The wound healing of deep partial-thickness burn in Bama miniature pigs is accelerated by a higher dose of hUCMSCs

doi:10.21203/rs.3.rs-4492376/v1

Download PDF

Research Article

The wound healing of deep partial-thickness burn in Bama miniature pigs is accelerated by a higher dose of hUCMSCs

https://doi.org/10.21203/rs.3.rs-4492376/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Deep partial-thickness burns have a significant impact on both the physical and mental health of patients. Our previous study demonstrated human Umbilical Cord Mesenchymal stem cells (hUCMSCs) could enhance the healing of severe burns in small animal burn models, such as rats. Furthermore, our team has developed a deep partial-thickness burn model in Bama miniature pigs, which can be utilized for assessing drug efficacy in preclinical trials for wound healing. Therefore, this study further determine the optimal dosage of hUCMSCs in future clinical practice by comparing the efficacy of low-to-high doses of hUCMSCs on deep partial-thickness burn wounds in Bama miniature pigs.

Materials and methods

The male Bama miniature pigs (N = 8, weight: 23–28 kg and length: 71–75 cm) were used to establish deep partial-thickness burn models, which were used a continuous pressure of 1 kg and contact times of 35 s by the invented electronic burn instrument at 100℃ to prepare 10 round burn wounds with diameter of 5 cm according to our previous report. And then, 0×10^7, 1×10^7, 2×10^7, 5×10^7 and 1×10^8 doses of hUCMSCs were respectively injected into burn wounds of their corresponding groups. After treatment for 7, 14 and 21 days, the burned wound tissues were obtained for histological evaluation, including Hematoxylin and eosin (HE) for histopathological changes and total inflammatory cells infiltration, immunohistochemistry for neutrophil (MPO+) infiltration and microvessel (CD31+) quantity, and masson staining for collagen deposition. And the levels of lipopolysaccharide (LPS), inflammatory factors TNF-α, IL-1β, IL-10 and angiogenesis factors angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), as well as collagen type-I and type-III of the burned wound tissues were quantified by ELISA.

Results

All of doses hUCMSCs can significantly increase wound healing rate and shorten healing time of the deep partial-thickness burn pigs in a dose-dependent manner. Furthermore, all of doses hUCMSCs can significantly promote epithelialization and decreased inflammatory reaction of wound, including infiltration of total inflammatory cells and neutrophil, and levels of LPS, and proinflammatory factors TNF- α and IL-1β, while the level of anti-inflammatory factor IL-10 increased compared to the burn group. Meanwhile, the amounts of microvessel, expression of Ang-2 and VEGF were increased in all of doses hUCMSCs group than those in the burn group. Furthermore, the collagen structure was disordered and partially necrotized, and ratios of collagen type-I and type-III were significantly decreased in burn group (4:1 in normal skin tissue), and those of all hUCMSCs groups were significantly improved in a dose-dependent manner. In a word, 1×10^8 dose of hUCMSCs could regenerate the deep partial-thickness burn wounds most efficaciously compared to other dosages and the burn control groups.

Conclusion

This regenerative cell therapy study using hUCMSCs demonstrates the best efficacy toward a high dose, that is dose of 1×10^8 of hUCMSCs was used as a reference therapeutic dose for treating for 20 cm² deep partial-thickness burns wound in future clinical practice.

MSCs

burn wound

inflammation

angiogenesis

collagen

Burn injuries are the fourth most common type of trauma in the world, second only to the injuries caused by motor vehicle, falls and interpersonal violence [1, 2].The incidence rate of burns is on the rise each year, presenting a significant threat to the health and safety of individuals. The key for burn treatment is to repair and reconstruct burn wound as soon as possible. The faster the wound heals, the less serious the scar will be. Patients with deep burns can be treated with surgery, but this procedure comes with the risk of secondary trauma and can significantly increase the psychological and financial burden on patients. Deep partial-thickness burns or deep second-degree burns with fewer residual cutaneous adnexa belong to depth-burns that require a long time to heal, thus forming hypertrophic scars leading to serious complications such as unbearable itching and pain, disfigurement and functional impairment [3–5]. These impairments also result in negative psychological issues such as low self-esteem, anxiety, irritability, and more, significantly impacting the quality of life and potentially leading to social isolation.[6] Therefore, deep partial-thickness burns have always been the focus and challenge of research in this field.

In recent years, there have been some advances in treating burn wounds, but definitive therapy and effective drugs are not yet available for deep partial-thickness burns. The human umbilical cord mesenchymal stem cells (hUCMSCs) with high proliferation, multidirectional differentiation, low immunogenicity, without tumorigenesis and ethical issues are considered as an excellent candidate for cell-based therapy and regenerative medicine. Local or systemic administration of hUCMSCs with varying doses and delivery frequencies can effectively address COVID-19, ARDS, sepsis and other critical illnesses, as along with traumatic brain injury (TBI), acute kidney injury (AKI), acute lung injury (ALI), large area burns and other severe organ injuries [7–13]. Furthermore, the existing evidence on MSC biology has advanced the development of specific guidelines and quality control methods, ultimately enabling the clinical application of hUCMSCs[14–16]. To obtain the best therapeutic effect, the delivery dosage and frequency of MSCs administration should be considered comprehensively. For instance, Gertraud Eylert et al seeded between 2×10^2 and 2×10^6 cells/cm² of UCMSCs on the DRT (Integra® is one of the most recognized scaffolds worldwide) forming biocomposites, subsequently grafted it onto full-thickness burn excised wounds. They found the biocomposites with 5×10^3 and 1×10^7 cells regenerated the full-thickness burn excised wounds most efficaciously[17]. And recently, endovascular of UCMSCs 4.8 to 8.6×10^7 cells significantly increase in neo vessels, accompanied by complete or gradual ulcer healing in the diabetic foot ulcer patients[18]. Our previous study also validated that systemic administration of 5×10^6 hUCMSCs twice a week significantly enhanced wound healing and addressed dysfunctions of vascular endothelial barrier in rats with 50% TBSA (total body surface area) full-thickness burns [19]. Furthermore, our team established a deep partial-thickness burn model in Bama miniature pigs[20], which can be used for assessing drug efficacy in preclinical trials for wound healing. Therefore, based on the therapeutic benefits and preclinical trials models mentioned above, this study further validated the ideal dosage of hUCMSCs for future clinical practice by comparing the efficacy of varying doses of hUCMSCs on deep partial-thickness burn wounds in Bama miniature pigs.

Animal care

All the experiments adhered to procedures consistent with the International Guiding Principles for Biomedical Research Involving Animals issued by the Council for the International Organizations of Medical Sciences (CIOMS) and ARRIVE (Animal Research: Reporting of In Vivo Experiments) 2.0 guidelines. The study was approved by the Institutional Animal Care and Use Committee at The Fourth Medical Center of PLA General Hospital (the ethics approval ID 2021KY033-KS001). A total of 8 ordinary male Bama miniature pigs were purchased from the Beijing Strong Century Minipigs Breeding Base. The minipigs were housed at the Laboratory Animal Center of The Fourth Medical Center of PLA General Hospital (Beijing, China), and kept on a 12:12-h light–dark cycle, at room temperature maintained between 20 and 22 ℃ with a humidity range of 50–60%, for 1 week before the experiment for adaptation.

Cell culture

The primary human umbilical cord MSCs were purchased from GENESIS STEM CELL Co., Ltd in China, cultured in Mesenchymal Stem Cell Medium (MSCM, ScienCell, USA) and humidified in a 5% CO2 incubator at 37°C. In the following passages, abbreviation of human umbilical cord MSC was hUCMSCs and its passages 3–8 were used for all the following experiments as previously described (Liu et al., 2013).

Establishment of deep partial-thickness burn in Bama miniature pigs

The present study is a one-way, one-period, self-control comparative trial enrolled 8 wild-type pigs with 10 burn wounds on each back and receiving hUCMSCs injection treatment.When the 8 male single-caged Bama miniature pigs reached a weight of 23–28 kg and had a mean body length of 71–75 cm, they were used to make deep partial-thickness burn models according to our previous method[20]. Before the experiment, the hair of each pig was shaved and depilated on the back. Each pig was then anesthetized with an intramuscular injection of 1 mg of Ketamine and Sumianxin II. The anesthesia was maintained with 3% Pentobarbital sodium (0.2 mL/kg) as needed. In this study, 10 round burn wounds with a diameter of 5 cm were prepared separately on both sides of each pig’s back by applying a continuous pressure of 1 kg perpendicular to the spine and contact times of 35 s using the invented electronic burn instrument (ZL201920043476.1) at 100 ℃. The burn wounds were histologically confirmed using our published protocol [20]. The 10 burn wounds were randomly divided into 5 groups: burn group (0×10^7 cells/1mL hUCMSCs were locally subcutaneous injected into burn wounds); hUCMSCs-1 group (1×10^7 cells/1mL hUCMSCs subcutaneous injection); hUCMSCs-2 group (2×10^7 cells/1mL hUCMSCs subcutaneous injection); hUCMSCs-3 group (5×10^7 cells/1mL hUCMSCs subcutaneous injection); hUCMSCs-4 group (1×10^8 cells/1mL hUCMSCs subcutaneous injection). Each group contained 16 burn wounds in total and paired two burn wounds was used as one mixed sample (n = 8) on one pig. Paired two burn wounds were randomized using a computer based random order generator. And local treatment and every other day of delivery frequency of hUCMSCs is based on clinical practice. Then, all burn wounds were wrapped by sterilized gauze and secure the outermost layer with an elastic animal compression jacket. After that, the pigs were kept in cages and free to drink and eat. The physiological state and burn wounds of the Bama miniature pigs were then closely monitored and observed.The animals were excluded if died before the end. All the animals were anesthetized with Pentobarbital sodium for samples collection, and received euthanasia with intravenous barbiturate (90mg/kg body weight) at the study end.

Specimen collection

After hUCMSCs treatment for 7, 14 and 21 days, the burned wound tissues on both sides of pig’s back were cut to the muscular layer. The cut site was in the middle of the burn wound. Each sample was divided into small pieces, and one piece was fixed in 4% paraformaldehyde for Hematoxylin and eosin (HE) and Masson staining, as well as immunohistochemical analysis; also, another piece was stored in liquid nitrogen for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10), lipopolysaccharide (LPS), angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), collagen type-I and type-III detections using the ELISA kits.

HE staining

After fixation with 4% paraformaldehyde for 24 h at room temperature, the specimens were embedded in paraffin and sectioned in a plane perpendicular to the incision (Leica, German). Five-micrometer-thick sections were prepared, deparaffinized in dimethylbenzene, and rehydrated. Preparative sections were stained with HE in accordance with standard procedures. The sections finally were placed under an optical microscope (Olympus, USA) to observe the histopathological changes and total inflammatory cells infiltration.

Masson staining

The pre-treatment of the sections of burn tissue was consistent with that of HE staining. The pre-treated sections were immersed in potassium dichromate overnight at room temperature and then stained in hematoxylin solution for 3–5 min. After being washed with flowing distilled water for 1–3 min, the blue was reversed by immersing them in ethanol-hydrochloric acid solution. Subsequently, the sections were transferred into a dye solution composed of ponceau and acid fuchsin for 10–15 min, phosphomolybdic acid aqueous solution for 3–5 min, aniline blue staining solution for 6 min, glacial acetic acid (1% v/v) for 2 min, and 95% ethanol and xylene for 5 secs. The slides were dried in a ventilated place, and then covered with a cover slip with neutral balsam. After drying, the sections were observed collagen deposition under an optical microscope (Olympus, USA).

Immunohistochemical staining

About 5 µm sections were treated with pepsin for 20 min, 3% hydrogen peroxide methanol was used to block peroxidase for 10 min, and then the specimens were blocked with PBS containing 5% normal horse serum. They were further incubated with specific antibodies (monoclonal mouse antibodies against rat MPO or CD31; diluted at 1:200; Abcam, United Kingdom), followed by incubation with the corresponding secondary antibody and the PAP (peroxidase–anti-peroxidase) complex, and exposure using DAB (3,39-diaminobenzidine). The neutrophils and capillaries in wound tissues were counted in 5 randomly selected fields of the each slide by an experienced and independent cell scientist in a blinded manner.

Enzyme-linked immunosorbent assay (ELISA)

The total protein was extracted from frozen tissues and quantified by BCA method. The levels of TNF-α, IL-1β, IL-10, LPS, Ang-2, VEGF, collagen types I and III were examined with a double-antibody sandwich ELISA kit (eBioscience, USA) following the manufacturer’s instructions. The OD value was detected on a multi-detection microplate reader (Molecular Devices, USA).

Histology scoring system

In fact, monitoring wound progression over time is a critical aspect for studies focused on evaluating the efficacy of potential novel therapies. Histopathological analysis of wound biopsies can provide significant insight into healing dynamics, so we conducted histological scorings of deep partial-thickness burn wounds of all groups according to parameters in histology scoring system [21]. These parameters included re-epithelization, epithelial thickness index, keratinization, granulation tissue thickness, remodeling, and the scar elevation index.

Statistical analysis

Statistical analysis was performed using SPSS (21.0, IBM, Armonk, NY, USA). Data are presented as the mean ± SEM, and were compared with 95% confidence interval using a repeated measure two-way analysis of variance (ANOVA), one-way ANOVA, or Student’s t-test. Statistical significance was set at p < 0.05.

A higher dose of hUCMSCs immensely accelerated healing of burn wound

Wound healing was assessed by gross observation and photography at 0, 7, 14 and 21 days after the low-to-high doses of hUCMSCs treatments (Fig. 1A). At 0 day, all burned wounds were the same size and pale in color without fluid exudation. After awakening from anesthesia, the food intake, water intake, and body temperature of these burned pigs were all normal. At 7 days after treatment, there is a small amount of scabs dissolution but no obvious infection on the burn wounds of all groups. Compared with burn group, the low-to-high doses of hUCMSCs can promote partial healing around the wound in a dose-dependent manner, and 1×10^8 dose of hUCMSCs was most efficacious in all treatment groups. At 14 and 21 days after treatment, scabs on the wound thickened and hardened, and their color turns black in burn group. By contrary, the scabs of hUCMSCs-1, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups appeared red and white and had no exudation and infection. Furthermore, the low-to-high doses of hUCMSCs can significantly promote re-epithelialization of the deep partial-thickness burn wounds in a dose-dependent manner, and the therapeutic effect with 1×10^8 dose of hUCMSCs was the best.

Further, the healing times and healing rates of the deep partial-thickness burn wounds were also evaluated. As shown in Fig. 1B, the wound healing times in burn, hUCMSCs-1, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups were 30.29 ± 0.95 days, 23.81 ± 0.47 days, 22.36 ± 0.82 days, 21.92 ± 0.33 days, and 17.52 ± 0.62 days respectively. It was notable that the low-to-high doses of hUCMSCs can significantly shorten healing times of the deep partial-thickness burn wounds in a dose-dependent manner. The wound healing time of 1×10^8 dose of hUCMSCs was still the shortest compared to other treatment groups (p < 0.05). The complete healing of wounds, i.e., with good re-epithelization and a residual wound area of 1%, were evaluated via Image J software. At 7, 14 and 21 days after treatment, the healing rates of the hUCMSCs-1, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups were significantly higher than that of the burn group with a dose-dependent manner (Fig. 1C). Similarly, the wound healing rate of 1×10^8 dose of hUCMSCs was still the highest at every time-point compared to other treatment groups (p < 0.05).

A higher dose of hUCMSCs immensely alleviated structure damage of burn wound

To further determine the effect of the low-to-high doses of hUCMSCs on structure damage of burn wounds, we evaluated their histopathological changes using HE staining (Fig. 2). 7 days after treatment, the epidermis and dermis of the deep partial-thickness burn were necrotic, and the collagen fibers were also disordered and degenerated. Compared with the burn group, the low-to-high doses of hUCMSCs administration could significantly improve burn-induced structural damages in a dose-dependent manner. Furthermore, total numbers of inflammatory cells in wound of the burn group increased significantly on the 14th day than that on the 7th day, meanwhile, the increasing trend recovered to some extent by day 21. However, total inflammatory cells infiltrations in hUCMSCs-1, hUCMSCs-2, hUCMSCs-3, and hUCMSCs-4 groups were all significantly less than those in the burn group at the corresponding time point, with a certain dose dependence.

It is worth noting that the low-to-high doses of hUCMSCs administration could remarkably alleviated structure damage and promoted re-epithelialization, epidermal maturation and collagen structure recovery of burn wound in a dose-dependent manner, and the best therapeutic effect was still achieved by 1×10^8 dose of hUCMSCs.

A higher dose of hUCMSCs immensely regulated inflammation in burn wound

Essentially, inflammation is a kind of protective reaction against burn factors. Proper inflammatory reaction is beneficial to anti-impairment, while excessive inflammation is very harmful for wound repair. Therefore, the immunohistochemical staining of MPO in this part was employed to examine the degree of neutrophil infiltration. As shown in Fig. 3A, neutrophil infiltration all increased significantly in wound of the burn group at 7, 14, 21 days after treatment separately, which was the most deteriorated on the 14th day. By contrary, neutrophil infiltrations in hUCMSCs-1, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups decreased significantly, all of which were significantly lower than those in the burn group with a certain dose dependence as well. The result of the quantitative analysis is presented in the corresponding histogram (Fig. 3B).

Moreover, the contents of LPS (main ingredient of bacterial endotoxin), pro-inflammatory cytokines, such as TNF-α and IL-1β, and anti-inflammatory cytokine, such as IL-10 were examined by ELISA. Compared with those in the burn group, the contents of LPS, TNF-α and IL-1β in wounds of hUCMSCs-1, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups markedly decreased, while IL-10 content increased significantly at the corresponding time-point with a certain dose dependence (Fig. 2C-E).

The above results indicated that the low-to-high doses of hUCMSCs treatments regulated the inflammatory reaction in deep partial-thickness burn wounds, in which the injection of 1×10^8 hUCMSCs was found to have the best anti-inflammatory effect.

A higher dose of hUCMSCs immensely promoted neovascularization in burn wound

As we all know, the blood supply is the key for wound healing, and especially in the inflammation phase and proliferation phase. Therefore, neovascularization in deep partial-thickness burn wounds was evaluated by the immunohistochemistry assay. At 7 and 14 days after treatment, wound neovascularization occurred in all burn and hUCMSCs treatment groups. The neovascularization was quantified by counting the microvessels. And the microvessel numbers in hUCMSCs-1, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups significantly increased more than that in the burn group and were dose-dependent, in which the number of new microvessels was the most in hUCMSCs-4 group (Fig. 4A). It is worth noting that the microvessel numbers in burn group on the 21th were remarkably increased than that on the 7th and14th days. On the contrary, the microvessel numbers in hUCMSCs-3 and hUCMSCs-4 groups on the 21th were decreased than those on the 7th and14th days, but these newborn vessels tend to mature and have complete three-dimensional tubular structures that allow blood flow to pass through. The results of the quantitative analysis are presented in the corresponding histogram (Fig. 4B).

Considering the significant promoting role of Ang-2 and VEGF in angiogenesis, their contents in burn wounds of all groups were detected by ELISA. Compared to the burn group, the Ang-2 and VEGF contents in wounds of the low-to-high doses of hUCMSCs groups all increased significantly in a dose-dependent manner, in which their contents were most drastic increase in hUCMSCs-4 group at 7 and 14 days after treatment (Fig. 4C and 4D).

In general, the low-to-high doses of hUCMSCs could promote neovascularization of deep partial-thickness burn wounds, and a higher dose of 1×10^8 hUCMSCs was found to have the best therapeutic effect.

A higher dose of hUCMSCs immensely improving collagen arrangement and types I and III deposition ratio in burn wound

Masson staining was further performed to observe the collagen fiber structure changes and re-epithelialization qualities of the burn wounds. Consistent with the HE staining results, the Masson staining results also showed that the epidermis and dermis tissues were necrotic, and the collagen fibers were severely destroyed in wound of the burn group, but their arrangement becomes more and more regular and orderly accompanied by gradual increasing doses of hUCMSCs administration at 7 day after treatment. By the day 14, the necrotic epidermis has partly peeled off, and there was massive necrotic collagen and infiltration of inflammatory cells in the burn group. It was worth noting that new granulation tissue grew into the burn wound and began to re-epithelialize, as well as collagen fibers in dermis was still disordered in hUCMSCs-1 group. But the effects of hUCMSCs on collagen arrangement regularity and re-epithelialization quality were increasing by gradual increasing doses of hUCMSCs administration. Up to day 21, the burn group's wounds also began to re-epithelialize, but collagen structure was disordered and irregular. Compared with that in the burn group, the re-epithelialize quality in epidermis and arrangement of collagen fibers in dermis in all hUCMSCs groups became better and better by gradual increasing doses of hUCMSCs administration, and the best therapeutic effect was still achieved by 1×10^8 dose of hUCMSCs (Fig. 5A).

Moreover, the collagen types I and III are the main collagen types of healthy skin and the ratio of collagen types I and III determined progress of burn wound repair. In general, the ratio of collagen types I and III is 4:1 in normal skin tissue, but their ratio sharply decreased in skin wounds of burn group. Compared with those in the burn group, the ratioes of collagen types I and III in all hUCMSCs groups increased significantly in a dose-dependent manner at 7,14 and 21 days after treatment. Similarly the best therapeutic effect was still achieved by 1×10^8 dose of hUCMSCs admistration (Fig. 5B).

A higher dose of hUCMSCs immensely raising histology score of burn wound

Figure 6 and Table 1 provide all parameters of histological scores. Firstly, re-epithelization scores were all 1 in burn group, hUCMSCs-1 group and hUCMSCs-2 group, as well as 1–2 in hUCMSCs-3 group, but 2 in hUCMSCs-4 group. Secondly, scores of epithelial thickness index (ETI) and granulation tissue thickness were all 0–1 in burn group, hUCMSCs-1 group and hUCMSCs-2 group, as well as 1–2 in hUCMSCs-3 group, but 2 in hUCMSCs-4 group. Thirdly, keratinization scores were all 0–2 in burn group, hUCMSCs-1 group, hUCMSCs-2 group, hUCMSCs-3 group, but 2 in hUCMSCs-4 group. Fourthly, remodeling scores were all 1 in burn group, hUCMSCs-1 group, hUCMSCs-2 group and hUCMSCs-3 group, but 2 in hUCMSCs-4 group. Fifth, the scar elevation index (SEI) scores were all 0 in burn group, hUCMSCs-1 group, hUCMSCs-2 group, hUCMSCs-3 group, but 2 in hUCMSCs-4 group. Collectively, total scores of all parameters in burn group, hUCMSCs-1 group, hUCMSCs-2 group, hUCMSCs-3 group and hUCMSCs-4group were 2–6, 2–6, 2–6, 4–9 and 12, respectively. Score 12 in the histology scoring system represented complete and prefect healing of wound, and only 1×10^8 dose of hUCMSCs got the score 12 in all groups. And total score of each group also represents its different degree and quality of healing progression in deep partial-thickness burn wound of Bama miniature pigs. Although the scoring system provides important quantitative criteria for wound healing progress, it still has certain limitations and lacks detailed scoring.

In clinical practice, the treatment of deep partial-thickness burn wounds has always been an very challenging issue [10, 17, 22]. In recent years, MSCs have garnered global attention and been widely used for promoting wound healing in a various types of injuries [23–26], particularly hUCMSCs with some advantages of high proliferation, multidirectional differentiation, low immunogenicity, convenient material selection might ultimately contribute to the clinical application of deep burn wounds through self-multipotency property [27–29]. In addition, recent trials have also confirmed the efficacy of hUCMSCs in several other conditions such as neural, liver, kidney, bone, heart diseases, wound healing and immune-mediated disorders [18, 30–38]. Therefore, determining cell dosage for clinical trials is essential to enhance success rate and minimize therapy failure probability. Our study with a wide dose range fills a gap with respect to dosage and discusses the effects of future cell-based therapy. And in our low-to-high hUCMSCs-dose treatment deep partial-thickness burn in Bama miniature pigs model [16], where we evaluated 12 wound healing parameters, and our data show that a high dose of 1×10^8 cells/20cm² regenerates the burn wounds most efficaciously, followed by some even lower doses of 5×10^7, 2×10^7, 1×10^7cells/20cm².

Generally, skin wound healing, triggered by tissue injury, involves four stages: hemostasis, inflammation, proliferation, and maturation. MSCs can support all stages of the wound healing process. The preclinical results indicate that a high dose of 1×10^8 hUCMSCs therapy improved macroscopic wound healing by promoting faster epithelialization, a more appropriate inflammatory response, and reduced scarring [39–41]. Several studies have shown that restricted inflammation of burn is beneficial, and excessive or persistent inflammation incites wound tissue destruction, even nonhealing [42]. The hUCMSCs demonstrated the capacity to suppress the immune response in conditions abundant in inflammatory cytokines, such as SIRS, infections, and even sepsis, in both preclinical and clinical trials [43, 44]. And our results further confirmed that hUCMSCs significantly reduced inflammatory cell infiltration, decreased LPS, TNF-α and IL-1β contents, and increased IL-10 content, showing that hUCMSCs markedly regulate inflammatory reaction of burn wounds in Bama miniature pigs. There is one limitation that systematic inflammation in pigs has a little influence on wound local inflammation.

Furthermore, our data also confirmed that the most beneficial therapy of high dose of 1×10^8 hUCMSCs with pro-angiogenic and fibroproliferative effects increased neo vascularization and collagen formation, as well as reduced fibrosis [8, 39, 45]. Angiogenesis is a crucial step in burn wound healing process, as the growth of new blood vessels supplies abundant oxygen and nutrients, and ultimately nourishing the growing tissues in the wound [46]. Angiopoietin (Ang) and VEGF are very important vascular growth factors. VEGF plays a key role in angiogenesis by stimulating endothelial cell proliferation, migration and organization into tubules[16, 45]. Ang-2, the best characterized angiopoietins, is ligands for the Tie2 receptor tyrosine kinase, which is present on endothelial cells and endothelial progenitor cells. Both two growth factors collectively induce new blood vessel formation and modulate maturation of neovascularization [16, 47]. And our findings indicate that injecting hUCMSCs into the deep partial-thickness burn wound results in elevated levels of Ang-2 and VEGF, potentially contributing to the process of neovascularization. Furthermore, collagen fibres involved in the formation of granulation tissue to fill the wound defects and set the stage for epidermal cell coverage [39, 48]. Collagen type I and type III depositions and their ratios in wounds are important determinant of the wound healing process [40]. Our results also demonstrated that hUCMSCs significantly enhance collagen deposition and increase collgen types I and III ratio of burn wounds, and ultimately expedite the re-epithelialization progress of the deep partial-thickness burn wounds.The clinical application of hUCMSCs in regenerative medicine encompasses the infusion method, dosing and frequency of dilivery, and other relevant factors. Barczewska et al found that three intrathecal injections of 3×10^7 UCMSCs improved ALSFRS in Amyotrophic lateral sclerosis (ALS) patients [30]. Wang and coworkers have found that intravenous injection of 0.5×10^7 cells/kg UCMSCs is feasible and effective, as well as well tolerated in patients with primary biliary cirrhosis (PBC) [31]. In a different clinical study [37], the intramedullary (4×10^7 cells) and intravenous (2×10^8 cells) infusion of UCMSCs combined with teriparatide demonstrated positive outcomes in individuals with osteoporotic vertebral compression fractures. It also reported that UCMSCs (1×10^8 cells cells) significantly decreased the WOMAC and could be a potentially new regenerative treatment for patients with knee OA [36]. Therefore, our study covered the dose range of hUCMSCs mentioned in the above literature to compare and analyze their efficacy, and local treatment and delivery frequency of hUCMSCs is based on clinical practice.

This regenerative cell therapy study using hUCMSCs, which have a significant and beneficial impact on burn wound healing stages, including the inflammation phase, angiogenesis and collagen accumulation, shows the best efficacy toward a high dose, that is dose of 1×10^8 of hUCMSCs was used as a reference therapeutic dose for treating for 20 cm² deep partial-thickness burns wound in future clinical practice. Moreover, the high dose of 1×10^8 treatment is also safe and free from any other side effects.

Ethics approval and consent to participate

The project “An application study of new materials and techniques in the treatment of burns with different depths” was approved by the Institutional Animal Care and Use Committee at The Fourth Medical Center of PLA General Hospital (the ethics approval ID 2021KY033-KS001, Date approval: Nov. 29th, 2021 ).

Consent for publication

The manuscript did not contain any individual person’s data.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by research projects of Youth Independent Innovation Science Foundation of PLA General Hospital (22QNCZ031) and National Natural Science Foundation of China (81701900).

Authors' contributions

LL, XH, JZ, SL: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing and financial support. PQ, YZ, HY, YK, Y Yin, JC, Y Yu, HJ, HY, and JC: provision of study material, data analysis, and interpretation. LL, XH, JZ, SL contributed equally to this study. All authors read and approved the final manuscript.

Acknowledgements

The authors declare that they have not used Artificial Intelligence in this study.

GBD Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015; 385:117-171.
Norton R, Kobusingye O. Injuries. N Engl J Med. 2013; 368:1723-1730.
Legrand M, Dépret F, Mallet V. Management of Burns. N Engl J Med. 2019; 381:1188-1189.
Chen J, Wang H, Mei L, et al.A pirfenidone loaded spray dressing based on lyotropic liquid crystals for deep partial thickness burn treatment: healing promotion and scar prophylaxis,” J Mater Chem B.2020;8:2573-2588.
Piccolo NS, Piccolo MS, Piccolo MT.Fat grafting for treatment of burns, burn scars, and other difficult wounds. Clin Plast Surg.2015;42: 263-283.
Robert R, Meyer W, Bishop S, et al. Disfiguring burn scars and adolescent self-esteem. Burns.1999;25:581-585.
Batsali AK, Kastrinaki MC, Papadaki HA, et al. Mesenchymal stem cells derived from Wharton's Jelly of the umbilical cord: biological properties and emerging clinical applications. Curr Stem Cell Res Ther. 2013; 8:144-155.
Xiang E, Han B, Zhang Q, et al. Human umbilical cord-derived mesenchymal stem cells prevent the progression of early diabetic nephropathy through inhibiting inflammation and fibrosis. Stem Cell Res Ther. 2020;11: 336.
Cheng L, Wang S, Peng C, et al. Human umbilical cord mesenchymal stem cells for psoriasis: a phase 1/2a, single-arm study. Signal Transduct Target Ther. 2022;7:263.
Jeschke MG, van Baar ME, Choudhry MA, et al., Burn injury. Nat Rev Dis Primers.2020; 6:11.
Chu M, Wang H, Bian L, et al., Nebulization Therapy with Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes for COVID-19 Pneumonia. Stem Cell Rev Rep.2022;18: 2152-2163.
Iglesias M, Butrón P, Torre-Villalvazo I, et al. Mesenchymal Stem Cells for the Compassionate Treatment of Severe Acute Respiratory Distress Syndrome Due to COVID 19. Aging Dis.2021; 12:360-370.
Shi W, Nie D, Jin G, et al. BDNF blended chitosan scaffolds for human umbilical cord MSC transplants in traumatic brain injury therapy. Biomaterials.2012; 33:3119-3126.
Margiana R, Markov A, Zekiy AO, et al. Clinical application of mesenchymal stem cell in regenerative medicine: a narrative review. Stem Cell Res Ther.2022; 13:366.
Saeedi P, Halabian R, Imani Fooladi AA. A revealing review of mesenchymal stem cells therapy, clinical perspectives and Modification strategies. Stem Cell Investig.2019; 6:34.
Fam NP, Verma S, Kutryk M, et al. Clinician guide to angiogenesis. Circulation. 2003;108:2613-2618.
Eylert G, Dolp R, Parousis A, et al., Skin regeneration is accelerated by a lower dose of multipotent mesenchymal stromal/stem cells-a paradigm change. Stem Cell Res Ther.2021;12: 82.
Qin HL, Zhu XH, Zhang B, et al. Clinical Evaluation of Human Umbilical Cord Mesenchymal Stem Cell Transplantation After Angioplasty for Diabetic Foot. Exp Clin Endocrinol Diabetes.2016;124:497-503.
Liu L, Yin H, Hao X, et al., Down-Regulation of miR-301a-3p Reduces Burn-Induced Vascular Endothelial Apoptosis by potentiating hMSC-Secreted IGF-1 and PI3K/Akt/FOXO3a Pathway. iScience.2020; 23:101383.
Hao X, Chi Y, Bai H, et al., Establishment of a Bama miniature pig burn model with different burn depths. Gland Surg. 2022;11:1647-1655.
van de Vyver M, Boodhoo K, Frazier T, et al. Histology Scoring System for Murine Cutaneous Wounds.Stem Cells Dev.2021; 30: 1141-1152.
Gibson ALF, Carney BC, Cuttle L, et al. Coming to Consensus: What Defines Deep Partial Thickness Burn Injuries in Porcine Models?. J Burn Care Res.2021;42:98-109.
Jo H, Brito S, Kwak MB, et al. Applications of Mesenchymal Stem Cells in Skin Regeneration and Rejuvenation. Int J Mol Sci.2021;22:5.
Liu L, Yu Y, Hou Y, et al. Human umbilical cord mesenchymal stem cells transplantation promotes cutaneous wound healing of severe burned rats. PLoS One.2014; 9:e88348.
Kim KH, Blasco-Morente G, Cuende N, et al. Mesenchymal stromal cells: properties and role in management of cutaneous diseases. J Eur Acad Dermatol Venereol.2017; 31:414-423.
Rodgers K, Jadhav SS. The application of mesenchymal stem cells to treat thermal and radiation burns. Adv Drug Deliv Rev. 2018; 123:75-81.
Liu Y, Zeng BH, Shang HT, et al. Bama miniature pigs (Sus scrofa domestica) as a model for drug evaluation for humans: comparison of in vitro metabolism and in vivo pharmacokinetics of lovastatin. Comp Med. 2008;58:580-587.
Soleymaninejadian E, Pramanik K, Samadian E. Immunomodulatory properties of mesenchymal stem cells: cytokines and factors. Am J Reprod Immunol. 2012; 67:1-8.
Wang CW, Yang LY, Chen CB, et al. Randomized, controlled trial of TNF-α antagonist in CTL-mediated severe cutaneous adverse reactions. J Clin Invest. 2018; 128:985-996.
Barczewska M, Maksymowicz S, Zdolińska-Malinowska I, et al. Umbilical Cord Mesenchymal Stem Cells in Amyotrophic Lateral Sclerosis: an Original Study. Stem Cell Rev Rep.2020;16:922-932.
Wang L, Li J, Liu H, et al. Pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis. J Gastroenterol Hepatol.2013. 28 :85-92.
Zhang YC, Liu W, Fu BS, et al. Therapeutic potentials of umbilical cord-derived mesenchymal stromal cells for ischemic-type biliary lesions following liver transplantation. Cytotherapy. 2017;19:194-199.
Gao LR, Chen Y, Zhang NK, et al. Intracoronary infusion of Wharton's jelly-derived mesenchymal stem cells in acute myocardial infarction: double-blind, randomized controlled trial. BMC Med. 2015;13:162.
Bartolucci J, Verdugo FJ, González PL, et al. Safety and Efficacy of the Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells in Patients With Heart Failure: A Phase 1/2 Randomized Controlled Trial (RIMECARD Trial [Randomized Clinical Trial of Intravenous Infusion Umbilical Cord Mesenchymal Stem Cells on Cardiopathy]). Circ Res. 2017;121:1192-1204.
Matas J, Orrego M, Amenabar D, et al. Umbilical Cord-Derived Mesenchymal Stromal Cells (MSCs) for Knee Osteoarthritis: Repeated MSC Dosing Is Superior to a Single MSC Dose and to Hyaluronic Acid in a Controlled Randomized Phase I/II Trial. Stem Cells Transl Med. 2019;8:215-224.
Dilogo IH, Canintika AF, Hanitya AL, et al. Umbilical cord-derived mesenchymal stem cells for treating osteoarthritis of the knee: a single-arm, open-label study. Eur J Orthop Surg Traumatol. 2020;30: 799-807.
. Shim J, Kim KT, Kim KG, et al. Safety and efficacy of Wharton's jelly-derived mesenchymal stem cells with teriparatide for osteoporotic vertebral fractures: A phase I/IIa study. Stem Cells Transl Med. 2021;10:554-567.
. Huang J, Li Q, Yuan X, et al. Intrauterine infusion of clinically graded human umbilical cord-derived mesenchymal stem cells for the treatment of poor healing after uterine injury: a phase I clinical trial. Stem Cell Res Ther. 2022;13:85.
. Bowers S, Franco E. Chronic Wounds: Evaluation and Management.Am Fam Physician. 2020;101:159-166.
Iyyam Pillai S, Palsamy P, Subramanian S, et al. Wound healing properties of Indian propolis studied on excision wound-induced rats. Pharm Biol. 2010;48:1198-1206.
Ashida A, Murata H, Mikoshiba Y, et al. Successful treatment of rheumatoid vasculitis-associated skin ulcer with a TNF-α antagonist. Int J Dermatol. 2014; 53:e154-156.
Hu X, Liu L, Wang Y, et al. Human Umbilical Cord-Derived Mesenchymal Stem Cells Alleviate Acute Lung Injury Caused by Severe Burn via Secreting TSG-6 and Inhibiting Inflammatory Response. Stem Cells Int.2022; 2022:8661689.
Abbaszadeh H, Ghorbani F, Derakhshani M, et al. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles: A novel therapeutic paradigm. J Cell Physiol.2020; 235:706-717.
Putra A, Ridwan FB, Putridewi AI, et al. The Role of TNF-α induced MSCs on Suppressive Inflammation by Increasing TGF-β and IL-10. Open Access Maced J Med Sci. 2018; 6:1779-1783.
Arnold F, West DC. Angiogenesis in wound healing. Pharmacol Ther. 1991; 52:407-422.
Shpichka A, Butnaru D, Bezrukov EA, et al. Skin tissue regeneration for burn injury. Stem Cell Res Ther. 2019; 10: 94.
Yancopoulos GD, Davis S, Gale NW, et al. Vascular-specific growth factors and blood vessel formation. Nature. 2000;407: 242-248.
Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol.2007;127:998-1008.

Table 1 is available in the Supplementary Files section.

Table1.docx
Table 1. The influence of the low-to-high doses of hUCMSCs on the histology scoring of the deep partial-thickness burn wound in Bama miniature pigs. There are 6 parameters, including re-epitheliazation, epidermal thickness index (ETI), keratinization, granulation tissue, remoding and scar evevation index (SEI) and corresponding scroring criteria in the histology scoring system. For all quantitative parameters, measurements should be taken at ﬁve positions. And scores of burn group and the low-to-high doses of hUCMSCs treatment groups were all presented in the table at 21 day after treatment.
renamedab228.pdf

Download PDF

Reviewers agreed at journal
14 Jul, 2024
Reviewers invited by journal
13 Jul, 2024
Editor assigned by journal
01 Jul, 2024
First submitted to journal
28 Jun, 2024
Editorial decision: Major Revision
19 Jun, 2024

You are reading this latest preprint version

The wound healing of deep partial-thickness burn in Bama miniature pigs is accelerated by a higher dose of hUCMSCs

Status:

Version 1

Abstract

Background

Materials and methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Animal care

Cell culture

Establishment of deep partial-thickness burn in Bama miniature pigs

Specimen collection

HE staining

Masson staining

Immunohistochemical staining

Enzyme-linked immunosorbent assay (ELISA)

Histology scoring system

Statistical analysis

Results

A higher dose of hUCMSCs immensely accelerated healing of burn wound

A higher dose of hUCMSCs immensely alleviated structure damage of burn wound

A higher dose of hUCMSCs immensely regulated inflammation in burn wound

A higher dose of hUCMSCs immensely promoted neovascularization in burn wound

A higher dose of hUCMSCs immensely raising histology score of burn wound

Discussion

Conclusion

Declarations

References

Table 1

Supplementary Files

Status:

Version 1