The authors thank Dr. Ashish D Wadhwani. (Assistant Professor and Head, Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, Ooty, India) for assisting in In vitro cell viability studies.
The authors would like to thank the Department of Science and Technology - Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi, for supporting our department.
The authors also like to thank Mr. Sanju. S and Mr. Sriram. Narukulla&#160;M. Pharm students for their support in the video shoot.
This research was supported by the JSS Academy of Higher Education &#38; Research (JSSAHER).

All the animal procedures performed were approved by the institutional animal ethical committee of JSS College of Pharmacy, Ooty, India.
1. Preparation of DOX loaded porous scaffolds by freeze-drying method
<ol>
	<li>Add 1.2 g of COL to 100 mL of water (e.g., Millipore) and keep aside for swelling.</li>
	<li>Stir the swollen COL dispersion at 2000 rpm overnight to ensure complete dissolution of COL.</li>
	<li>Prepare CS solution by dissolving approximately 0.8 g of CS in 100 mL of 1% acetic acid.</li>
	<li>Stir the CS solution overnight at 2000 rpm to ensure uniform dispersion.</li>
	<li>Mix DOX (1% w/v), followed by CS solution, to the COL solution, and stir for 30 min.</li>
	<li>Filter the obtained physical mixture using a muslin cloth to remove the particulate matter.</li>
	<li>Deep freeze the obtained filtrate at -85 &#176;C &#177; 4 &#176;C for about 24 h.</li>
	<li>Lyophilize the deep freeze mixture at -85 &#176;C &#177; 4 &#176;C for 72 h.</li>
	<li>Store the obtained scaffolds in a desiccator for further analysis 16,17.</li>
</ol>
2. Crosslinking of scaffold
<ol>
	<li>Dissolve MES (0.488 g) in 50 mL of water.</li>
	<li>Soak 50 mg of the DOX loaded scaffold in 20 mL of the MES buffer for 30 min.</li>
	<li>Mix 19.5 mL of MES buffer with 0.1264 g of EDC and 0.014 g of NHS in a separate beaker.</li>
	<li>Immerse the scaffold in the buffer mixture for 4 h to achieve crosslinking 16.</li>
	<li>Store the DOX loaded crosslinked (CL) and non-crosslinked scaffolds (NCL) for further evaluation.</li>
</ol>
3. Characterization of scaffolds
<ol>
	<li>Morphological examination using a scanning electron microscopy (SEM)
	<ol>
		<li>Characterize the scaffolds for morphological analysis using SEM (1 cm &#215; 1 cm &#215; 0.5 cm).</li>
		<li>Stain the cross-section and exterior surface of the scaffold with the delicate layer of gold (~150 &#197;).</li>
		<li>Capture the photographic image at the excitation voltage of 5 kV and 10 kV.</li>
		<li>Place the samples in aluminum stubs and enclose them with the gold at approximately 9 V.</li>
		<li>Measure the scaffold using SEM with the increased resolution at 10 kV.</li>
	</ol>
	</li>
	<li>Porosity determination
	<ol>
		<li>Measure the porosity of the scaffolds using the liquid displacement method (ethanol) 18.</li>
		<li>Calculate the porosity of the scaffolds using the below formulae. 
		<img alt="Equation 1" src="/files/ftp_upload/62184/62184eq01.jpg" /> 
		Ww = Wet weight of the scaffold 
		Wd = Dry weight of the scaffold 
		Wv = Volume of the scaffold</li>
	</ol>
	</li>
	<li>Determining the water absorption capacity
	<ol>
		<li>Measure the dry weight of the scaffold.</li>
		<li>Incubate the weighed scaffold at 37 &#176;C for 24 h in phosphate buffer saline (PBS) pH 7.4.</li>
		<li>Remove the excess PBS over the scaffold using filter paper.</li>
		<li>Measure the water absorption capacity using the below formulae 17. 
		<img alt="Equation 2" src="/files/ftp_upload/62184/62184eq02.jpg" /> 
		WS = Percentage of water absorption 
		W1=Wet weight of the scaffold 
		W0= Dry weight of the scaffold</li>
	</ol>
	</li>
	<li>Scaffold degradation
	<ol>
		<li>Incubate the scaffold (1cm x 1cm) at 37 &#176;C for 7 days in a PBS of pH 7.4 containing lysozymes.</li>
		<li>Wash the scaffold to remove any adhered ions on the surface.</li>
		<li>Freeze dry the washed scaffold 17.</li>
		<li>Calculate the rate of degradation using formulae. 
		<img alt="Equation 3" src="/files/ftp_upload/62184/62184eq03.jpg" /> 
		Ww = Initial weight of the scaffold 
		Wd = Weight of the scaffold after freeze-drying</li>
	</ol>
	</li>
	<li>In vitro release studies
	<ol>
		<li>Determine the release behavior of the DOX from the scaffold using the dialysis sack method.</li>
		<li>Disperse the scaffold in a few milliliters of simulated wound fluid (pH 7.4) and transfer it into a dialysis bag.</li>
		<li>Tightly close the ends of the membrane bag and immerse in the 500 mL of simulated wound fluid solution.</li>
		<li>Stir the wound fluid solution containing the dialysis bag at 200-250 rpm.</li>
		<li>Collect the supernatant solution and replace it with an equal quantity of fresh buffer solution at definite time intervals.</li>
		<li>Determine the percentage of DOX release from the scaffolds in the supernatant solution using a UV-visible spectrometer at 240 nm.</li>
	</ol>
	</li>
</ol>
4. In vitro anti-bacterial studies
<ol>
	<li>Determine the minimum inhibitory concentration (MIC) of the CL and DOX-CL scaffolds against the S. aureus, S. epidermis, E. coli, P. aeruginosa using the micro-broth dilution method.</li>
	<li>Prepare the bacterial cultures using Mueller-Hinton broth at a ratio of 1:1000 to obtain 0.5 McFarland turbidity.</li>
	<li>Add D-glucose (800 mg/dL) to the bacterial cultures for hyperglycation 19,20.</li>
	<li>Mince and solubilize the CL and DOX-CL in DMSO (negative control).</li>
	<li>Serially dilute the hyperglycated bacterial suspension (100 &#181;L) and test samples (100 &#181;L of scaffolds solution) in 96 well plate.</li>
	<li>Incubate the plate at 37 &#176;C for 20-24 h.</li>
	<li>Record the absorbance at a wavelength of 600 nm 21.</li>
</ol>
5. In vitro biocompatibility studies
<ol>
	<li>Evaluate the biocompatibility of the prepared scaffolds using MTT [(3-(4, 5 dimethyl thiazole-2 yl) -2, 5-diphenyl tetrazolium bromide)] assay.</li>
	<li>Sterilize the scaffolds of standard dimension and place them in 24 well plates.</li>
	<li>Add 3T3-L1 cells to the 24 well plate and incubated for 72 h.</li>
</ol>
6. In vivo animal studies
<ol>
	<li>Induction of DM and excision wound
	<ol>
		<li>Feed the animal with a high-fat diet for two weeks and administer a single dose of streptozotocin (STZ) (50 mg/kg body weight) in citrate buffer solution intraperitoneally to Wistar albino rats (180-200 g) for the induction of type-2 diabetes.</li>
		<li>Choose the animals with a constant blood glucose of 250 mg/dL for the study.</li>
		<li>Randomize the selected animals for the induction of excision wounds.</li>
		<li>Anesthetize the diabetic rats using diethyl ether (5 mL was added to the priorly saturated anesthesia chamber) and confirm using the toe pinch method and mucous membrane color.</li>
		<li>Shave the dorsal area (Dorsal thoracic, lumbar region) using an aseptic trimmer and blades (A40).</li>
		<li>Sterilize the shaved area with an alcoholic swab.</li>
		<li>Excise the skin (2 x 2 cm2 and a depth of 1 mm) with an aseptic surgical A40 blade on the shaved area to create an open wound.</li>
		<li>Divide the animals into three groups (Group 1- Disease control (Control), Group 2- CL scaffold (Placebo), Group 3- DOX CL scaffold), each group consisting of 6 rats.</li>
		<li>Affix the CL and DOX CL scaffolds using surgical tape and cover the control group with sterile gauze for 21 days.</li>
		<li>Trace the wound area on a sterile OHP sheet and measure the percentage reduction of the wound using the grid method on days 0, 7, 14, and 21 for all groups.</li>
		<li>Calculate the percentage wound reduction using the below formulae. 
		<img alt="Equation 4" src="/files/ftp_upload/62184/62184eq04v2.png" /></li>
	</ol>
	</li>
</ol>
7. Histopathological studies
<ol>
	<li>Isolate the healed wound area on days 7, 14, and 21, store in formalin solution (10%).</li>
	<li>Section the tissues using a microtome to obtain a thickness of 6 &#181;m.</li>
	<li>Mount the sections on a glass slide and stain using Hematoxylin and eosin 17.</li>
	<li>Capture the images under 40x magnification using a digital microscope.</li>
</ol>
8. Hydroxyproline estimation
<ol>
	<li>Isolate the healed wound area on days 0, 7, 14, and 21 for evaluation.</li>
	<li>Estimate the hydroxyproline content using the procedure described by Reddy G et al., 1996 22.</li>
</ol>
9. Elisa test
<ol>
	<li>Estimate the MMP-9 levels using the Elisa kit as per the manufacturer&#39;s instructions.</li>
	<li>Isolate the tissue samples from the healed wound area on day 21 and mince using a tissue homogenizer.</li>
	<li>Centrifuge the obtained homogenate and collect the supernatant.</li>
	<li>Dilute the supernatant at 100-fold using assay buffer.</li>
	<li>Scan the plate using a multiple plate reader.</li>
</ol>
10. Statistical analysis
<ol>
	<li>Represent the obtained outcomes as Mean &#177; SD.</li>
	<li>Perform the statistical analysis using Graph pad prism v5.01.</li>
	<li>Attain the statistical significance using One Way Analysis of Variance (ANOVA) and Dunnet&#39;s post hoc test.</li>
	<li>Consider the values with p&#8804;0.05 as significant.</li>
</ol>

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The authors declare that they have no competing financial interests.

The main objective of this study was to determine the effect of DOX loaded COL-CS scaffold on DW healing in rats. CL and NCL were prepared and evaluated in terms of morphology, swelling index, in vitro release kinetics, and biocompatibility.
Characterization of the DOX loaded NCL and CL scaffold 
The prepared scaffolds were found to be porous with interconnected pores. These interconnected pores assure the porous, spongy nature that helps in the proper diffusion of oxygen...

Diabetes mellitus (DM) is a condition where the body&#39;s failure to deliver insulin or react to its outcomes in abnormal digestion of straightforward sugars brings about an upsurge of blood glucose 1. The most consecutive and crushing entanglement of DM is the diabetic wound (DW). Roughly 25% of patients with DM have the opportunity to build up a DW in their lifetime 1. The hindered healing of DW is accredited to a triopathy of DM: immunopathy, vasculopathy, and neuropathy. Whenever DW is left untreated, it may result in gangrene development, therefore prompting the removal of the concerned organ 2.
Plenty of treatments, such as instructing the patients (inspect wound daily, cleanse the wound, avoid activities that creates pressure on the wound, periodic glucose monitoring, etc.), controlling their blood glucose, wound debridement, pressure offloading, medical procedure, hyperbaric oxygen therapy, and advanced therapies are in practice&#160;3,4. The majority of these medications fail to address all prerequisites vital for DW care in light of the multifactorial pathophysiological conditions and unexpected expenses related to these medicines&#160;5. Even though the DW pathogenesis is multifactorial, the persistent inflammation with inappropriate tissue management is stated to be the actual reason for delayed healing in DWs 5,6.
Augmented levels of inflammatory and pro-inflammatory mediators in DW result in diminished growth factors responsible for delayed wound healing 2,6. Improper extracellular matrix (ECM) formation in DWs is accredited to increased levels of matrix metalloproteinases (MMPs) accountable for the rapid degradation of formed ECM. In MMPs, MMP-9 is reported as a major intermediary responsible for prolonged inflammation and rapid ECM degradation 7. It is stated that local treatment with an anti-inflammatory drug that decreases the elevated levels of MMP-9 re-establishes cutaneous homeostasis, framework arrangement and prompts better healing of DWs 8,9.
Doxycycline (DOX), an MMP-9 inhibitor, was chosen to suppress the elevated levels of MMP-9, a major inflammatory mediator responsible for persistent inflammation in DWs 10,11,12. In addition, DOX possess antioxidant (produce free hydroxy and phenoxy radicals capable of binding with reactive oxygen species) 13 and anti-apoptotic (inhibit caspase expression and mitochondrial stabilization) 14 activities that are essential for the treatment of DW. The arrangement of frameworks containing DOX, collagen (COL), and chitosan (CS) was chosen. The choice of COL depends on the way that it helps in providing the necessary framework responsible for mechanical strength and tissue regeneration 15. On the other hand, CS is structurally homologous to glycosaminoglycan, associated with several wound healing phases. It is also reported that CS holds significant anti-bacterial property 15. Hence, the COL/CS scaffold of DOX is formulated to suppress the prolonged inflammation, followed by supporting the matrix formation for successful wound healing in DM conditions.

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

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

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.

Characterization of the DOX loaded NCL and CL scaffold 
On visual examination, the NCL and CL scaffold was found to be cream in color. Besides, both the scaffolds appear to be like a sponge, stiff and inelastic when examined physically. SEM images of the NCL and CL scaffolds are shown in Figure 1. From the figure, it was clear that there was a decrease in pore size after crosslinking by forming intermolecular linkages. Also, the NCL and CL scaffolds porosity were found ...

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

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

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 ...

doxycycline-loaded-collagen-chitosan-composite-scaffold-for

Research

JoVE Journal

Bioengineering

4.3K Views.  JSS Academy of Higher Education & Research. 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. 

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

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

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

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

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 ...

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

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

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

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

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

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

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

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

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

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

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

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

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

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

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

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

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

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

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

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

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

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

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

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

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

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

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

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

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