D.O.Z. is supported by a grant awarded from The Geneva Foundation. S.N. was supported by a Postdoctoral Fellowship Grant from the Pittsburgh Tissue Engineering Initiative.

1. Isolating Adipose-Derived Stem Cells (ASC)
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Isolate rat perirenal and epididymal adipose and wash with sterile Hank's buffered salt solution (HBSS) containing 1% fetal bovine serum (FBS) as previously described6.</li>
	<li>Mince the tissue and transfer 1-2 g into 25 mL of HBSS containing 1% FBS into a 50 mL tube and centrifuge at 500 g for 8 min at room temperature.</li>
	<li>Collect the free-floating adipose tissue layer and transfer to 125 mL erlenmeyer flask and treat with 25 mL of collagenase type II (200 U/mL) in HBSS for 45 min at 37 &deg;C on an orbital shaker (125 rpm).</li>
	<li>Carefully remove the liquid fraction (below oil and adipose layer) and filter sequentially through a 100-&mu;m and 70-&mu;m nylon mesh filter. Centrifuge the filtrate at 500 g for 10 min at room temperature, aspirate the supernatant and wash the pellet twice with 25 mL HBSS.</li>
	<li>Resuspend the cell pellet in 50 mL of growth medium (MesenPRO RS Basal Medium) supplemented with MesenPRO RS Growth Supplement, antibiotic-antimycotic (100 U/mL of penicillin G, 100 &mu;g/mL streptomycin sulfate, and 0.25 &mu;g/mL Amphotericin B), and 2 mM L-glutamine and pipet cells into 2 T75 flasks (25 mL/flask).</li>
	<li>Culture the ASC in a 5% CO2 humidified incubator at 37 &deg;C (Passage 2-4 ASC are used for all experiments).</li>
</ol>
2. Preparing Chitosan Microspheres (CSM)
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>CSM are prepared by a water-in-oil emulsification process along with an ionic coacervation technique using our previous protocol6. Emulsify an aqueous solution of chitosan (6 mL of 3%w/v chitosan in 0.5 M acetic acid) in a 100 mL of an oil phase mixture consisting of soya oil, n-octanol (1:2 v/v) and 5% sorbitan-mono-oleate (span 80) emulsifier, using overhead (1700 rpm) and magnetic stirring (1000 rpm) simultaneously, in opposite directions. This dual method of mixing ensures that micelles formed early on before cross-linking occurs can remain in solution and not settle to the bottom. Furthermore, the magnetic stir bar aids in de-aggregating chitosan during micelle formation and rigidization.</li>
	<li>The mixture is continuously stirred for approximately 1 hour until a stable water-in-oil emulsion is obtained. Ionic crosslinking is initiated with the addition of 1.5 mL of 1% w/v potassium hydroxide in n-octanol every 15 min for 4 h (24 mL total).</li>
	<li>After completion of the crosslinking reaction, slowly decant the oil phase of the mixture containing CSM and immediately add the spheres to 100 mL of acetone. Wash the spheres with acetone until the oil phase is completely removed.</li>
	<li>Dry the recovered spheres in a vacuum desiccator and analyze without further processing. The average CSM particle size, surface area per milligram and the unit cubic volume was determined using particle size analyzer.</li>
	<li>For subsequent experiments, wash CSM three times with sterile water to remove residual salts and sterilize with absolute alcohol.</li>
</ol>
3. Determining the Number of Free Amino Groups in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Determine the number of free amino groups present in CSM after ionic crosslinking by using the trinitro benzenesulfonic (TNBS) acid assay of Bubins and Ofner7. Incubate 5 mg of microspheres with 1 mL of 0.5% TNBS solution in a 50 mL glass tube for 4 h at 40 &deg;C and hydrolyze with the addition of 3 mL of 6N HCl at 60 &deg;C for 2h.</li>
	<li>Cool the samples to room temperature, and extract the free TNBS by adding 5 mL of deionized water and 10 mL of ethyl ether.</li>
	<li>Warm a 5 mL aliquot of the aqueous phase to 40 &deg;C in a water bath for 15 min to evaporate any residual ether, cool to room temperature, and dilute with 15 mL of water.</li>
	<li>Measure the absorbance at 345 nm with a spectrophotometer using TNBS solution without chitosan as blank and the chitosan used for CSM preparation to determine the total number of amino groups. Estimate the number of free amino groups of the CSM relative to chitosan.</li>
</ol>
4. Loading ASC in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Equilibrate 5 mg of sterilized CSM from section 2.5 in sterile HBSS overnight and add to an 8-&mu;m pore size membrane culture plate insert (24-well plate).</li>
	<li>After the CSM have settled onto the membrane, carefully aspirate the HBSS and add 300 &mu;L of growth medium to the inside of the insert and 700 &mu;L of growth media to the outside of the insert.</li>
	<li>Resuspend ASC at the appropriate concentration (1 x 104 to 4 x 104) in 200 &mu;L of growth medium and seed over the CSM inside the culture plate insert. The final volume of medium within the culture insert, after seeding, is 500 &mu;L.</li>
	<li>Incubate the ASC seeded on CSM for 24 h in a 5% CO2 humidified incubator at 37 &deg;C.</li>
</ol>
5. Determining the Percentage of ASC Loading and Cell Viability in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>After incubation, collect the ASC-loaded CSM in a sterile 1.5- ml microcentrifuge tube without disturbing the cells that have migrated into the insert membrane.</li>
	<li>Remove the residual medium and add 250 &mu;L of fresh growth medium to the tube.</li>
	<li>To each tube add 25 &mu;L MTT [3-(4,5-dimethylthiozole-2-yl)-2,5-diphenyltetrazolium bromide] solution (5 mg/mL) and incubate for 4 h in a 5% CO2 humidified incubator at 37 &deg;C.</li>
	<li>After incubation, remove the medium, add 250 &mu;L of dimethyl sulfoxide, and vortex the mixture for 2-5 min to solubilize the formazan complex.</li>
	<li>Centrifuge the CSM at 2700 x g for 5 min, and determine the supernatant absorbance measured at 570 nm using 630 nm as reference.</li>
	<li>Determine the cell number associated with the CSM relative to the absorbance value obtained from known numbers of viable ASC.</li>
</ol>
6. Characterizing ASC-CSM-Embedded Collagen Gel
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Mix ASC-loaded CSM (5 mg containing &asymp; 2 x 104 cells) with type 1 collagen (7.5 mg/mL) extracted from rat tail tendon according the method of Bornstein8 and fibrillate after adjusting the pH to 6.8 using 2 N NaOH.</li>
	<li>Add the fibrillated collagen-ASC-CSM mixture to a 12-well plate and incubate for 30 min at in a 5% CO2 humidified incubator at 37 &deg;C.</li>
	<li>After complete fibrillation, incubate the collagen-ASC-CSM gels for up to 14 days in a 5% CO2 humidified incubator at 37 &deg;C.</li>
	<li>Release and migration of cells from CSM into the gel were observed using standard microscopy techniques.</li>
</ol>
7. Representative Results
In the present study, we have developed an in vitro strategy to deliver stem cells from chitosan microspheres (CSM) into a collagen gel scaffold. Porous CSM of uniform size (175-225 &mu;m in diameter) and composition were prepared and used as cell carriers (Figure 2). Upon incubating ASC with the CSM, the cells attached at a concentration of 2 x 104 cells/5mg of CSM. The cells were capable of spreading over the microsphere, while extending filopodia into the porous crevices of the microsphere (Figure 3). Once the cell-loaded CSM were mixed with the collagen gel, the cells immediately began migrating into the gels (Figure 4).
<img alt="Table 1" src="/files/ftp_upload/3624/3624table1.jpg" /> 
Table 1. Biological advantages for the use of chitosan and collagen in a stem cell delivery system.
<img alt="Figure 1" src="/files/ftp_upload/3624/3624fig1.jpg" /> 
Figure 1. Schematic depicting the overall strategy for the dual use of ASC-CSM loaded collagen scaffolds. Figures 2, 3, and 4 are annotated within the schematic to assist with interpretation of the images.
<img alt="Figure 2" src="/files/ftp_upload/3624/3624fig2.jpg" /> 
Figure 2. Schematic representation depicting the process of seeding stem cells onto chitosan microspheres. The process involves co-culturing ASC with CSM in an 8- &mu;m pore size membrane culture plate insert. After 24 hours, the microspheres are removed from the insert and are ready for embedding into a biomaterial matrix.
<img alt="Figure 3" src="/files/ftp_upload/3624/3624fig3.jpg" /> 
Figure 3. Morphological characterization of CSM-loaded with ASC. Panel A depicts a light micrograph of ASC-loaded CSM, while panel B shows the same field of view superimposed with an image obtained by confocal fluorescence microscopy. ASC were preloaded with calcein AM (green). Panel C depicts an image of an SEM image of an unloaded microsphere, while panel D shows cells loaded onto the microsphere (asterisks). The TEM image in panel E shows a cross-section of an unloaded microsphere. A multitude of pores and crevices are located throughout the microsphere. Panel F shows a cross-sectioned microsphere with cells (arrows) attached and extending filopodia into the crevices. Original magnifications: A &amp; B= 70X; C= 500x , D= 2,000x; E&amp;F= 2,500x.
<img alt="Figure 4" src="/files/ftp_upload/3624/3624fig4.jpg" /> 
Figure 4. Migration of ASC from the CSM into the three-dimensional collagen scaffold. Panels A and B depict the CSM with cells migrating away from microsphere and into the collagen matrix on day 3 (A, arrows). Panel B shows a similar culture after 12 days. Transmission electron microscopy (TEM) images are depicted in C, D, and E. Asterisks in C and D show a microsphere that has been cross-sectioned with cells migrating away from the microsphere (arrows). Higher magnification of panel D is depicted in panel E, and shows cell filopodia attached to the collagen fibrils (inset). Original magnification: A&amp;B = 100x; C&amp;D = 6,000x; E = 20,000x, inset = 150,000x.
<img alt="Figure 5" src="/files/ftp_upload/3624/3624fig5.jpg" /> 
Figure 5. Schematic depicting the vast uses of CSM in regenerative medicine and drug delivery.

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No competing financial interests exist. 
Disclaimers
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the U.S. Government. The authors are employees of the U.S. Government, and this work was prepared as part of their official duties. All work was supported by the U.S. Army Medical Research and Materiel Command. This study was conducted under a protocol reviewed and approved by the US Army Medical Research and Materiel Command Institutional Review Board, and in accordance with the approved protocol.

A major hurdle in stem cell-based therapy is developing efficient methods for delivery of cells to the specified regions for repair. Due to patient to patient variability, the tissue type, injury size and depth; the methodology of delivering stem cells must be determined on a case-by-case basis. Although embedding stem cells within a matrix and delivering them to the wound site appears to be a next logical approach for tissue engineering, some technical hurdles remain. This includes the ability of the embedded cel...

<table border="1" >
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Hanks BalancedSalt Solution (HBSS)</td>
 <td>Gibco</td>
 <td>14175</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Collagenase Type II</td>
 <td>Sigma-Aldrich</td>
 <td>C6685</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>70-&mu;m nylon mesh filter</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>100-&mu;m nylon mesh filter</td>
 <td>BD Biosciences</td>
 <td>352360</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>MesenPRO Growth Medium System</td>
 <td>Invitrogen</td>
 <td>12746-012</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>T75 Tissue Culture Flask</td>
 <td>BD Biosciences</td>
 <td>137787</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Chitosan</td>
 <td>Sigma-Aldrich</td>
 <td>448869</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Acetic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>320099</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>N-Octanol</td>
 <td>Acros Organics</td>
 <td>150630025 </td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Sorbitan-Mono-oleate</td>
 <td>Sigma-Aldrich</td>
 <td>S6760</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Potassium Hydroxide</td>
 <td>Sigma-Aldrich</td>
 <td>P1767</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher Scientific</td>
 <td>L-4859</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>270741</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Trinitro Benzenesulfonic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>P2297</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Hydrochloric Acid</td>
 <td>Sigma-Aldrich</td>
 <td>320331</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Ethyl Ether</td>
 <td>Sigma-Aldrich</td>
 <td>472-484</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>8-&mu;m Tissue Culture Plate Inserts</td>
 <td>BD Biosciences</td>
 <td>353097</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>1.5-ml Microcentrifuge Tubes</td>
 <td>Fisher</td>
 <td>05-408-129</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>MTT Reagent</td>
 <td>Invitrogen</td>
 <td>M6494</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D8779</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Qtracker Cell Labeling Kit (Q tracker 655)</td>
 <td>Molecular probes</td>
 <td>Q2502PMP</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Type 1 Collagen</td>
 <td>Travigen</td>
 <td>3447-020-01</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Sodium Hydroxide</td>
 <td>Sigma-Aldrich</td>
 <td>S8045</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>12-Well Tissue Culture Plates</td>
 <td>BD Biosciences</td>
 <td>353043</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf</td>
 <td>5417R</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Orbital Shaker</td>
 <td>New Brunswick Scienctific</td>
 <td>C24</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Humidified Incubator with Air-5% CO2</td>
 <td>Thermo Scientific</td>
 <td>Model 370</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Overhead Stirrer</td>
 <td>IKA</td>
 <td>Visc6000</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Magnetic Stirrer</td>
 <td>Corning</td>
 <td>PC-210 </td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Vacuum Desiccator</td>
 <td>-</td>
 <td>-</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Particle Size Analyzer</td>
 <td>Malvern</td>
 <td>STP2000 Spraytec</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Water Bath</td>
 <td>Fisher Scientific</td>
 <td>Isotemp210</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Spectrophotometer</td>
 <td>Beckman</td>
 <td>Beckman Coulter DU800UV/Visible Spectrophotometer</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Diagger</td>
 <td>3030a</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Microplate Reader</td>
 <td>Molecular Devices</td>
 <td>SpectraMax M2</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Light/Fluorescence Microscope</td>
 <td>Olympus</td>
 <td>IX71</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Confocal Microscope</td>
 <td>Olympus</td>
 <td>FV-500 Laser Scanning Confocal Microscope</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Scanning Electron Microscope</td>
 <td>Carl Zeiss MicroImaging</td>
 <td>Leo 435 VP</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Transmission Electron Microscope</td>
 <td>JEOL</td>
 <td>JEOL 1230</td>
 <td>Equipment</td>
 </tr>
 </tbody>
 </table>

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.

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

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

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.

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 ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Research

JoVE Journal

Bioengineering

16.7K 조회수.  United States Army Institute of Surgical Research. 현재 줄기 세포 치료에서 주요 장애물은 호스트 조직으로 이들 세포를 전달하는 가장 효과적인 방법을 결정합니다. 지방 파생 줄기 세포들이 multipotency을 유지할 수 있도록하면서 여기에서, 우리는 효과적이고 접근 간단 키토산 기반의 전달 방법을 설명합니다.

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

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.

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

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

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

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

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

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.

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

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) &amp; crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling &amp; degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p&#8804;0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

The prepared DOX-CL scaffold satisfied the prerequisites of an ideal DW dressing in mechanical strength, porosity, water absorption, degradation rate, sustained release, anti-bacterial, biocompatibility, and anti-inflammatory properties, which are considered to be essential for the recovery of damaged tissue in DWs.

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

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...