room temperature for 2 hr will yield a solution of SLS collagen.
Typically, SLS crystallites will not be visible under an optical microscope. An AFM is required to confirm the presence of SLS crystallites. In a typical sample, a random 100 &times; 100 &mu;m2 scan by AFM will usually show many dots. Zooming into a 10 &times; 10 &mu;m2 scan will usually show several SLS crystallites (Figure 6a). And a 2 &times; 2 &mu;m2 scan (Figure 6b) shows the finer structure of an SLS crystallite.
<img alt="Figure 1" src="/files/ftp_upload/4417/4417fig1.jpg" /> 
 Figure 1. The three structurally different forms of collagen fibrils that can be formed in vitro from collagen monomer. AFM images depicting collagen monomer and the three higher order collagen structures all on the same size scale (2 &times; 2 &mu;m2).
<img alt="Figure 2" src="/files/ftp_upload/4417/4417fig2.jpg" /> 
 Figure 2. Digital image at ~200&times; magnification by optical DIC microscopy of native collagen fibrils on mica (a) untouched and (b) thresholded and sharpened to highlight the fibrils.
<img alt="Figure 3" src="/files/ftp_upload/4417/4417fig3.jpg" /> 
 Figure 3. AFM images of native collagen fibrils on mica. Intermittent contact mode AFM amplitude images are shown.
<img alt="Figure 4" src="/files/ftp_upload/4417/4417fig4.jpg" /> 
 Figure 4. (a) 0.5 &times; 1 &mu;m2 intermittent contact mode AFM height image (vertical scale = 100 nm) and (b) longitudinal cross-section of a native collagen fibril.
<img alt="Figure 5" src="/files/ftp_upload/4417/4417fig5.jpg" /> 
 Figure 5. AFM images of FLS collagen fibrils on mica. (a) 10 &times; 10 &mu;m2 contact mode AFM deflection image, (b) 2 &times; 2 &mu;m2 contact mode AFM deflection image and (c) longitudinal cross-section of a FLS collagen fibril.
<img alt="Figure 6" src="/files/ftp_upload/4417/4417fig6.jpg" /> 
 Figure 6. AFM images of SLS collagen on mica. Intermittent contact mode AFM amplitude images are shown.

Watch this Scientific Journal Video about In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen at JoVE.com

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

in-vitro-synthesis-native-fibrous-long-spacing-segmental-long-spacing

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Bioengineering

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

Targeting the Rat&#039;s Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...