The authors thank the Affiliated Hospital of Xuzhou Medical University for its cooperation, including the recruitment and follow-up of patients with diabetic foot wounds. The authors also thank the patients who participated in the patient needs survey during the design of this study.
The author(s) announce the receipt of the following financial support for research, authorship, and/or publication of this article: National Natural Science Foundation of China 82172224, Postgraduate Research &#38; Practice Innovation Program of Jiangsu Province (SJCX22-1271), and&#160;the Innovation &#38; Technology Commission (Health@InnoHK).

This prospective, single-center, randomized, controlled clinical study was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (XYFY2021-KL124-02). The study started in July 2021 and will continue until July 2023; 60 patients were recruited in this experiment. All patients signed an informed consent that allowed the researchers to use their clinical materials and biological data.
1. Recruitment of patients
<ol>
	<li>Inclusion criteria:
	<ol>
		<li>Ensure that the patients&#39; ages are between 18 years and 80 years (inclusive), regardless of gender.</li>
		<li>Ensure that the patients with type 2 diabetes and diabetic foot ulcers meet the 1999 WHO diagnostic criteria14 and have a glycated hemoglobin (HbA1c) level &#8804;10% as detected during the screening period or within 3 months prior to randomization.</li>
		<li>Confirm that the ankle-brachial index15 of the target limb is at least 0.8 without intermittent claudication.</li>
		<li>Ensure that each patient&#39;s ulcer has the following characteristics: 
		Grade 1 or grade 2 according to the Wagner grading system16 for ulcers. 
		Location in the foot, ankle, or front shin. 
		Cross-sectional area after debridement of 2-5 cm2. 
		&#8203;No pus or necrotic substance visible to the naked eye at least 4 weeks before randomization.</li>
		<li>Do not include patients undergoing routine treatment of other wounds in the study.
		<ol>
			<li>If multiple wounds are present, select the wound that meets the inclusion criteria and has the largest intervention and assessment area.</li>
			<li>If two or more wounds are similarly large, select the one with the most severe grade.</li>
			<li>If there are two or more wounds with the same area and grade, select the one with the longest wound duration.</li>
		</ol>
		</li>
		<li>Participation in this clinical study is voluntary. Ensure that the patients cooperate with the physicians conducting the study and sign an informed consent form.</li>
	</ol>
	</li>
	<li>Exclusion criteria:
	<ol>
		<li>Exclude patients with clear surgical indications: vascular occlusion, bone exposure, abscess, osteomyelitis.</li>
		<li>Exclude patients with revascularization or angioplasty within 3 months prior to enrollment.</li>
		<li>Exclude patients with hepatic impairment; specifically, exclude patients with ALT (aspartate aminotransferase) and AST (alanine aminotransferase) levels three times higher than the upper limit of normal.</li>
		<li>Exclude patients with blood creatinine levels more than two times above the upper limit of normal; serum albumin &#60;2.0 g/dL; immunosuppressive drug treatment; various malignant tumors; and pregnancy, breastfeeding, or a recent birth plan.</li>
		<li>Exclude patients with contraindications, allergies, or known allergies to any component of stem cell preparation products.</li>
		<li>Exclude patients in other situations that cause the investigator to believe the patient should not participate in this study:
		<ol>
			<li>Exclude patients if it is discovered after admission that they do not meet the most important admission criteria.</li>
			<li>Exclude patients with serious adverse reactions, patients who request to withdraw from the study, and patients whose legal guardian requests for them to withdraw from the study.</li>
			<li>Exclude patients with a lack of medication or effective post-enrollment observational data.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Randomization and blinding
	<ol>
		<li>Randomize the patients recruited from the outpatient department of the Affiliated Hospital of Xuzhou Medical University on a 1:1 basis into the stem cell therapy group and the conventional wound treatment group.</li>
		<li>Conduct a double-blind trial for the treatment. Ensure a third party measures the wound area of each patient.</li>
	</ol>
	</li>
</ol>
2. Preoperative treatment
<ol>
	<li>Perform routine pretreatment examinations for the patients after enrollment, including a routine blood test, urinalysis, a routine stool assessment, a biochemical function assessment, a coagulation function assessment, virology, etc. Ensure that patients are followed up in the same hospital to ensure the accuracy of the tests.</li>
	<li>Include patients who meet the inclusion criteria and have signed the written informed consent. Assign each subject randomly to either the stem cell therapy group or the conventional wound treatment group in a 1:1 ratio using a proportionate sampling technique.</li>
</ol>
3. Treatment procedures
NOTE: Patients in both groups receive systematic routine wound dressing changes every 3 days. For the stem cell treatment group, patients receive local injections of stem cells four times (on day 1, day 8, day 15, and day 22 after enrollment). In the conventional wound treatment group, the patients are treated with silver ion dressing four times (on day 1, day 8, day 15, and day 22 after enrollment). The specific steps are as follows.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65045/65045fig01.jpg" /> 
Figure 1: Treatment process. In each group, 30 patients will undergo treatment strictly following the protocol described in this study. <a href="https://www.jove.com/files/ftp_upload/65045/65045fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Stem cell therapy group
	<ol>
		<li>After locally disinfecting the wound with povidone-iodine and removing the necrotic tissue from the wound surface with surgical scissors, use a syringe to aspirate 50 mL of physiological saline to clean the wound surface inside and out.</li>
		<li>Measure the wound area using the sterile foil edge hook method17. Apply the sterile film transparent dressing with its coordinate grid to the wound surface, and outline the shape of the wound surface along its edge with a marker. Calculate the wound area according to the area of each coordinate grid of the film.</li>
		<li>Inject the human umbilical cord mesenchymal stem cells (2 x 105 cells/cm2; 0.4 mL/cm2) into the periphery and the base of the wound (injection spacing: 0.75 cm; injection volume: 0.1 mL/site). 
		NOTE: The number of injections for each patient depends on the wound area.</li>
		<li>Trim the sterile dressing to appropriate size, cover it over the wound surface, and then bandage it.</li>
	</ol>
	</li>
	<li>Conventional wound treatment group
	<ol>
		<li>After locally disinfecting the wound with iodophor and removing the necrotic tissue from the wound surface with surgical scissors, use a syringe to aspirate 50 mL of physiological saline to clean the wound surface inside and out.</li>
		<li>Measure the wound area using the sterile foil edge hook method17. Apply the sterile film transparent dressing with its coordinate grid to the wound surface, and outline the shape of the wound surface along its edge with a marker. Calculate the wound area according to the area of each coordinate grid of the film.</li>
		<li>Trim the silver ion dressing to an appropriate size, cover it over the wound surface, and then bandage it.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65045/65045fig02.jpg" /> 
Figure 2: Schematic diagram of foot ulcer area measurement. <a href="https://www.jove.com/files/ftp_upload/65045/65045fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65045/65045fig03.jpg" /> 
Figure 3: Schematic of the human umbilical cord mesenchymal stem cell injection. <a href="https://www.jove.com/files/ftp_upload/65045/65045fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Observation indicators
<ol>
	<li>Obtain the demographic data of the subjects.</li>
	<li>Record the concomitant treatments; specifically, record the wound status (location, area, depth, infection, and ischemia), the wound healing rate, the healing time, and complications.</li>
	<li>Perform laboratory tests. Take the wound secretion for swabs and bacteriological culture. Conduct routine blood tests and tests for liver and kidney function.</li>
</ol>
5. Follow-up
<ol>
	<li>Examine the patients on the first day of therapy and during the follow-up visits 15 days and 30 days after the last therapy.</li>
	<li>Conduct follow-ups using both outpatient and telephone appointments to record the treatment effects, condition changes, recovery situation, and current living status.</li>
</ol>
6. Outcomes efficacy assessment
<ol>
	<li>Conduct the clinical evaluation according to the following criteria.
	<ol>
		<li>Calculate the primary outcome index: 
		The 30 day wound healing rate = (original wound area &#8722; unhealed wound area at 30 days)/original wound area &#215; 100%.</li>
		<li>Calculate the secondary outcome index
		<ol>
			<li>Wound healing time: Define the time of wound closure as the time at which the wound is completely re-epithelialized.</li>
			<li>Calculate the complete healing rate: 
			The complete healing rate = the number of complete healed cases/the total number of cases &#215; 100%</li>
		</ol>
		</li>
		<li>Calculate the surgical intervention rate: 
		Surgical intervention rate = the number of surgical interventions/the total number of cases &#215; 100%. 
		NOTE: Subjects who underwent one of the following procedures are recorded as patients with surgical interventions: debridement and drainage, skin grafting, adjacent skin flap, distal skin flap, non-anastomotic skin flap, and amputation.</li>
		<li>Perform the bacterial culture18 according to the standard clinical inspection procedures, and record bacterial culture results of &#8805;1 pathogen as positive. Calculate the pathogen positive rate: 
		Pathogen positive rate = the number of cases detected pathogen positive/the total number of cases &#215; 100%.</li>
	</ol>
	</li>
</ol>
7. Safety procedures
<ol>
	<li>In the case of an adverse event (any disease, new symptoms, signs, or laboratory examination abnormalities or the deterioration of the original symptoms and signs occurring during the stem cell clinical research, regardless of whether the event is related to the clinical research drug or not), take the necessary measures for treatment and rescue.</li>
	<li>Track and investigate all the adverse events and the treatment process, and record the results in detail until properly resolved or until the condition is stable. If a test is abnormal and has clinical significance, follow it up until it returns to normal.</li>
</ol>

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The authors have no conflict of interest to declare.

DFUs are a major global public health problem and a key cause of lower limb amputations and poor health-related quality of life19,20. At present, clinical management is still dominated by conventional debridement, hyperbaric oxygen therapy, vacuum sealing drainage (VSD), and conservative management. Larger wounds often require the transplantation of skin and skin flaps. Many patients suffer from long-term and repeated illnesses that cause severe physical and ment...

Diabetes mellitus is a disease that affects individuals worldwide, and the World Health Organization (WHO) predicts that the number of people with diabetes mellitus will increase from 285 million in 2010 to 439 million in 20301. Diabetic foot ulcers (DFUs) are one of the most serious complications of diabetes and are main contributors to non-traumatic lower-extremity amputations across the world2,3,4,5.
Recently, stem cells have flourished as a therapy due to their pluripotency, self-renewal, and ability to promote the secretion of regenerative cytokines6,7. A previous clinical experiment showed that fat-derived stem cell gels had a positive effect on the treatment of foot ulcers in chronic diabetes8. The authors verified the effectiveness of using stem cells to treat diabetic wounds in 59 patients. At week 12, the rate of complete wound closure in the treatment and control groups was 82% and 53%, respectively, which indicates that stem cells are effective for the treatment of refractory diabetic wounds. Overall, the ability of stem cells to regenerate, replace, repair, and differentiate has given infinite hope to the life science community9.
In 2008, Dulchavsky et al.10 used grafts containing autologous bone marrow stromal cells (BMSCs) to treat 20 cases of non-healing wounds of different causes. Histological examinations showed that the skin wounds of 18 patients were completely re-epithelialized. Similarly, the use of allogeneic non-diabetic mesenchymal stem cells (MSCs) in gelatin scaffolds for the treatment of nonunion diabetic wounds can promote angiogenesis, increase re-epithelialization, and reduce the ulcer area11. However, there are few cases of stem cell treatment of diabetic foot wounds in domestic and foreign clinical research studies; most are only case reports or exploratory clinical research lacking a strict experimental design, and there are few large samples with good design or randomized controlled clinical trials. As stem cells are not common drugs or biological products, the preparation methods and quality controls differ between studies. Data from one study may not fully reflect the safety of all stem cells from the same species. Therefore, we further summarized the relevant basic research and preclinical experiments with human umbilical cord mesenchymal stem cells (HUCMSCs) and systematically evaluated the safety and efficacy of their clinical application. On this basis, the injection of mesenchymal stem cells from the human umbilical cord to repair diabetic foot wounds was developed as a treatment. This study aims to verify the efficacy and safety of stem cell repair of diabetic foot wounds in clinical use.
In summary, stem cell therapy has a broad spectrum of applications and great potential and represents a promising new medical treatment method. With the continued support of the National Natural Science Foundation of China (No. 81571901, No. 81501671, and No. 82172224), we conducted a series of studies on the treatment of diabetic wounds with HUCMSCs. We have published more than 20 related articles in the Journal of Investigative Dermatology, Stem Cell Research and Therapy, and Cell Death and Disease and have applied for three national invention patents, thus accumulating a large foundation of research12,13. Here, we provide a standard approach to evaluate the injection of HUCMSCs for diabetic foot wound repair. This standard procedure has been approved by Chinese drug clinical trials (Trial registration number: Chinese drug clinical trials: MR-32-21-015759, [Initial Release: 10/20/2021]).

<table><tbody><tr><td>Silver Iin Wound Dressing</td><td>Shandong Cheerain Medical Co.,Ltd.&amp;nbsp;</td><td>20152640521</td><td>Sterile silver ion dressing for medical use (Type F) 10 cm x 10 cm</td></tr><tr><td>Human Umbilical Cord Mesenchymal Stem Cells Injection</td><td>Shandong Qilu Cell Therapy Engineering Technology Co., Ltd.</td><td>32183185-X</td><td>Main components: human umbilical cord mesenchymal stem cells. Pharmacological effect: non-specific immunomodulator can enhance the secretion of growth factor, vasoactive factor and anti-inflammatory factor, improve the anti infection ability of human body, eliminate inflammation, promote angiogenesis and ulcer healing.</td></tr><tr><td>Sterile mesh film transparent dressing</td><td>Smith &amp;amp; Nephew</td><td>20162644490</td><td>Sterile mesh film transparent dressing (used for wound area measurement) 6 cm x 7 cm</td></tr></tbody></table>

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.

At present, our research is still in the patient recruitment phase, and we have now completed three patients in the HUCMSCs treatment group and three patients in the control group with silver ion dressings, giving a total of six patients with chronic diabetic foot wounds. The average size of the ulcer area of a patient in the HUCMSCs treatment group was 3.5 cm2, and this was reduced to 2.6 cm2, 1.8 cm2, and 1.25 cm2 on the 8th, 15th, and 22nd days after treatment with HUCMSCs (...

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

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 ...

prospective-randomized-controlled-study-human-umbilical-cord

Research

JoVE Journal

Bioengineering

2.8K Views.  Affiliated Hospital of Xuzhou Medical University. Ons protocol is bedoeld om een uniform model te bieden voor systeemcelinjectie om diabetische zweren te behandelen om de veiligheid en effectiviteit van deze behandeling te evalueren. We bieden een onderzoeksstandaard voor stamcellen voor de behandeling van diabetische zweren, die de voordelen heeft van de hoge consistenties en de herhaalbaarheid. Diabetische zweer is slechts een van de vertegenwoordigers van veel voorkomende refractaire zweren.We hopen via dit protocol stamcellen toe ...

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

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Developmental Biology

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord Wharton's jelly, as well as from the umbilical arteries and umbilical vein. They are known as perivascular stem cells obtained from umbilical arteries (UCA-PSCs), perivascular stem cells obtained from the umbilical vein (UCV-PSCs), and mesenchymal stem cells obtained from Wharton's jelly (WJ-MSCs). UCA-PSCs and UCV-PSCs are pericytes derived from perivascular regions that are progenitors of MSCs. Isolation and culture of the three kinds of cells is an important source for studying stem cell transplantation and repair. The present protocol focuses on the isolation and culture of cells through mechanical separation, adherent culture, and cell crawling out. Through this technique, the three different types of stem cells can be derived. Cell surface markers were detected by flow cytometry. The stem cells were detected for multilineage differentiation potential by adipogenic, osteogenic, and neural-like differentiation, which is consistent with the phenotype of MSCs. This experimental protocol expands the source of UC-MSCs. In addition, the cell isolation method provides a basis for further study of regenerative medicine and other applications.

The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton's jelly.

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord ...

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

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

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 ...

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

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

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