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. 2024 Nov 19;15:437. doi: 10.1186/s13287-024-04063-x

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

Lingying Liu 1,2,4,✉,#, Xingxia Hao 2,#, Jing Zhang 2,#, Shaozeng Li 3,#, Shaofang Han 1, Peipei Qian 1, Yong Zhang 1,✉, Huaqing Yu 1, Yuxin Kang 4, Yue Yin 2, Weiouwen Zhang 1, Jianmei Chen 5, Yang Yu 6, Hua Jiang 7, Jiake Chai 8, Huinan Yin 8, Wei Chai 9
PMCID: PMC11575178  PMID: 39563365

Abstract

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 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 HE staining for histopathological changes, immunohistochemistry for neutrophil (MPO+) infiltration and microvessel (CD31+) quantity, as well as Masson staining for collagen deposition. The levels of 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/type-III of the 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 inflammatory cells and levels inflammatory factors. Meanwhile, the amounts of microvessel 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 groups and the burn group.

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 20 cm2 deep partial-thickness burns wound in future clinical practice.

Graphical abstract

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Keywords: MSCs, Burn wound, Inflammation, Angiogenesis, Collagen

Introduction

Burn injuries are the fourth most common type of trauma in the world, second only to 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 [3]. Thus, during healing forming hypertrophic scars leads to serious complications such as unbearable itching and pain, disfigurement and functional impairment [4, 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 an excellent candidate for cell-based therapy and regenerative medicine. A study revealed that hUCMSCs containing a tissue engineering construct xenotransplant in rabbits were used to successfully repair full-thickness cartilage defects in the femoral patellar groove, and no immune rejection was detected after 16 months of xenograft hUCMSC repair and regeneration [7]. More importantly, local or systemic administration of hUCMSCs with varying doses and delivery frequencies can effectively address COVID-19, ARDS, sepsis and other critical illnesses, along with traumatic brain injury (TBI), acute kidney injury (AKI), acute lung injury (ALI), large area burns and other severe organ injuries [8–14]. 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 [15–17].

To obtain the best therapeutic effect, the delivery dosage and frequency of MSCs administration should be considered comprehensively. For instance, Eylert et al. used biocomposites with between 2 × 10^2 and 2 × 10^6 cells/cm2 of UCMSCs to treat full-thickness burn excised wounds. They found biocomposites with the range from 5 × 10^3 to 1 × 10^7 cells regenerated the wounds most efficaciously [18]. Another recent study showed that endovascular of UCMSCs 4.8 to 8.6 × 10^7 cells significantly increase in neovessels, accompanied by complete or gradual ulcer healing in diabetic foot ulcer patients [19]. 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 [20]. Furthermore, our team established a deep partial-thickness burn model in Bama miniature pigs [21], which can be used for assessing drug efficacy in preclinical trials for wound healing. And in clinical studies, the dosage range for UCMSCs to exert effective therapeutic effects is from 1 × 10^7 to 1 × 10^8 [18–22]. Therefore, based on the therapeutic benefits and preclinical trial 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.

Materials and methods

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

Around 5 × 10^6 of the primary (P0) human umbilical cord MSCs were purchased from GENESIS STEM CELL Co., Ltd in China [23], first inoculated to T75 for 2D culture in Mesenchymal Stem Cell Medium (MSCM, ScienCell, USA) and humidified in a 5% CO2 incubator at 37 °C. And approximately 5 × 10^7 cells can be harvested by 2D culturing to the third generation (P3). The abbreviation of human umbilical cord MSC was hUCMSCs.

To realize large-scale expansion of hUCMSCs to meet the requirements of this study, we further used FloTrix miniSpin bioreactor, a three-dimensional (3D) cell culture system. And 1 × 10^7 of P3 cells and 0.6 g of porous microcarriers 3D TableTrix® (CytoNiche Biotech, China) were added to a sterile 500 mL spinner flask (miniSPIN-SSF500; CytoNiche Biotech, China) with a final volume of 200 mL and 4 sterile spinner flasks were simultaneously used. Spinner flasks were then placed on a 3D FloTrix mini-SPIN system inside the 37℃, 5% CO2 incubator and agitation speeds could be set to 40 rpm for 4 days [24]. At last, all passages 3–8 cells from 2D and 3D culturing were harvested and concentrated, then counted to calculate the total cell number and cell viability in the GMP laboratories of our hospital. A total of 1.8 × 10^10 cells were used for all the following experiments.

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 [21]. 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 [25]. 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, hUCMSCs-2, hUCMSCs-3 and hUCMSCs-4 groups (1 × 10^7, 2 × 10^7, 5 × 10^7, 1 × 10^8 cells/1mL hUCMSCs subcutaneous injection, respectively). Each group contained 16 burn wounds in total and paired two burn wounds were used as one mixed sample on one pig. Paired two burn wounds were randomized using a computer-based random order generator. Specifically, as shown in the Graphical Abstract, the distance between two burn wounds in the same treatment group is 6–7 cm, while the distance between two wounds in different treatment groups is 8–10 cm. Each round burn wound is divided into 8 equal parts by 4 straight lines passing through the center of the circle. The 0.2 ml of hUCMSCs were first subcutaneously injected at the center of the circle, then 0.1 ml of hUCMSCs were injected sequentially into the midpoint of each line. In fact, the distance between the injection points of two burn wounds is greater than 10 cm in the same treatment group or between different treatment groups to avoid interference among the treatments. When the operator suddenly feels a fall or relaxation during deep injection, the needle of the syringe is slightly retracted before starting to inject hUCMSCs, and the process of injecting cells is also very difficult. In particular, this position is the actual location for subcutaneous injection of the hUCMSCs. Furthermore, the local treatment and every other day of delivery frequency of hUCMSCs is based on clinical practice. Then, all burn wounds were wrapped with 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. All animals survived and did not die until the endpoint. They were anesthetized with Pentobarbital sodium for sample collection, and received euthanasia with intravenous barbiturate (90 mg/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 the pig’s back were cut to the muscular layer. The cut site was the surrounding area of center of the round burn wound and the size was approximately 0.3 cm×0.3 cm. Each specimen 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), angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), collagen type-I and type-III detections using the porcine TNF-α ELISA Kits (JHN92498), porcine IL-1β ELISA Kits (JHN92268), porcine IL-10 ELISA Kits (JHN92244), porcine VEGF ELISA Kits (JHN92517), porcine Ang-2 ELISA Kits (JHN92028) (Jinhengnuo, China) and rabbit polyclonal antibodies against porcine Collagen I + Collagen III (ab24135) (Abcam, United Kingdom).

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 cell 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 s. 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 of MPO (ab134132) and CD31 (ab28364) (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 numbers of neutrophils and capillaries in wound tissues were counted in 5 randomly selected fields of 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, Ang-2, VEGF, collagen types I and III were examined using the above ELISA kits 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 [25]. 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 were presented as the mean ± SEM, and using a repeated measure two-way analysis of variance (ANOVA), one-way ANOVA, or Student’s t-test. 95% confidence intervals were calculated and differences were considered statistically significant at *p < 0.05, **p < 0.01 and #p < 0.05, ##p < 0.01.

Results

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 turned black in burn group. By contrast, 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.

Fig. 1.

Fig. 1

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The effect of the low-to-high doses of hUCMSCs on wound healing of the deep partial-thickness burn in Bama miniature pigs. A: The 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. B: The burn wound healing time of burn group and low-to-high doses of hUCMSCs groups was recorded and presented in the histogram. C: The wound healing rate was at 0, 7, 14 and 21 days after the low-to-high doses of hUCMSCs treatments is presented in the corresponding histogram. Values are represented as mean ± SD (n = 8). Asterisk (*) and double asterisk (**) stand for p < 0.05 and p < 0.01 compared with burn group, respectively. A single (#) and double (##) stand for p < 0.05 and p < 0.01, respectively

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, 20.92 ± 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 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 hUCMSCs treatment groups (p < 0.05). The complete healing of wounds, i.e., with good re-epithelization and a residual wound area of 1%, was 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 in 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 cell 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.

Fig. 2.

Fig. 2

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The effect of the low-to-high doses of hUCMSCs on histopathological changes of the deep partial-thickness burn in Bama miniature pigs using HE staining

It is worth noting that the low-to-high doses of hUCMSCs administration could remarkably alleviate 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. The proper inflammatory reaction is beneficial to anti-impairment, while excessive inflammation is very harmful to 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. Neutrophil infiltrations of all treatment groups 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).

Fig. 3.

Fig. 3

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The effect of the low-to-high doses of hUCMSCs on inflammations of the deep partial-thickness burn in Bama miniature pigs. A: The neutrophil infiltration was detected though MPO immunohistochemistry staining, and the red arrows indicated the infiltrated neutrophils in burn wound. B: The results of the quantitative analysis were presented in the corresponding histogram. C-E: The contents of TNF-α, IL-1β and IL-10 in burn wound of all groups were detected after hUCMSCs treatment for 7, 14 and 21 days, respectively. Values are represented as mean ± SD (n = 8). Asterisk (*) and double asterisk (**) stand for p < 0.05 and p < 0.01 compared with burn group, respectively. A single (#) and double (##) stand for p < 0.05 and p < 0.01, respectively

Moreover, the contents of 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 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. 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 (red arrow) was the most in hUCMSCs-4 group (Fig. 4A). It is worth noting that the microvessel numbers in burn group on the 21st were remarkably increased than that on the 7th and 14th days. On the contrary, the microvessel numbers in hUCMSCs-3 and hUCMSCs-4 groups on the 21st were decreased than those on the 7th and 14th days, but these newborn vessels tend to mature and have complete three-dimensional tubular structures (blue arrow). This situation allow blood flow to pass through.The results of the quantitative analysis are presented in the corresponding histogram (Fig. 4B).

Fig. 4.

Fig. 4

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The effect of the low-to-high doses of hUCMSCs on neovascularization of the deep partial-thickness burn wound in Bama miniature pigs. A. The microvessels were detected though CD31 immunohistochemistry staining, and the red and blue arrows indicated the newborn blood capillary in the deep partial-thickness burn wound of Bama miniature pigs. B. The results of the quantitative analysis were presented in the corresponding histogram. C-D. The contents of Ang-2 and VEGF in wound tissues were evaluated by ELISA. Values are represented as mean ± SD (n = 8). Asterisk (*) and double asterisk (**) stand for p < 0.05 and p < 0.01 compared with burn group, respectively. A single (#) and double (##) stand for p < 0.05 and p < 0.01, respectively

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 D).

In general, 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 epidermis and dermis tissues in Masson staining were necrotic, and the collagen fibers were severely destroyed in wound of the burn group. But increasing doses of hUCMSCs get their arrangement becomes more orderly at 7 day treatment. By day 14, the necrotic epidermis had 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 the dermis was still disordered in the hUCMSCs-1 group. However, the effects of hUCMSCs on collagen arrangement regularity and re-epithelialization quality were increased by gradual increasing doses of hUCMSCs administration. Up to day 21, the burn group’s wounds also began to re-epithelialize, but the collagen structure was disordered and irregular. Compared with that in the burn group, the re-epithelialize quality in the epidermis and arrangement of collagen fibers in the dermis in all hUCMSCs groups became better and better by gradually increasing doses of hUCMSCs administration, and the best therapeutic effect was still achieved by 1 × 10^8 dose of hUCMSCs (Fig. 5A).

Fig. 5.

Fig. 5

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The effect of the low-to-high doses of hUCMSCs on collagen deposition and arrangement of the deep partial-thickness burn wound in Bama miniature pigs. A: The collagen fiber structure changes and re-epithelialization qualities were assessed by Masson staining at 7, 14 and 21 days after the low-to-high doses of hUCMSCs treatments. B. The contents of type-I and type-III collagen were detected by ELISA and their ratio is presented in the corresponding histogram. Values are represented as mean ± SD (n = 8). Asterisk (*) and double asterisk (**) stand for p < 0.05 and p < 0.01 compared with burn group, respectively. A single (#) and double (##) stand for p < 0.05 and p < 0.01, respectively

Moreover, collagen types I and III are the main collagen types of healthy skin and the ratio of collagen types I and III determined the 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 the burn group. Compared with those in the burn group, the ratios 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; Table 1 provide all parameters of histological scores. Firstly, re-epithelization scores were all 1 in the burn group, hUCMSCs-1 group and hUCMSCs-2 group, as well as 1–2 in the hUCMSCs-3 group, but 2 in the 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 the hUCMSCs-3 group, but 2 in the 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 the burn group, hUCMSCs-1 group, hUCMSCs-2 group and hUCMSCs-3 group, but 2 in the hUCMSCs-4 group. Fifth, the scar elevation index (SEI) scores were all 0 in the burn group, hUCMSCs-1 group, hUCMSCs-2 group and hUCMSCs-3 group, but 2 in the hUCMSCs-4 group. Collectively, the total scores of all parameters in the 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 perfect 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.

Fig. 6.

Fig. 6

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The influence of low-to-high doses of hUCMSCs on the histology scoring of the deep partial-thickness burn wound in Bama miniature pigs. Visual overview of the histology scoring system illustrating the various phases of wound healing (1–4) from the deep partial-thickness burn to either completely healed or scaring. The overlap of physiological processes that occur as part of healing, are indicated within each of the phases using a visual analog method. The approximate timeframe (days post wounding) that corresponds with each of the phases of healing is indicated at the top of the illustration. The histology score is based on set criteria using various quantitative and semi-quantitative measures to assess each of the physiological parameters in either HE or Masson or MPO + or CD31 + stained sections (refer to Table 1)

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

graphic file with name 13287_2024_4063_Tab1_HTML.jpg

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Discussion

In clinical practice, the treatment of deep partial-thickness burn wounds has always been an very challenging issue [10, 18, 24]. In recent years, MSCs have garnered global attention and have been widely used for promoting wound healing in various types of injuries [26–30]. Particularly, hUCMSCs might ultimately contribute to the clinical application of deep burn wounds through self-multipotency property [31–37]. 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 [19, 38–42]. 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 concerning dosage and discusses the effects of future cell-based therapy. In our low-to-high hUCMSCs-dose treatment in pigs model [17], 6 wound healing parameters were evaluated, and revealed a high dose of 1 × 10^8 cells/20cm2 regenerates the burn wounds most efficaciously, followed by doses of 5 × 10^7, 2 × 10^7, 1 × 10^7cells/20cm2. Moreover, this study is a self-controlled study and might have interference between the treatment groups. The influence on the different local adjacent organs needed further study.

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 high dose of 1 × 10^8 hUCMSCs therapy improved macroscopic wound healing via faster epithelialization, appropriate inflammation, and reduction of scarring [43–45]. Several studies have shown that restricted inflammation of burn is beneficial, and excessive or persistent inflammation incites wound tissue destruction, even nonhealing [46]. In both preclinical and clinical trials, the hUCMSCs were demonstrated to suppress the immune responses in inflammatory cytokines enriched diseases, such as SIRS, infections, and even sepsis [47, 48]. Similarly, our study also showed that hUCMSCs markedly regulated inflammation of burn wounds in pigs through reduced inflammatory cell infiltration, and decreased TNF-α and IL-1β levels, as well as increased IL-10 level.

Furthermore, our data also confirmed that the most beneficial therapy of a high dose of 1 × 10^8 hUCMSCs with pro-angiogenic and fibroproliferative effects increased neo-vascularization and collagen formation, as well as reduced fibrosis [9, 43, 49]. Angiogenesis is a crucial step in burn wound healing process. As the growth of new blood vessels supplies abundant oxygen and nutrients, angiogenesis provide these for nourishing the growing tissues in the wound [50]. Angiopoietin (Ang) and VEGF are significant vascular growth factors. VEGF plays a key role in angiogenesis by stimulating endothelial cell proliferation, migration and organization into tubules [17, 49]. Ang-2 is the best characterized angiopoietins, and is also ligands for the Tie2 receptor tyrosine kinase. Moreover, Ang-2 was proven to be present on endothelial cells and endothelial progenitor cells. Both two growth factors collectively induce new blood vessel formation and modulate maturation of neovascularization [17, 51]. And our findings indicate that injecting hUCMSCs resulted in elevated levels of Ang-2 and VEGF. Ang-2 and VEGF promotion potentially contributed to the process of neovascularization. Moreover, collagen fibers involved in the formation of granulation tissue to fill the wound defects and set the stage for epidermal cell coverage [43, 52]. Collagen type I and type III depositions and their ratios in wounds are important determinant of the wound healing process [44]. Our results also demonstrated that hUCMSCs significantly enhance collagen deposition and increase collagen 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 delivery, 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 [34]. 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) [34]. In a different clinical study [41], 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) significantly decreased the WOMAC and could be a potentially new regenerative treatment for patients with knee OA [40]. Therefore, our study covered the dose range of hUCMSCs mentioned in the above literatures to compare and analyze their efficacy. And the local treatment and delivery frequency of hUCMSCs is based on clinical practice.

Conclusion

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

Acknowledgements

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

Author contributions

Lingying Liu, Xingxia Hao, Jing Zhang, Shaozeng Li, conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing and financial support. Shaofang Han, Peipei Qian, Yong Zhang, Huaqing Yu, Yuxin Kang, Yue Yin, Weiouwen Zhang, Jianmei Chen, Yang Yu, Hua Jiang, Jiake Chai, Huinan Yin and Wei Chai: provision of study material, data analysis, and interpretation. Lingying Liu, Xingxia Hao, Jing Zhang, Shaozeng Li contributed equally to this study. All authors read and approved the final manuscript.

Funding

This work was supported by research projects of Youth Independent Innovation Science Foundation of PLA General Hospital (22QNCZ031), National Natural Science Foundation of China (81701900) and (U22A20355).

Data availability

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

Declarations

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 ), and the application of human umbilical cord mesenchymal stem cells (hUCMSCs) is one of the new techniques.

Consent for publication

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

Competing interests

The authors declare that they have no competing interests.

Footnotes

Liu et al. Different doses of hUCMSCs treated deep burn wound of pigs.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lingying Liu, Xingxia Hao, Jing Zhang and Shaozeng Li contributed equally to this work.

Contributor Information

Lingying Liu, Email: liulingyingyisheng@163.com.

Yong Zhang, Email: zhy-545@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

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

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