This work was supported by the UWS Research Grant.

1. Chitosan Adhesive Preparation
<ol>
 <li>The chitosan powder is soluble in acetic acid solution; for the preparation of a stock solution of acetic acid (2% v/v), add 10 ml glacial acetic acid to 490 ml deionized water (DI-H2O).</li>
 <li>For the preparation of a stock solution of Rose Bengal (RB) (0.01% w/v) in acetic acid, weigh 5 mg of RB in a vial wrapped with aluminum foil to avoid photobleaching. Add 0.5 ml of DI-H2O to dissolve the powder then add 49.5 ml of the acetic acid stock solution (described in section 1.1).</li>
 <li>For the preparation of chitosan solution (1.7% w/v), weigh 0.85 g of chitosan powder (medium MW, 85% degree of acetylation) and add to 50 ml of RB acetic acid solution. The control was prepared by dissolving the chitosan in acetic acid solution without RB.</li>
 <li>Add a magnetic stirrer to the chitosan mixture and stir at room temperature. To ensure complete dissolution of the components, stir the chitosan in acetic acid + RB for two weeks and the chitosan in acetic acid for 5 days (control). When chitosan is dissolved, the solution pH increases from ~2.6 to ~3.9. Note that RB is poorly soluble in acidic pH and it requires an increase in the pH of the solution and an extended stirring time to dissolve.</li>
 <li>Centrifuge the solutions at 3270 x g for 1 hr and collect the supernatant using, for example, a sterile 10 ml syringe. Make sure not to disturb the impurity pellet. During this step, the solution is cleaned from macroscopic impurities and insoluble matter.</li>
 <li>Inject the (centrifuged) chitosan solution on a flat perpex slab using a sterile syringe. The thickness of the dried film depends on the ratio between the dispensed amount of solution and the surface area over which the solution is spread. Ensure that the solution is free from air bubbles and evenly spread on the slab. To obtain thin films with ~13 &mu;m thickness, inject ~1 ml solution over 12 cm2 surface area (dimensions~3x4 cm, Figure 1A).</li>
 <li>Cover the Perspex slab with a screen, made of aluminum foil for example, to avoid photobleaching of the solution. Ensure that enough air ventilation is guaranteed to dry the solution under the screen.</li>
 <li>Leave the solution to dry at room temperature (25 &deg;C) and pressure (1 atm) till the resulting film is insoluble in water and does not swell macroscopically. This is typically achieved in 2 weeks.</li>
 <li>Detach the films carefully using a thin and flat spatula: these films can be easily peeled off the Perspex slab without damaging them.</li>
 <li>Measure the thickness of the films using a micrometer.</li>
 <li>Cut the adhesive in rectangular strips with a clean sharp scissor and place them between sterile glass slides to preserve their flat shape. Wrap the slides in aluminum foil for light shielding and store them at room temperature. Label the adhesives with or without RB as "rose adhesive" or "chitosan adhesive" respectively.</li>
 <li>The rose adhesive films can be sterilized using ethanol (90%) or with gamma radiation (0.1 KGy/h for 24 hr)4,5.</li>
</ol>
2. Adhesive Optical Attenuation
<ol>
 <li>Fix the rose adhesive inside a quartz cuvette.</li>
 <li>Record its attenuated spectrum in the range of 400 - 800 nm using a dual-beam spectrophotometer4.</li>
 <li>Assuming the validity of Beer's law, calculate the attenuation length of the adhesive as follows: I = I0e-Ax where I0 is the incident beam intensity, 1/A is the attenuation length, and x is the film thickness.</li>
 <li>Repeat steps 2.1 to 2.3 for the chitosan adhesive to serve as a control.</li>
</ol>
3. In Vitro Application: Laser Tissue Bonding on Intestine
<ol>
 <li>Harvest calf intestine immediately after animal euthanasia and store at -80 &deg;C.</li>
 <li>Prior to use, defrost and hydrate the tissue by immersing it in DI-H2O for 10 min.</li>
 <li>Bisect the tissue into sections (2 x 1 cm) using a #10 blade under an operating microscope (x10). Keep the sections moist using DI-H2O.</li>
 <li>Bring the incision stumps close together (end to end). Wipe excess water off the surface using cotton tips.</li>
 <li>Place a rose adhesive strip (10 x 6 mm) across the incision on the serosa layer with microforceps ensuring full contact with the intestine (Figure 2).</li>
 <li>The tissue adhesion of the rose adhesive is activated by a diode-pumped solid state laser, which is coupled to a multimode optical fiber (core diameter 200 &mu;m)4.</li>
 <li>Set the power level of the laser to 180 mW and irradiate the adhesive for ~6 min with a beam spot size of ~5 mm, using the parameters listed in Table 1. Spot-irradiate the adhesive ensuring that each spot is irradiated for ~5 seconds before moving the beam to the adjacent spot. This procedure guarantees that the laser beam scans the whole surface area of the adhesive several times.</li>
 <li>To assess the tissue bonding strength, clamp a sample to a calibrated single column tensiometer (Instron, MA, USA) using mechanical grips. Move the grips at 22 mm/min until the two tissue stumps separate4.</li>
 <li>Place a rose adhesive on the tissue as described in step 3.5. Do not irradiate the sample and measure the tissue bonding strength as described in step 3.8. This sample serves as a control.</li>
</ol>
4. In Vivo Application: Tibial Nerve Anastomosis
<ol>
 <li>Sedate the Wistar rat with isoflurane/O2 mixture (4% during induction, 2% thereafter)5. Subcutaneously inject Buprenex (0.03 mg/kg) to provide intra-operative analgesic activity and alleviate possible pain or discomfort.</li>
 <li>The following surgical procedure should be performed under an operating microscope (x10). Make an oblique skin incision about 3 cm long in the dorso-lateral part of the right thigh and expose the tibial nerve with a muscle-splitting approach through the gluteal muscles2.</li>
 <li>Partially dissect and trim the adventitia of the tibial nerve using straight micro-scissors and absorb excess water with sterile gauze or cotton tips.</li>
 <li>Cut the tibial nerve with micro-scissors.</li>
 <li>Position a sterile chitosan strip (dimensions 5 x 4 mm) underneath the tibial nerve using micro-forceps.</li>
 <li>Approximate the nerve stumps end-to-end with micro-forceps over the rose adhesive strip.</li>
 <li>The strip fully adheres to the nerve forming a collar, which can assist with the rotation of the nerve during the laser irradiation of the strip.</li>
 <li>Activate the rose adhesive with the laser as described in steps 3.6) and 3.7).</li>
 <li>Trim the redundant rose adhesive from the collar of the operated nerve.</li>
 <li>Close the muscles and skin with five 3-0 sutures5.</li>
 <li>Standard post-operative recovery procedures should be followed including daily inspection for dehiscence or pus formation and analgesic administration, if required.</li>
</ol>
5. Representative Results
The films that are obtained are bright rose in color, thin and have a smooth surface (Figure 1). They are also flexible and can be rolled into tubes of small diameter without causing tears or any other apparent damage (Figure 1B). The rose adhesive has two absorption peaks at 530 and 562 nm; the green laser is thus strongly absorbed by the adhesive and the corresponding attenuation length at 532 nm is ~12 &mu;m (Figure 3). In contrast, chitosan films without RB attenuates weakly the laser (1/A ~162 &mu;m), likely due to scattering. It appears from the spectra plot that no significant aggregation of RB occurs in the films.
The rose adhesive bonds firmly to the intestine upon laser irradiation (Figure 2) achieving a typical maximum load at failure of 0.9 &plusmn; 0.4 N (n=30). The adhesive strength was estimated as the maximum load divided by the adhesive surface area, namely, 15 &plusmn; 6 kPa (n=30). The non-irradiated rose adhesive bonded significantly less to tissue (2 &plusmn; 2 kPa, n=30, unpaired t-test p&lt;106). The in-vivo anastomosis of the tibial nerve was successfully achieved following the application and bonding of the rose adhesive in conjunction with the green laser. One week postoperatively, the operated nerve was in continuity and the repair strength was 17 &plusmn; 9 kPa (n=10).
<img alt="Figure 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> 
Figure 1. A) The RB-chitosan solution is casted on a flat Perspex slab (slab thickness~5 mm). Graph paper is placed underneath the slab to facilitate the dry-cast step. The water-insoluble film is obtained two weeks after casting. B) The rose adhesive is flexible and can be rolled into a small cylinder.
<img alt="Figure 2" src="/files/ftp_upload/4158/4158fig2.jpg" /> 
Figure 2. The rose adhesive is bonded to calf intestine after laser irradiation. Uniform irradiation is applied on the RB adhesive during tissue-bonding; however selected spots are shown in this picture to illustrate the photo-bleaching effect of the laser on the adhesive.
<img alt="Figure 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> 
Figure 3. The absorption spectrum (in arbitrary units) of the rose adhesive shows two peaks at 530 and 562 nm. The green laser (&lambda;= 532 nm) is thus strongly absorbed by the adhesive during the laser exposure. The chitosan adhesive without RB absorbs poorly the laser beam.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>n</td>
 <td>Area (mm2)</td>
 <td>Power (W)</td>
 <td>Time (s)</td>
 <td>Fluence (J/cm2)</td>
 <td>I (W/cm2)</td>
 <td>Max Load/Area (kPa)</td>
 </tr>
 <tr>
 <td>Adhesive+Laser</td>
 <td>30</td>
 <td>60 &plusmn; 10</td>
 <td>0.18 &plusmn; 0.03</td>
 <td>365 &plusmn; 5</td>
 <td>~110</td>
 <td>~0.9</td>
 <td>15 &plusmn; 1</td>
 </tr>
 <tr>
 <td>Adhesive</td>
 <td>30</td>
 <td>60 &plusmn; 10</td>
 <td>NA</td>
 <td>NA</td>
 <td>NA</td>
 <td>NA</td>
 <td>2 &plusmn; 2</td>
 </tr>
 </tbody>
</table>
Table 1. Legend. n, sample number; Area, surface area of the rose adhesive (mean &plusmn; maximum error); Power, laser power (mean &plusmn; maximum error); Time, irradiation time (mean &plusmn; maximum error); Fluence, average laser fluence; I, estimated irradiance; Max Load/Area, maximum load at failure of the repaired tissue divided by the adhesive surface area, (mean &plusmn; SE).

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No conflicts of interest declared.

The rose adhesive fabrication is based on a simple dry-cast process, though the dissolution of RB in acidic pH requires prolonged stirring of the chitosan solution. It is important to let the solution dry up until it becomes a water-insoluble film. This happens when the weight water content is ~10% in the dried film6. Insoluble films are usually obtained 2 weeks after dry-casting at standard conditions of temperature and pressure (~25 &deg;C and ~1 atm). The mechanism of tissue bonding is not clear yet, althou...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Rose Bengal</td>
 <td>Sigma-Aldrich</td>
 <td>632-69-9</td>
 <td></td>
 </tr>
 <tr>
 <td>Chitosan (medium MW)</td>
 <td>Sigma-Aldrich</td>
 <td>10318AJ</td>
 <td></td>
 </tr>
 <tr>
 <td>Glacial acetic acid</td>
 <td>Sigma-Aldrich</td>
 <td>08050051</td>
 <td>2% v/v in DI water</td>
 </tr>
 <tr>
 <td>Magnetic stirrer</td>
 <td>Heidolph</td>
 <td>MR Hei-Mix S</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Allegra X-12R</td>
 <td></td>
 </tr>
 <tr>
 <td>Spectrophotometer</td>
 <td>Varian </td>
 <td>Cary 4000 UV-Visible</td>
 <td></td>
 </tr>
 <tr>
 <td>Laser</td>
 <td>CNI Laser </td>
 <td>MGL-532 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micrometer</td>
 <td>Mitutoyo</td>
 <td>Series 227</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical 			microscope</td>
 <td>Zeiss </td>
 <td>OPMI</td>
 <td></td>
 </tr>
 </tbody>
</table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production3. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (&lambda;=532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 &plusmn; 6 kPa, (n=30). The adhesion strength drops to 2 &plusmn; 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Sutures are usually needed to repair tissue during surgical procedures. However, their application can be problematic as they are invasive and may damage tissue. The fabrication and application methods of a novel tissue adhesive are here reported. This adhesive film is laser-activated and does not require the use of sutures.

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

fabrication-application-rose-bengal-chitosan-films-laser-tissue

Research

JoVE Journal

Bioengineering

12.1K visualizações.  University of Western Sydney, NSW Australia. Suturas são normalmente necessários para reparar o tecido durante procedimentos cirúrgicos. No entanto, a sua aplicação pode ser problemática uma vez que são invasivos e pode danificar o tecido. Os métodos de fabricação e aplicação de um adesivo de tecido romance são aqui relatados. Esta película adesiva é activada por laser e não requer o uso de suturas.

Vídeo: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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.

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

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...