The authors thank Dr. Yukiko Koga of the Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, for providing practical advice. We also thank Mr. Hidekazu Murata of the Diabetic Center of Tokyo Women&#8217;s Medical University School of Medicine for excellent technical support. This study was supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program of the Project for Developing Innovation Systems &#8220;Cell Sheet Tissue Engineering Center (CSTEC)&#8221; from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women&#39;s Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.
1. Preparation of Animals, Instruments, Culture Media, and Dishes
<ol>
	<li>Prepare complete culture medium using minimum essential medium alpha containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Store this for several months at 4 &#176;C until use.</li>
	<li>Use adult male ZDF rats as a type 2 diabetic obesity model. Use the fat pad of male Lewis rats to isolate adipose tissue to prepare the cell sheets.</li>
</ol>
2. Isolation and Culture of rASCs
<ol>
	<li>Obtain adipose tissue from Lewis rats (8 - 33 weeks old, male) under general anesthesia using isoflurane.
	<ol>
		<li>Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauzes, a scalpel, a needle holder, operating scissors, a pair of forceps, and a 5-0 nylon suture. Sterilize the surgical instruments and supplies.</li>
		<li>Prepare a 100-mm Petri dish with 10 mL of sterile phosphate-buffered saline (PBS) to temporarily preserve the obtained adipose tissue. Weigh the Petri dish with PBS using a balance before starting surgery to measure the collected adipose tissue. Lay out the surgical instruments and supplies on a sterile drape.</li>
		<li>Anesthetize the rats by using isoflurane at 3 - 4% via a rodent mechanical ventilator and maintain the rats at 2 - 3% isoflurane during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat.</li>
		<li>While wearing sterile gloves, position the rat in a supine position on a sterile drape. Shave the operative area with an electric razor and clean the abdominal section of the rat using sterile gauze soaked in 70% ethanol.</li>
		<li>Create an approximately 5 cm-long skin incision with a scalpel in the lateral lower abdomen of the rat. Expose and dissect the external oblique muscle. Then, expose the epididymal adipose tissue surrounding the epididymis. Gently pull the epididymal adipose tissue out. 
		NOTE: The adipose tissue can be obtained on either side of the rat&#39;s abdomen.</li>
		<li>Excise only the epididymal adipose tissue, avoiding damage to the epididymis, testis, and epididymal blood vessels (Figure S1). Soak the excised adipose tissue in the PBS in the Petri dish to prevent dryness and weigh the tissue.</li>
		<li>Close the abdominal muscle with 5-0 VICRYL and the skin with a 5-0 nylon suture. Then, return each rat that has undergone surgery to a separate cage (one rat per cage). 
		NOTE: Monitor the rats until they fully recover.</li>
	</ol>
	</li>
	<li>Isolate rASCs from 2 - 3 g of adipose tissue (excised from a Lewis rat and processed according to a previously-reported method)27. Briefly, enzymatically digest the excised adipose tissue with 0.1% type A collagenase at 37 &#176;C for 1 h to obtain the stromal-vascular fraction (SVF; Figure S2).
	<ol>
		<li>Prepare several 100-&#181;m cell strainers, several 50-mL tubes, several 15 mL tubes, and two scalpels on a biological clean bench. Turn on a centrifuge and warm up a water bath to 37 &#176;C. In addition, prepare approximately 50 mL of sterile PBS in a 50 mL tube with 0.1% (final concentration) of type A collagenase.</li>
		<li>Mince the adipose tissue into small pieces using two scalpels.</li>
		<li>Move all minced adipose tissue to a 50-mL tube and add PBS to bring the total volume to 15 mL.</li>
		<li>Rest the 50-mL tube upright for 5 min. Discard the upper layer and collect the lower layer except for the debris.</li>
		<li>Add 0.1% of type A collagenase solution to the 50-mL tube with PBS containing the upper layer in order to enzymatically digest the adipose tissue. 
		NOTE: Warm approximately 20 mL of PBS per 2 - 3 g of adipose tissue in a 37 &#176;C water bath.</li>
		<li>Tilt and shake the 50 mL tube gently at 120 - 130 rpm for 1 h in a 37 &#176;C water bath.</li>
		<li>Rest the 50 mL tube upright for 5 min after shaking.</li>
		<li>Filter the solution using a 100 &#181;m cell strainer and pour the solution into a new 50 mL tube. Dispense the filtered solution into two 15 mL tubes.</li>
		<li>Centrifuge the solution in the 15 mL tubes for 5 min at 700 x g. Carefully remove the upper layer of supernatant and leave 5 mL of the lower layer of the solution. Do not aspirate the pellet.</li>
		<li>Add approximately 5 mL of complete culture medium to each 15 mL tube, up to 10 mL per 15 mL tube, and carefully re-suspend the pellet.</li>
		<li>Repeat steps 2.2.8 - 2.2.9. Then, obtain the SVF.</li>
	</ol>
	</li>
	<li>Prepare two 60 cm2 culture dishes. Collect the SVF after two centrifugations at 700 &#215; g for 5 min. Suspend the SVF and plate 10 mL of SVF solution on each 60-cm2 culture dish.</li>
	<li>Culture the dishes for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Prepare PBS and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA), storable for several months at 4 &#176;C until use.</li>
	<li>After initial plating (24 h), wash the cells with PBS 3 times to remove unattached cells and debris. Add fresh complete medium.</li>
	<li>Passage the cells that are nearly sub-confluent on day 3. Add 1 mL of 0.25% trypsin-EDTA and incubate the cells at 37 &#176;C in a 5% CO2 incubator for 3 - 5 min. 
	NOTE: Complete the trypsinization step within 10 min to prevent cell damage.</li>
	<li>Observe the cells under a light microscope after trypsinization to confirm that all the cells have thoroughly detached from the dishes. Then, add 9 mL of complete culture medium to the dishes.</li>
	<li>Count the number of cells using a hemocytometer.</li>
	<li>Subculture the cells at a density of 1.7 x 103 cells/cm2 every 3 days until passage 3. Culture the subcultured cells at 37 &#176;C in the humidified atmosphere of a 5% CO2 incubator.</li>
</ol>
3. Creation of rASC Sheets
<ol>
	<li>Prepare several 35-mm diameter temperature-responsive culture dishes. Pre-coat the dishes with FBS (or complete medium containing FBS) at 37 &#176;C in a 5% CO2 incubator for more than 1 h. 
	NOTE: A 35 mm diameter temperature-responsive culture dish is a commercially available product.</li>
	<li>Prepare a thermo-plate and incubate at 37 &#176;C for the following experimental procedures. 
	NOTE: Perform every procedure using temperature-responsive culture dishes on a 37 &#176;C thermo-plate to prevent the cells from spontaneously detaching from the dish.
	<ol>
		<li>Warm the complete culture medium used in the procedures to 37 &#176;C prior to experimentation.</li>
		<li>Seed passage 3 rASCs derived from Lewis rats onto a 35 mm diameter temperature-responsive culture dish at a density of 1.5 x 105 cells/dish for 3 days using a 2 mL total volume of complete culture medium. 
		NOTE: When rASCs are plated onto a new culture dish, the dish should be slowly rocked back and forth and left and right in an incubator to achieve uniform rASC seeding and a uniformly thick rASC sheet.</li>
		<li>Change 2 mL of the medium to 2 mL of complete culture medium containing 16.4 &#181;g/mL L-ascorbic acid phosphate magnesium salt n-hydrate (AA) on day 3 after seeding to the temperature-responsive culture dishes incubated on a 37 &#176;C thermo-plate. 
		NOTE: Dissolved AA can be stored at -30 &#176;C for months.</li>
		<li>Culture the cells for an additional 4 - 5 days and replace the medium every 2 days with fresh medium containing AA.</li>
		<li>Confirm the proliferation and generation of ASCs under a light microscope to determine whether gaps occurred between the cells in a contiguous cell sheet.</li>
	</ol>
	</li>
	<li>Transfer the cell sheets from the incubator to the benchtop for 15 - 20 min to cool them to room temperature.; observe the cells spontaneously detach as a contiguous cell sheet. Harvest the rASC sheet from the dish surface with a pair of forceps. 
	NOTE: Usually, rASC sheets can be handled with a pair of forceps. If necessary, a transfer membrane can be used to transfer a cell sheet from the culture dish to the wound site if the cell sheet is brittle and fragile.</li>
</ol>
4. Preparation of the Full-thickness Skin Defect Wound Model and Transplantation of rASC Sheets
<ol>
	<li>Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauze, 5-0 nylon suture, a scalpel, a periosteal raspatory, a needle holder, operating scissors, and a pair of forceps. Sterilize the surgical instruments and supplies. Lay out the surgical instruments and supplies on a sterile drape.</li>
	<li>Prepare PBS and maintain it at room temperature, along with some artificial skin and a non-adhesive dressing. Precut the artificial skin (15 x 10 mm) and non-adhesive dressing (20 &#215; 15 mm). Soak the inner collagen sponge layer of the artificial skin in saline before using the artificial skin. 
	NOTE: The artificial skin is made of two layers: an outer silicone sheet layer and an inner collagen sponge.</li>
	<li>Use ZDF rats (16 - 18 weeks old, male, 500 - 600 g) as a wound-healing model for type 2 diabetes and obesity.</li>
	<li>Anesthetize the ZDF rats by inhalation of 4% isoflurane using a rodent mechanical ventilator. Induce anesthesia by using isoflurane at 4 - 5% via a rodent mechanical ventilator and maintain it at 3 - 4% during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat via a toe pinch.</li>
	<li>While wearing sterile gloves, position the rat in the prone position on a sterile drape. Clean the head of the rat using sterile gauze soaked in 70% ethanol, shave the operative area with an electric razor, and clean the skin after shaving using sterile gauze soaked in 70% ethanol.</li>
	<li>Create a squared full-thickness skin defect (15 x 10 mm2) on the head of the anesthetized ZDF rats by removing the cutaneous tissue from the epidermis to the periosteum. Excise the skin and cutaneous tissue with a scalpel and remove the periosteum with a periosteal raspatory.</li>
	<li>Move the rASCs on the 35 mm temperature-responsive culture dish from the incubator to a room-temperature environment immediately prior to transplantation.</li>
	<li>Observe the rASCs on the 35 mm temperature-responsive culture dish spontaneously detach as a sheet from the dish surface after the formation of a wound. Harvest the rASC sheets after 7 days of culturing on temperature-responsive culture dishes. Remove the cultured medium and wash the rASC sheet with PBS three times. Soak the rASC sheet in PBS until transplantation to prevent desiccation.</li>
	<li>Place the rASC sheet immediately over the skull defect using a pair of forceps. If the cell sheet is brittle and fragile, a membrane can be used as a scaffold for transferring the cell sheet from the culture dish to the wound site.</li>
	<li>Cover the rASC sheet and defect with the artificial skin (15 x 10 mm2) and close the wound with approximately 10 stiches using the 5-0 nylon suture. To protect the wound, place a non-adhesive dressing (20 x 15 mm2) over the artificial skin, using 5-0 nylon sutures to keep the wound environment moist and to absorb exudates. 
	NOTE: The non-adhesive dressings will be removed by the ZDF rats within a few days after application. Therefore, it is necessary to regularly monitor the rats after transplantation. Usually, the non-adhesive dressing is replaced every 2 days under general anesthesia.</li>
	<li>Return each rat that has undergone surgery to a separate cage until they have fully recovered (one rat per cage). After an observation period, euthanize the rats using the approved method of isoflurane overdose.</li>
</ol>

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The following authors disclose financial relationships relevant to this publication: Teruo Okano is a founder and director of the board of Cell Seed Inc., which licenses technology and patents from Tokyo Women's Medical University, and Teruo Okano and Masayuki Yamato are stakeholders in Cell Seed Inc. Tokyo Women's Medical University receives research funds from Cell Seed Inc. The other authors declare that they do not have financial relationships relevant to this publication.

The most critical steps for successfully culturing an rASC sheet are as follows: 1) The temperature must be maintained at approximately 37 &#176;C during culturing on the temperature-responsive culture dishes. During the creation of an rASC sheet, every procedure was performed on a 37 &#176;C thermo-plate, and every reagent was warmed to 37 &#176;C to prevent the cells from spontaneously detaching from the dish31. 2) The recipient ZDF rats must be monitored to prevent the removal of the non-adhesive dressing, which is critical for the successful transplantation of rASC sheets. If the dressing is removed, a new non-adhesive dressing must be applied to prevent the transplanted ASC sheets from detaching from the wound site.
Using this procedure, rASC sheets were generally obtained within 5 - 7 days of seeding passage 3 cells on temperature-responsive culture dishes. The culture time required to generate an rASC sheet can be adjusted according to the initial cell density and the time at which the complete culture medium containing AA is applied. If an rASC sheet detaches from the dish during cell culturing, the rASC sheet should be remade, and additional dishes should be prepared in the event of cell detachment.
The limitations of this protocol are as follows: 1) Strict temperature management must be applied to maintain an approximate temperature of 37 &#176;C during the entire process when using temperature-responsive culture dishes. 2) After obtaining an rASC sheet, special medical devices must be used to maintain moist conditions, because the rASC sheet is sensitive to dry conditions. 3) Post-operative management, including daily observation of the condition of the recipient rat, is required.
Larger wounds with exposed bone are often observed clinically. For example, traffic accident trauma, burns, infected wounds, and damaged or necrotic wounds after debridement can develop into large wounds with exposed bone. Here, a clinical model of severe wounds was developed using a large, full-thickness skin defect with exposed bone in rats with type 2 diabetes and obesity. This model has the potential to be used as a standard model for evaluating wound healing in diabetic rats.
Artificial skin is a commercially available medical device for full-thickness skin defects after debridement, and recombinant basic fibroblast growth factor (bFGF) has been widely used for wound healing to promote angiogenesis and granulation. These two treatments have been used to achieve considerable therapeutic results, even for chronic wounds, such as diabetic wounds. It has been reported that ASCs secrete angiogeneic growth factors28, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and bFGF, which could contribute to neovascularization29,30 and accelerate wound healing. Our previous study confirmed that ASCs continuously secrete these growth factors31. Therefore, ASC sheets, combined with artificial skin, have the potential to be used as a new therapeutic option for accelerating vascularization and wound healing31, and these sheets might be applicable to the treatment of many types of intractable ulcers or burns in human clinical settings in the future.

The population of diabetic patients is increasing worldwide and reached 400 million in 20151; an estimated 15 - 25% of patients with diabetes are at risk from the progression of a lower-extremity diabetic ulcer2. Lower-extremity diabetic ulcers are intractable and might require a prolonged therapeutic period with rehabilitation training after complete recovery. A long therapy period often results in a significant reduction in patient quality of life. Thus, new therapies that decrease or prevent aggravation must be developed for the treatment of diabetic wounds. To evaluate diabetic wound healing, we optimized a diabetic ulcer wound-healing model in rats, which mimics practical clinical conditions, and evaluated whether transplanting adipose-derived stem cell (ASC) sheets using cell-sheet engineering accelerated wound healing.
Mesenchymal stromal cells (MSCs) exhibit an excellent potential for accelerating wound healing because of their self-renewal capacity, their immunomodulatory effects, and their ability to differentiate into various cell lineages3. ASCs are a type of MSC derived from adipose tissue, and they exhibit several advantages over MSCs derived from other tissues, including their angiogenic potential and paracrine activity4,5. Adipose tissue is relatively abundant in the human body, and its accessibility allows for collection using minimally invasive procedures. Therefore, ASCs have been used experimentally for wound-healing applications6,7.
Previous reports have shown that the direct injection of single-cell MSC suspensions into areas around wounds can accelerate wound healing8,9. However, despite reports of the acceleration of wound healing in diabetic ulcer models following the injection of single-cell suspensions, the survival time of transplanted cells at the wound site is not clear.
In this study, we applied cell-sheet engineering using temperature-responsive culture dishes. These dishes have the temperature-responsive polymer N-isopropylacrylamide covalently bound onto their surface10. The grafted polymer layer allows for temperature-controlled cell adhesion to or detachment from the surface of the culture dish. The surface of the dish becomes hydrophobic at 37 &#176;C, allowing cells to adhere and proliferate, whereas cells spontaneously detach from the surface when it becomes hydrophilic at temperatures below 32 &#176;C. Cultured cells can be harvested as a contiguous cell sheet with intact cell-to-cell junctions and extracellular matrices (ECMs) simply by reducing the temperature; thus, proteolytic enzymes that damage the ECM, such as trypsin, are not required11. Therefore, cell-sheet engineering can preserve cell-to-cell connections and improve the efficacy of cell transplantation.
In addition, cell-sheet transplantation increases cell survival rates when compared to cell injection12.In this protocol, Zucker diabetic fatty (ZDF) rats were selected as a type 2 diabetes and obesity model with delayed wound healing. ZDF rats spontaneously develop obesity at approximately 4 weeks. They then develop type 2 diabetes with obesity between 8 and 12 weeks of age, at which point they exhibit hyperglycemia associated with insulin resistance, dyslipidemia, and hypertriglyceridemia13. Delayed wound healing, reduced blood flow in peripheral blood vessels, and diabetic nephropathy are also observed14,15,16. Moreover, ZDF rats might be an appropriate model for studying the healing of intractable cutaneous ulcers, such as diabetic ulcers.
The differences between humans and rodents in wound-healing mechanisms are associated with anatomical differences in the skin. Wound healing in normal rats is based on wound contraction, whereas wound healing in humans is based on re-epithelialization and granulation tissue formation. Typically, wound splinting used in rodent models helps to minimize wound contraction and allows for the gradual formation of granulation tissue17, although wounds in nondiabetic rats are almost completely closed by contraction. However, diabetic wound contraction in ZDF rats is impaired, and wound healing primarily occurs through re-epithelialization and granulation tissue formation; thus, this process is more similar to human wound healing14.
Diabetic wounds with exposed bone after debridement are often encountered clinically. Previous studies have examined 12-mm diameter full-thickness skin wounds on the backs of athymic nude mice18,19 and 10-mm diameter full-thickness skin wounds on the backs of normal mice20. To develop a clinical model for severe diabetic wounds, larger (15 x 10 mm2) full-thickness skin defects with exposed bone and without the periosteum were created, as previously described21, in rats with type 2 diabetes and obesity.
Rat ASC (rASC) sheets from the ASCs of normal Lewis rats were created through the allogenic transplantation of ASC sheets. In clinical practice, autologous transplantation is unfeasible because diabetic patients with ulcers often exhibit severe diabetic complications, such as uncontrolled high blood glucose levels and high body mass indices, and these complications cause wound-healing disorders that increase the difficulty of obtaining adipose tissue from these patients. Furthermore, ASCs from animals with diabetes exhibit altered properties and impaired function22. Therefore, the protocol presented here describes the allogenic transplantation of rASC sheets from normal rats and the application of artificial skin to diabetic rats.
The bilayer artificial skin used in this protocol prevents the spontaneous contraction of the wounds, promotes the synthesis of a new connective tissue matrix, and resembles the true dermis23. In this protocol, artificial skin is placed on an rASC sheet and fixed with nylon threads to prevent wound contraction or enlargement resulting from loose rat skin. In addition, the artificial skin provides a three-dimensional framework for the ASC sheets, maintains a moist environment for the transplanted ASC sheets and wounds, and protects the wounds from infection and external forces. Finally, a non-adhesive dressing is placed over the wound to protect it from external impact, maintain a moist wound environment, and absorb exudate.
An rASC sheet is thin, flexible, and deformable and can be adhered to moving recipient sites, such as a beating heart24. Cell-sheet engineering has been used for the reconstruction of various tissues and can generate curative effects25,26. ASC sheets that exhibit clinical therapeutic potential might accelerate the healing of many types of wounds. Moreover, the allogeneic transplantation of ASC sheets, combined with the use of artificial skin, might be applicable to the treatment of intractable ulcers or burns, such as those observed in peripheral arterial disease or collagen disease, or they may be administered to patients who are undernourished or are using steroids. This approach increases the efficiency of transplanting ASCs. The wound-healing ZDF rat model produces a severe wound condition that resembles the human wound healing process and mimics clinical conditions in a small-sized experimental animal.

<table><tbody><tr><td>&amp;alpha;-MEM glutamax</td><td>Invitrogen</td><td>32571-036</td><td>Carlsbad, CA</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Japan Bioserum Co Ltd.</td><td>S1650-500</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Collagenase A</td><td>Roche Diagnostics</td><td>10 103 578 001</td><td>Mannheim, Germany</td></tr><tr><td>60 cm&lt;sup&gt;2&lt;/sup&gt; Primaria tissue culture dish</td><td>BD Biosciences</td><td>353803</td><td>Franklin Lakes, NJ</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffer Saline (PBS)</td><td>Life Technologies</td><td>1490-144</td><td></td></tr><tr><td>0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>L-ascorbic acid phosphate magnesium salt n-hydrate</td><td>Wako</td><td>013-19641</td><td></td></tr><tr><td>35-mm temperature-responsive culture dish (Upcell&lt;sup&gt;TM&lt;/sup&gt;)</td><td>CellSeed</td><td>NUNC-174904</td><td>Tokyo, Japan</td></tr><tr><td>Microwarm plate (MP-1000)</td><td>Kitazato Science Co., Ltd.</td><td>1111</td><td></td></tr><tr><td>Rodent mechanical ventilator</td><td>Stoelting</td><td>#50206</td><td>Wood Dale, IL</td></tr><tr><td>4% isoflurane</td><td>Pfizer Japan</td><td>114-13340-3</td><td>Tokyo, Japan</td></tr><tr><td>Artificial skin (Pelnac&lt;sup&gt;&amp;reg;&lt;/sup&gt;)</td><td>Smith &amp;amp; Nephew</td><td>PN-R40060</td><td>&amp;nbsp;Tokyo, Japan</td></tr><tr><td>Non-adhesive dressing (Hydrosite plus&lt;sup&gt;&amp;reg;&lt;/sup&gt;)</td><td>Smith &amp;amp; Nephew</td><td>66800679</td><td>Known as Allevyn non-adhessing&lt;sup&gt;&amp;reg;&lt;/sup&gt; in the United State</td></tr><tr><td>5-0 nylon suture</td><td>Alfresa</td><td>EP1105NB45-KF2</td><td></td></tr><tr><td>20 CELLSTAR TUBES</td><td>greiner bio-one</td><td>227 261</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning Incorporated</td><td>430791</td><td></td></tr><tr><td>14 GOLDMAN-FOX PERIOSTEAL</td><td>Hu-Friedy</td><td>P14</td><td>Chicago, IL</td></tr></tbody></table>

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.

This protocol attempted to establish a new cell-based therapy for intractable diabetic wounds. Briefly (as illustrated in Figure 1), allogeneic rASC sheets were created from normal rats using cell-sheet engineering and were then transplanted using a bilayer of artificial skin onto a full-thickness skin defect on a diabetic rat. Light microscope images of a good example of an rASC sheet (Figure 2A) and a bad example of an rASC sheet (Figure 2B) are shown in Figure 2. When ASCs are plated onto a new culture dish, the dish should be slowly rocked back and forth and left and right in an incubator to achieve uniform rASC seeding and a uniformly-thick rASC sheet (Figure 2A). If the rASCs cannot be uniformly attached and cultured on the surface of the culture dish, the sheet cannot be collected as a contiguous ASC sheet (Figure 2B). Figure 3 shows ASC sheets that have been harvested as a contiguous cell sheet at room temperature because the ASCs were uniformly attached to the dish surface. Usually, rASC sheets can be handled with a pair of forceps. If necessary, a transfer membrane can be used to transfer a cell sheet from the culture dish to the wound site, such as if the cell sheet is brittle and fragile. 
Figure 4 depicts the ZDF rats used as a diabetic wound-healing model and the transplantation of allogeneic rASC sheets combined with artificial skin. An rASC sheet is soft and flexible, adjustable in size, and capable of being extended to every corner of the wound site with a pair of forceps (Figure 4A-F). The rASC sheet-covered defect was also covered with artificial skin (15 x 10 mm2) and sutured with approximately 10 stitches using a 5-0 nylon suture (Figure 4G). To protect the wound, a moist wound environment was maintained and exudates were absorbed, a non-adhesive dressing (20 x 15 mm2) was placed over the artificial skin, and 5-0 nylon sutures were applied (Figure 4I). The non-adhesive dressing is often removed by the ZDF rats within several days of application. Therefore, the rats must be monitored after transplantation. Usually, the non-adhesive dressing is replaced every 2 days under general anesthesia. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54539/54539fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The macroscopic photographs in Figure 5 are the representative results of the transplantation of an rASC sheet. In our previous study, the average wound area in the rASC sheet transplantation group (Figure 5B) was significantly smaller than in the control group (Figure 5A). For the controls, only artificial skin was used to cover the wound, without the transplantation of an rASC sheet. These images were taken on the 14th day after the creation of the wound (n = 6 in each group)31.
<img alt="Figure 1" src="/files/ftp_upload/54539/54539fig1.jpg" /> 
Figure 1: Schematic of the Experimental Transplantation Procedure. Schematic of the experimental transplantation procedure performed with an allogeneic rat adipose-derived stem cell (rASC) sheet and artificial skin in a rat wound-healing model of type 2 diabetes and obesity. (A) Rat adipose tissue was surgically excised from normal Lewis rats. rASCs were isolated and seeded onto a 60 cm2 culture dish and cultured at 37 &#176;C in a 5% CO2 incubator for 7 - 8 days. (B) rASCs were subcultured every 2-3 days, and passage 3 rASCs were seeded onto a 35 mm diameter temperature-responsive culture dish. The cells were cultured in complete medium containing 16.4 &#181;g/mL L-ascorbic acid phosphate magnesium salt n-hydrate (AA) at 37 &#176;C in a 5% CO2 incubator for 7 - 8 days. The rASCs were harvested as a contiguous rASC sheet by reducing the temperature to 20 &#176;C. (C) rASC sheets transplanted onto a 15 x 10 mm2 full-thickness skin defect with exposed bone on the heads of rats exhibiting diabetes and obesity (Zucker diabetic fatty (ZDF) rats) used as a wound-healing model. (D) An rASC sheet was placed on the skull directly over the defect and covered )with a 15 x 10 mm2 sheet of bilayer artificial skin, which was sutured into place with 10 nylon (5-0) sutures. Diabetes 2015;64: 2723-2734; with permission. Diabetes (c) copyright (2015) by the American Diabetes Association. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54539/54539fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54539/54539fig2.jpg" /> 
Figure 2: Light Microscope Images of ASCs. Light microscope images of ASC proliferation to the edge of culture dishes, without gaps between the ASCs. (A) An rASC sheet with a uniform thickness in all directions 7 days after the start of culturing (A). (B) An rASC sheet without uniform seeding. A contiguous rASC sheet cannot be obtained on day 7 after the start of culturing (B). Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54539/54539fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54539/54539fig3.jpg" /> 
Figure 3: Time-lapse Images of rASC Sheet Statues at Room Temperature. Time-lapse images of the status of an rASC sheet at room temperature. rASCs on a 35 mm diameter temperature-responsive culture dish spontaneously and gradually detached from the dish surface at room temperature (approximately 20 &#176;C) and were harvested as a contiguous sheet. (A) Approximately 5 min after moving the temperature-responsive culture dish to room temperature. (B) Approximately 10 min after moving the 35 mm diameter temperature-responsive culture dish to room temperature.(C) Approximately 20 - 30 min after moving the temperature-responsive culture dish to room temperature. This is a good-quality rASC sheet (C). (D-E) rASC sheet status approximately 20 - 30 min after moving the temperature-responsive culture dish to room temperature. This rASC sheet is of average quality (D). (F-G) rASC sheets are usually handled with a pair of forceps. If the cell sheet is brittle and fragile, a membrane can be used as a scaffold for transferring the cell sheet from the culture dish to the wound site. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54539/54539fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54539/54539fig4.jpg" /> 
Figure 4: Time-series Images of Wound Creation and rASC Sheet Transplantation with Artificial Skin and a Non-adhesive Dressing 
Time series images of wound creation and rASC sheet transplantation with artificial skin and a non-adhesive dressing. (A) The heads of ZDF rats were shaved with an electric razor. After shaving the body hair, check marks (15 x 10 mm2) were drawn using an oily hydrophobic pen. (B) A full-thickness skin defect (15 x 10 mm2) was created on the head of an anesthetized ZDF rat by removing cutaneous tissue from the epidermis to the periosteum. The skin and cutaneous tissue were excised with a scalpel, and the periosteum was removed with a periosteal raspatory. Using gauze moistened with sterile saline, pressure was applied to stop the bleeding after excision. (C) rASC sheet transplantation. An rASC sheet was placed over the defect immediately above the skull of the rat using a pair of forceps. (D-G) Adjusting rASC sheet extension to match wound size. The rASC sheet is flexible, adjustable, and can be extended to every corner of the wound site using a pair of forceps. For wider wounds, two or three flexible rASC sheets can be stacked. (H) Suturing the artificial skin covering the rASC sheet. The defect and the transplanted rASC sheet were covered with artificial skin (15 x 10 mm2), which was sutured with 10 stitches using 5-0 nylon sutures. (I) Suturing of the non-adhesive dressing (20 x 15 mm2) to the wound site covered with artificial skin. To protect the wound, non-adhesive dressing (20 x 15 mm2) was placed over the artificial skin with 5-0 nylon sutures. Diabetes 2015;64: 2723-2734; with permission. Diabetes (c) copyright (2015) by the American Diabetes Association. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54539/54539fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54539/54539fig5.jpg" /> 
Figure 5: Macroscopic Images of Full-thickness Skin Defects. Macroscopic photographs of full-thickness skin defects without the transplantation of an rASC sheet (A) and with the transplantation of an rASC sheet (B) 14 days after creation of the wound. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54539/54539fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

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.

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 ...

creation-transplantation-an-adipose-derived-stem-cell-asc-sheet

Nanyang Technological University

Research

JoVE Journal

Medicine

11.4K Views.  Tokyo Women's Medical University School of Medicine. Adipeus-afgeleide cellen van de stam (ASC's) gemakkelijk kunnen worden geïsoleerd en geoogst van het vet van de normale ratten. ASC bladen kunnen worden gemaakt met behulp van cellaag engineering en in Zucker vette diabetesratten exposeren full-dikte huid gebreken met blootgestelde bot en vervolgens bedekt met een dubbelgelaagde van kunstmatige huid kunnen worden getransplanteerd.

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

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

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 ...

Bioengineering

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

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 ...

Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with dressing changes, hyperbaric oxygen, and vacuum sealing drainage are the main conservative treatments in clinical practice, and large wounds often require skin grafts or skin flap grafts. However, the treatment effects are not ideal, and many complications exist. Due to its complex pathogenesis, long treatment time, significant associated difficulties, and high disability rate, diabetic foot ulcers cause a heavy burden to patients, society, and medical care. According to our previous study, the pharmacological effects of human umbilical cord blood stem cells include nonspecific immune regulation; increased secretion of growth factors, vasoactive factors, and anti-inflammatory factors; enhanced anti-infectious ability of the human body; elimination of inflammation; and promotion of angiogenesis and ulcer healing. These effects suggest stem cells may be useful as an autologous or allogeneic treatment for refractory wounds. Therefore, we are conducting a clinical trial to treat refractory diabetic wounds with human umbilical cord stem cells in our clinic for diabetic foot ulcer patients who meet the inclusion criteria.

The present protocol describes a prospective, randomized, controlled clinical study that evaluates a human umbilical cord mesenchymal stem cell injection for the treatment of chronic diabetic foot ulcers.

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

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 ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...

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

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