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

Published: March 3, 2023 doi: 10.3791/65045
* These authors contributed equally


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 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.


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]).

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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

  1. Inclusion criteria:
    1. Ensure that the patients' ages are between 18 years and 80 years (inclusive), regardless of gender.
    2. 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 ≤10% as detected during the screening period or within 3 months prior to randomization.
    3. Confirm that the ankle-brachial index15 of the target limb is at least 0.8 without intermittent claudication.
    4. Ensure that each patient'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.
      ​No pus or necrotic substance visible to the naked eye at least 4 weeks before randomization.
    5. Do not include patients undergoing routine treatment of other wounds in the study.
      1. If multiple wounds are present, select the wound that meets the inclusion criteria and has the largest intervention and assessment area.
      2. If two or more wounds are similarly large, select the one with the most severe grade.
      3. If there are two or more wounds with the same area and grade, select the one with the longest wound duration.
    6. Participation in this clinical study is voluntary. Ensure that the patients cooperate with the physicians conducting the study and sign an informed consent form.
  2. Exclusion criteria:
    1. Exclude patients with clear surgical indications: vascular occlusion, bone exposure, abscess, osteomyelitis.
    2. Exclude patients with revascularization or angioplasty within 3 months prior to enrollment.
    3. 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.
    4. Exclude patients with blood creatinine levels more than two times above the upper limit of normal; serum albumin <2.0 g/dL; immunosuppressive drug treatment; various malignant tumors; and pregnancy, breastfeeding, or a recent birth plan.
    5. Exclude patients with contraindications, allergies, or known allergies to any component of stem cell preparation products.
    6. Exclude patients in other situations that cause the investigator to believe the patient should not participate in this study:
      1. Exclude patients if it is discovered after admission that they do not meet the most important admission criteria.
      2. 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.
      3. Exclude patients with a lack of medication or effective post-enrollment observational data.
  3. Randomization and blinding
    1. 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.
    2. Conduct a double-blind trial for the treatment. Ensure a third party measures the wound area of each patient.

2. Preoperative treatment

  1. 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.
  2. 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.

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.

Figure 1
Figure 1: Treatment process. In each group, 30 patients will undergo treatment strictly following the protocol described in this study. Please click here to view a larger version of this figure.

  1. Stem cell therapy group
    1. 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.
    2. 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.
    3. 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.
    4. Trim the sterile dressing to appropriate size, cover it over the wound surface, and then bandage it.
  2. Conventional wound treatment group
    1. 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.
    2. 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.
    3. Trim the silver ion dressing to an appropriate size, cover it over the wound surface, and then bandage it.

Figure 2
Figure 2: Schematic diagram of foot ulcer area measurement. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Schematic of the human umbilical cord mesenchymal stem cell injection. Please click here to view a larger version of this figure.

4. Observation indicators

  1. Obtain the demographic data of the subjects.
  2. Record the concomitant treatments; specifically, record the wound status (location, area, depth, infection, and ischemia), the wound healing rate, the healing time, and complications.
  3. Perform laboratory tests. Take the wound secretion for swabs and bacteriological culture. Conduct routine blood tests and tests for liver and kidney function.

5. Follow-up

  1. Examine the patients on the first day of therapy and during the follow-up visits 15 days and 30 days after the last therapy.
  2. Conduct follow-ups using both outpatient and telephone appointments to record the treatment effects, condition changes, recovery situation, and current living status.

6. Outcomes efficacy assessment

  1. Conduct the clinical evaluation according to the following criteria.
    1. Calculate the primary outcome index:
      The 30 day wound healing rate = (original wound area − unhealed wound area at 30 days)/original wound area × 100%.
    2. Calculate the secondary outcome index
      1. Wound healing time: Define the time of wound closure as the time at which the wound is completely re-epithelialized.
      2. Calculate the complete healing rate:
        The complete healing rate = the number of complete healed cases/the total number of cases × 100%
    3. Calculate the surgical intervention rate:
      Surgical intervention rate = the number of surgical interventions/the total number of cases × 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.
    4. Perform the bacterial culture18 according to the standard clinical inspection procedures, and record bacterial culture results of ≥1 pathogen as positive. Calculate the pathogen positive rate:
      Pathogen positive rate = the number of cases detected pathogen positive/the total number of cases × 100%.

7. Safety procedures

  1. 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.
  2. 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.

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Representative Results

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 (Figure 4A-D), respectively. The average size of the ulcer of a patient in the control group before treatment with the silver ion dressing was 1.25 cm2. On the 8th, 15th, and 22nd days after treatment with silver ion dressing treatments (Figure 5A-D), the average ulcer size was reduced to 0.875 cm2, 0.8 cm2, and 0.75 cm2, respectively. The ulcer areas of the patients in both groups improved, and there were no complications such as wound infection or abnormal laboratory examinations. The existing patient data have been summarized in Table 1.

Figure 4
Figure 4: Representative photographs. (A-D) HUCMSCs treatment group on day 1, day 8, day 15, and day 22 after treatment. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Representative photographs. (A-D) Conventional treatment group on day 1, day 8, day 15, and day 22 after treatment. Please click here to view a larger version of this figure.

No. Sex Age Group Ulcer area (cm2)
(years) Day 1 Day 8 Day 15 Day 22
1 Male 69 1 3.5 2.6 1.8 1.25
2 Female 70 1 4.5 3.25 2.5 1.75
3 Female 69 1 3.5 2.5 1.25 1
Overall 69.3 3.83 2.78 1.85 1.33
4 Female 73 2 1.25 0.875 0.8 0.75
5 Male 70 2 5.5 4.55 4 3.5
6 Male 67 2 4 3.5 3 2.75
Overall 70 3.58 2.98 2.60 2.33
Group 1: Stem cell therapy group
Group 2: Conventional wound treatment group

Table 1: Clinical information of the six patients in the study.

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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 mental pain and severely limit their quality of life21. This means that there is a dire need for more effective therapies for DFUs.

Stem cell therapy has the potential as a novel therapeutic strategy in the treatment of many diseases22. Stem cells, important seed cells of regenerative medicine, are known for their ability for self-renewal, repair of damaged tissue, and multidirectional differentiation23. HUCMSCs have been the most frequently studied, and they are immunologically naive, less immunogenic, and non-tumorigenic24. These characteristics make HUCMSCs an ideal source of stem cells. Moreover, HUCMSCs can effectively secrete several growth factors such as VEGF, HGF, and bFGF25, as well as the anti-inflammatory factors IL-10, IL-4, and TGF-β26. They also have excellent proliferation and differentiation potential for tissue repair following injury27. These stem cells can significantly improve the proliferation and migration ability of human skin fibroblasts, human vein endothelial cells, and human keratinocytes. At the same time, HUCMSCs have the advantages of having convenient materials, no ethical disputes, and safe transplantation28.

However, while animal experiments and preclinical studies have promising results, a practical application of HUCMSCs to be used in the clinic remains lacking. As our clinical trials proceed, we believe that the efficacy and safety of HUCMSCs in the treatment of DFUs will be fully validated. Besides, this research will fill in the gap in terms of the clinical application of HUCMSCs and could form the groundwork for evaluating their clinical efficacy and side effects.

The critical step of this experiment is to inject a fixed amount of HUCMSCs into the wound at a fixed point and a fixed time. Moreover, long-term follow-up is required for patient laboratory tests, such as tests of blood glucose indicators.

This research is the first clinical research project on stem cell therapy for diabetic foot wounds in China. HUCMSCs have the following advantages in treating diabetic foot wounds. Compared with other stem cell biological agents, HUCMSCs have higher biosafety, stronger secretion ability, and more efficient treatment of diabetic wounds. Furthermore, this approach is generalizable to other wound healing therapies.

However, this study has some limitations. This experiment is a single-center clinical trial, and the number of patients is relatively small. Besides, the study population is primarily Asian, so the results cannot be fully extrapolated to other populations. In addition, the high cost might inevitably limit the wider applicability of this method.

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The authors have no conflict of interest to declare.


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 & Practice Innovation Program of Jiangsu Province (SJCX22-1271), and the Innovation & Technology Commission (Health@InnoHK).


Name Company Catalog Number Comments
Silver Iin Wound Dressing Shandong Cheerain Medical Co.,Ltd.  20152640521 Sterile silver ion dressing for medical use (Type F) 10 cm x 10 cm
Human Umbilical Cord Mesenchymal Stem Cells Injection Shandong Qilu Cell Therapy Engineering Technology Co., Ltd. 32183185-X 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.
Sterile mesh film transparent dressing Smith & Nephew 20162644490 Sterile mesh film transparent dressing (used for wound area measurement) 6 cm x 7 cm



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Human Umbilical Cord Mesenchymal Stem Cell Injection Diabetic Foot Ulcers Prospective Study Randomized Study Controlled Study Stem Cell Therapy Safety And Effectiveness Evaluation Research Standard High Consistency Repeatability Immune Rejection Disinfecting The Wound Site Necrotic Tissue Removal Saline Cleaning Wound Area Measurement Transparent Dressing Application
Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers
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Zhang, J., Zhao, B., Wei, W., Wang,More

Zhang, J., Zhao, B., Wei, W., Wang, D., Wang, H., Zhang, A., Tao, C., Li, X., Li, Q., Jin, P. Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers. J. Vis. Exp. (193), e65045, doi:10.3791/65045 (2023).

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