Name: doxycycline loaded collagen-chitosan composite scaffold for the accelerated healing of diabetic wounds
Uploaded: 2021-08-21T14:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds at JoVE.com

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><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&amp;amp;D Systems</td><td>RMP900</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; (&lt;em&gt;E. coli&lt;/em&gt;)</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>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt; (&lt;em&gt;P. aeruginosa&lt;/em&gt;)</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>&lt;em&gt;Staphylococcus aureus&lt;/em&gt; (&lt;em&gt;S. aureus&lt;/em&gt;)</td><td>National Collection of Industrial Microorganisms, Pune, India</td><td>NCIM 5022</td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus epidermis&lt;/em&gt; (&lt;em&gt;S. epidermis&lt;/em&gt;)</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&amp;ndash;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 ...

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Research

JoVE Journal

Bioengineering

4.4K 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 ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order 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 ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Developmental Biology

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Medicine

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Immunology and Infection

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.

This technique provides a guide to inflicting ex vivo wounds, performing laser capture microdissection and quantifying changes in gene expression related to poor wound healing processes in diabetes using clinically relevant human tissue.

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one ...

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

Summary

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Chapters in this video

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

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

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

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Protocol to Create Chronic Wounds in Diabetic Mice

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes