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CB-MNCs@ CS/HEC/GP promote wound healing in aged murine pressure ulcer model

Stem Cell Research & Therapy volume 16, Article number: 52 (2025) Cite this article

Abstract

Background

Non-healing pressure ulcers impose heavy burdens on patients and clinicians. Cord blood mononuclear cells (CB-MNCs) are a novel type of tissue repair seed cells. However, their clinical application is restricted by low retention and survival rates post-transplantation. This study aims to investigate the role of thermo-sensitive chitosan/hydroxyethyl cellulose/glycerophosphate (CS/HEC/GP) hydrogel encapsulated CB-MNCs in pressure ulcer wound healing.

Methods

Pressure ulcers were induced on the backs of aged mice. After construction and validation of the characterization of thermo-sensitive CS/HEC/GP hydrogel, CB-MNCs are encapsulated in the hydrogel, called CB-MNCs@CS/HEC/GP which was locally applied to the mouse wounds. Mouse skin tissues were harvested for histological and molecular biology analyses.

Results

CB-MNCs@CS/HEC/GP therapy accelerated pressure ulcer wound healing, attenuated inflammatory responses, promoted cell proliferation, angiogenesis, and collagen synthesis. Further investigation revealed that CB-MNCs@CS/HEC/GP exerted therapeutic effects by promoting changes in cell types, including fibroblasts, endothelial cells, keratinocytes, and smooth muscle cells.

Conclusion

CB-MNCs@CS/HEC/GP enhanced the delivery efficiency of CB-MNCs, preserved the cell viability, and contributed to pressure ulcer wound healing. Thus, CB-MNCs@CS/HEC/GP represents a novel therapeutic approach for skin regeneration of chronic wounds.

Introduction

Pressure ulcers (PUs) are a frequent public health problem worldwide, growing prevalence as the population ages in recent years [1]. PUs are torturous, costly and negatively impacts the quality of life of the patient and their caregivers. There are more cases as life expectancy increases. Despite numerous measures used to prevent and cure pressure ulcers, either some cases are resistant to treatment, or the healing process is slower than expected, prevalent among the elderly and critically ill patients [2]. Thus, effective management of PUs is crucial.

CB-MNCs, harvested from neonatal umbilical cord blood, are composed mainly of lymphocytes and monocytes, and are rich in various types of stem cells including hematopoietic stem cells, endothelial progenitor cells, lymphoid progenitor cells, embryonic-like stem cells, and mesenchymal stem cells [3]. Due to their low immunogenicity, allogeneic CB-MNCs do not trigger an immune response and can survive longer in the body. These primitive stem cells from cord blood have high proliferative and differentiation potential, allowing them to integrate into lesion sites and differentiate into necessary cell types, thereby replacing damaged cells and reconstructing functional cellular networks, which helps in restoring tissue function [4]. Recent studies have confirmed the safety and potency of CB-MNCs in treating various diseases [5, 6], yet research on their use in PUs is still lacking.

CB-MNCs, due to their abundant source, relatively low cost, and multiple differentiation potentials, have become potential seed cells for tissue repair. Immune cells within CB-MNCs can secrete immunoregulatory and chemotactic factors, such as interleukins and Vascular Endothelial Growth Factor, reducing inflammation and promoting wound healing. The endothelial progenitor cells can differentiate into endothelial cells, contributing to capillary network formation and secreting angiogenic factors to enhance angiogenesis [7, 8]. However, using CB-MNCs in wound healing is faced with challenges. Factors such as mechanical damage, inflammation, edema, immune responses, and pH changes at wound site can significantly reduce the survival rates of transplanted cells [9]. Additionally, leakage of cell suspensions leads to a loss of transplanted cells, further reducing their concentration and impacting therapeutic outcomes [10]. Current strategies involve combining seed cells with biomaterials to extend their retention at the wound site without affecting their biological activity, which has become a focal point in developing seed cell-based therapeutic approaches.

Hydrogel can mimic the microenvironment of stem cells, providing an appropriate spatial environment for their growth. Additionally, hydrogel can effectively promote the release of growth factors and other nutrients, demonstrating tremendous potential in three-dimensional culture of stem cells [11]. Studies had shown that engineering fibrin hydrogels promoted the wound healing potential of mesenchymal stem cells spheroids [12]. Moreover, hydrogel-combined with bone marrow mesenchymal stem cells or adipose-derived stem cells significantly alleviates inflammation and promotes epithelial cell proliferation, re-epithelialization, and wound closure in severe wounds such as diabetic ulcers and burns [13, 14]. However, there is no research confirming the effect of CB-MNCs@CS/HEC/GP therapy on PU wound.

CS/GP hydrogel exhibits excellent biocompatibility and serves as a good carrier for cells and drugs. Particularly, the unique thermosensitive effect of CS/GP allows for the preparation of injectable hydrogels. These hydrogels remain liquid at room temperature but quickly transform into a gel state upon transplantation to the damaged site due to temperature increase, effectively filling the wound [15]. Compared to traditional implantation methods, this thermo-sensitive hydrogel can more easily cover irregular wounds. However, it has some drawbacks, such as the requirement of high GP concentration to rapidly form a gel at physiological temperature, which may exert certain toxicity to the body and cells [16, 17]. Incorporating HEC into CS/GP hydrogel not only reduces the usage of GP but also enhances the performance of the gel [18]. HEC, prepared by the reaction of alkaline cellulose with ethylene oxide, possesses properties such as thickening, suspension, adhesion, emulsification, dispersion, film formation, moisture retention, and colloidal protection, with wide applications in medical and food fields [19]. Adding HEC helps to reduce the negative impact of high osmotic pressure of GP on cells, thereby improving the overall biocompatibility and safety of the hydrogel. However, the optimal ratio of HEC has yet to be determined.

Therefore, we aim to explore a wound-friendly hydrogel carrying CB-MNCs to promote PUs healing. In this study, a series of novel thermo-sensitive hydrogels were obtained by mixing different ratios of HEC and CS/GP, and the hydrogel was characterized accordingly. CB-MNCs were encapsulated in the hydrogel, and their role and mechanism in promoting wound healing of PUs in mice were explored through histological and molecular biology methods.

Materials and methods

Statement

The work has been reported in line with the ARRIVE guidelines 2.0.

Materials

HEC (Cat No. 09368-100G), GP (Cat No. 50020-100G) and D-gal (Cat No. G5388-100G) were purchased from Sigma-Aldrich. Chitosan (Cat No. MS0810-100G), deacetylation ≥ 95%, molecular weight ~ 500,000, was purchased from Maokangbio. Diluted hydrochloric acid (Cat No. QBWECD433034), 0.1 mol/L, was purchased from Codow. Calcein-AM/PI Double Stain Kit (Cat No. 40747ES76) and Cell Counting Kit-8 (CCK8, Cat No. 40203ES60) were purchased from Yeasen. Modified Massons Trichrome Stain Kit (Cat No. G1346), Crystal Violet Ammonium Oxalate Solution 0.1% (Cat No. G1063), and Penicillin–Streptomycin- Amphotericin B Solution (Cat No. P7630) were purchased from Solarbio. DAB chromogenic reagent kit (Cat No. G1212-200T) were purchased from Servicebio. TSA Fluorescence Triple Staining Kit (Cat No. RK05903) was purchased from ABclonal. Anti-beta-galactosidase (GLB1, Cat No. 15518–1-AP), anti-P53 (Cat No. 10442–1-AP), anti-Interleukin-10 (IL-10, Cat No. 60269–1-IG), anti-Tumor necrosis factor-α (TNF-α, Cat No. 60291–1-IG), anti-vimentin (Cat No. 60330–1-IG), anti-collagen1 (Cat No. 14695–1-AP), anti-CD31 (Cat No. 28083–1-AP), anti-keratin5 (Cat No. 28506–1-AP), anti-alpha-smooth muscle actin (α-SMA, Cat No. 14395–1-AP) and anti-GAPDH (Cat No. 80570–1-RR) were purchased from Proteintech. anti-CD11c (Cat No. 97585S) and anti-CD206 (Cat No. 24595S) were purchased from Cell Signalling. Anti-human nucleoli (Cat No. ab190710) and DAPI(Cat No. ab104139) were purchased from Abcam. Alexa fluor 488 (Cat No. A23220), Alexa fluor 594 (Cat No. A23410) and anti-Ki67 (Cat No. ABM40064) were purchased from Abbkine. Stripping Buffer (Cat No. CW0056M) was purchased from CWBIO. Roswell Park Memorial institute 1640 (RPMI 1640, Cat No. C11875500BT) was purchased from Gibco. Phosphate-buffered saline (PBS, Cat No. MA0015), fetal bovine serum (FBS, Cat No. PWL114) and 0.25% trypsin digest contained EDTA (Cat No. MA0233) were purchased from Meilunbio. Transwell cell chamber (Cat No. 3422) was purchased from corning. Isoflurane (Cat No. R510-22–10) was purchased from RWD.

Synthesis of CS/HEC/GP

CS (400 mg) and HEC (80 mg) were sterilized under ultraviolet light for 30 min on the clean bench. They were then dissolved in 18 mL of 0.1 M hydrochloric acid and stirred with a magnetic stirrer for 24 h. Impurities were removed by centrifuging at 14,000 rpm for 20 min, and the supernatant (CS/HEC solution) was collected. Subsequently, GP powder was dissolved in distilled water to prepare the GP solution. After pre-cooling both solutions at 4 °C for 10 min, the GP solution (2 mL, 300 mg/mL) was gradually added dropwise into the CS-HEC solution, stirring continued until a clear CS/HEC/GP solution formed. Finally, this solution was placed in a 37 °C water bath to prepare the CS/HEC/GP, with final concentrations of CS, HEC, and GP being 2%, 0.4%, and 3% (W/V), respectively.

Characterization of physical properties of hydrogel

Detection of gelling time and water content of hydrogel

1 mL of the prepared hydrogel precursor solution (before gelling) was transferred into a 5 mL EP tube, which was then placed in a constant temperature water bath at 37 °C. The formation of the gel was checked every 30 s by slightly tilting or completely inverting the EP tube. If the hydrogel maintained its shape for more than 60 s during tilting or inversion, it was considered that the gel had formed, and the time required for gelation was recorded. After gelation, the hydrogel was removed from the water bath and the surface moisture was absorbed with filter paper. The wet weight (Ww) of the hydrogel was then measured. The hydrogel was subsequently freeze-dried, and its dry weight (Wd) was measured. Finally, the water content of the hydrogel was calculated using the formula MC = [(Ww—Wd) / Ww] × 100%.

Degradation characteristics of hydrogel in vitro

After gel formation, the hydrogel mass was weighed and recorded as W0. The hydrogel was then immersed in PBS at 37 °C for constant temperature soaking. On days 3, 7, and 14, the surrounding water of hydrogel was absorbed with filter paper. The mass of the hydrogel was recorded again as Wt. The mass retention rate “L” of the hydrogel was calculated using the formula L = (W0—Wt) / W0 × 100%.

Toxicity analysis of hydrogel

The cytotoxicity of the hydrogel was evaluated using the CCK-8 assay. The hydrogel was soaked in RPMI 1640 medium at 37 °C in an incubator for 24 h, and the supernatant was collected. CB-MNCs were seeded in a 96-well plate at a density of 3 × 104 cells/mL and cultured with the supernatant for 24 h. Afterwards, 100μL of solution containing 10% CCK-8 was added, and the mixture was incubated in the dark for 2 h. The absorbance was measured at 450 nm.

CB-MNCs extraction and characterization

CB-MNCs were provided by Shandong Qilu Stem Cell Engineering Company Limited. In brief, human umbilical cords from full-term, complication-free deliveries were used, and umbilical cord blood was collected from the umbilical vein immediately after birth. Specifically, the umbilical cord blood samples were received and disinfected, the blood bags were sealed and disinfected using a heat sealer. The umbilical cord blood was transferred to 50 mL centrifuge tubes and centrifuged at 900 g for 15 min. The upper layer of plasma was refrigerated for later use. The lower layer of blood cells was mixed evenly with saline and then separated by centrifugation at room temperature through a Ficoll layer to isolate white blood cells. The collected white cell layer was washed and resuspended in saline, followed by cell counting, diameter distribution measurement, cell viability assessment, endotoxin testing, and surface antigen identification. The cells were mixed with plasma in a specific ratio in cryoprotectant, precooled to 4 °C, and then cryopreserved in liquid nitrogen. After cell resuscitation, the viability of the revived cells was assessed using the trypan blue exclusion test. Under a microscope, live cells (unstained) and dead cells (stained) were counted. The survival rate was calculated as follows: Survival Rate (%)=Number of Live Cells/Total Number of Cells×100.

CB-MNCs cultured inside hydrogel and tested for survival status

After recovery, CB-MNCs were resuspended in the precursor solution of the hydrogel to adjust the cell density to 1 × 106 cells/mL. The mixture was promptly aliquoted into 24-well plates and placed in a 37 °C cell culture incubator for 10 min to allow gelation. After gelation, RPMI 1640 medium containing 10% FBS was added, and the plates were further incubated at 37 °C with 5% CO2. The medium was changed once after 30 min and subsequently every 24 h. Live/dead fluorescence staining was performed on the CB-MNCs cultured within the hydrogel at 1, 3, 7, and 14 days.

Transwell experiment to Detect of CB-MNCs migration rate

To evaluate the effect of CS/HEC/GP on CB-MNCs migration, Transwell migration assays were performed with two groups: the CB-MNCs control group and the CB-MNCs@CS/HEC/GP experimental group. CB-MNCs were resuspended in the CS/HEC/GP precursor solution at a density of 5 × 105 cells/mL. A 100 μL aliquot of the cell mixture was placed in the upper Transwell chamber and allowed to gel at 37 °C for 10 min. After gelation, 100 μL of RPMI 1640 medium was added to the upper chamber, and 500 μL of RPMI 1640 with 10% FBS was added to the lower chamber. In the control group, CB-MNCs were directly suspended in RPMI 1640 and added to the upper chamber. Both setups were incubated at 37 °C with 5% CO2, and migrated cells in the lower chamber were counted at 3, 6, 12, and 24 h.

Establishment and treatment of PUs model in aged mice

Animal care and experimental procedures were approved by the Ethics Committee of the School of Nursing and Rehabilitation, Shandong University (Approval No: 2023-D-013). Forty-eight 8-week-old male C57BL/6JNifdc mice from Vital River were selected to evaluate the therapeutic effects of CB-MNCs and CB-MNCs@CS/HEC/GP.

The mice were housed in the animal facility of the Center for Model Animal Research, Shandong University, under controlled conditions: 22–25 °C ambient temperature, 12-h light/dark cycle, and access to adequate food and water. An aged mouse model was established through daily subcutaneous injections of D-galactose (1000 mg/kg in 0.9% saline) for 8 weeks [20]. The control group (n = 24) received an equivalent volume of saline. PUs were induced on the backs of the mice following a method similar to previous studies [21, 22]. Briefly, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg), and hair was removed from the back using depilatory cream. Four circular ceramic magnets (12 mm diameter) were then applied to the back skin for 12 h to create one cycle of ischemia–reperfusion (IR). After three IR cycles and a 7-day recovery, PUs were formed.

Exclusion criteria included: (1) no significant increase in GLB1 and P53 expression after 8 weeks of D-gal injection; (2) failure to develop second-degree or higher PUs after IR cycles; (3) fused or irregularly sized PUs after IR cycles. Mice showing severe infection, persistent pain, or distress were humanely euthanized (by placing them in a chamber with an isoflurane-soaked cotton ball, followed by cervical dislocation).

To minimize potential confounders, the following strategies were employed: (1) Cage locations were randomized within the facility to avoid location-based biases; (2) The order of treatments was randomized. For example, to control for wound site effects on healing, we rotated treatment applications across different PUs sites on different mice. Specifically, the CB-MNCs@CS/HEC/GP group received 100 μL of the CB-MNCs/CS/HEC/GP mixture (1 × 105 cells/μL) applied to each wound, while the CB-MNCs group received 100 μL of the CB-MNCs/PBS mixture with the same cell concentration. The CS/HEC/GP group received 100 μL of the CS/HEC/GP mixture alone, and the control group was treated with 100 μL of PBS.

Wound area was measured and recorded daily over a two-week observation period, and wound imaging was performed at specific time points. The wound closure rate was calculated using ImageJ software (National Institutes of Health) with the formula: Wound closure rate (%) = (A0—At) / A0 × 100%, where A0 is the initial wound area and At is the wound area at the time of measurement.

Immunohistochemistry and immunofluorescence staining

Skin wound tissues were collected on post-treatment days 3, 7, and 14, washed with PBS, fixed in 4% formaldehyde, dehydrated through a graded series of ethanol, embedded in paraffin, and sectioned for histological and immunohistochemical analyses. Hematoxylin and eosin (HE) staining was performed on sections from day 14 samples to observe general tissue morphology, while Masson’s trichrome staining was conducted on sections from day 3 and day 7 samples to evaluate collagen deposition and wound remodeling.

Immunohistochemical analysis was performed on day 7 sections, which were rehydrated, blocked, and incubated with anti-IL-10 (1: 250; Proteintech) and anti-TNF-α (1: 400; Proteintech) overnight at 4 °C, followed by incubation with HRP-labeled secondary antibodies at room temperature. DAB substrate was used for color development, and hematoxylin was applied for counterstaining.

Immunofluorescence staining was conducted on sections from days 3 and 7 to assess inflammatory cell markers using anti-CD11c (1: 200; Proteintech) and anti-CD206 (1: 800; Proteintech), as well as to evaluate newly formed blood vessels in regenerating tissue using anti-CD31 (1: 400; Proteintech). Additional markers, including Ki67 (1: 250; Abbkine), vimentin (1: 1000; Proteintech), α-SMA (1: 1600; Proteintech), Keratin5 (1: 500; Proteintech), and Nucleoli to human (1: 100; Abcam), were analyzed on day 7 sections. Alexa Fluor 488 and Alexa Fluor 594 (Abbkine) labeled secondary antibodies and DAPI (Beyotime Biotechnology) were used for visualization. Quantitative analysis of positive staining cells was conducted using ImageJ software (ver. 1.53q).

Western blotting

Seven days post-treatment, skin tissue samples were collected, snap-frozen in liquid nitrogen, and stored at -80 °C for protein extraction. Upon thawing on ice, the tissue was minced and homogenized in RIPA buffer containing a protease inhibitor. The tissues were lysed using an ultrasonicator and then incubated on ice for 30 min. This was followed by centrifugation at 12,000 rpm for 15 min, and the supernatant was collected for protein extraction. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h and then incubated overnight at 4 °C with antibodies against Collagen (1: 2000; Proteintech), CD31 (1: 4000; Proteintech), Keratin5 (1: 3000; Proteintech), α-SMA (1: 4000; Proteintech), and GAPDH (1: 4000; Proteintech). The membrane was then incubated with HRP-linked secondary antibodies for 1 h. ECL chemiluminescent substrate was used for visualization, and chemiluminescence imaging equipment was used for detection.

Statistical analysis

Data were presented as the mean ± standard deviation (SD) of at least five replicates. All statistical analyses were performed using SPSS software (version 27.0). For comparisons between two groups, independent two-sample t-tests were conducted to determine if there were significant differences between the means of the groups. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was used. Normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was tested using Levene's test. If these assumptions were met, one-way ANOVA was conducted, followed by the Least Significant Difference (LSD) test for post-hoc analysis to identify specific group differences. Three-way ANOVA was utilized to evaluate the interaction effects among multiple factors, including cell therapy (CB-MNCs vs. Non-CB-MNCs), hydrogel application (CS/HEC/GP vs. Non- CS/HEC/GP), and age factors (aged vs. young). This analysis helped to identify both the main effects and interaction effects among the factors. Simple main effects were further analyzed when significant interactions were found. Statistical significance was defined as a p-value < 0.05. Results from ANOVA and t-tests are reported with corresponding p-values. If the assumptions of normality or homogeneity of variances were violated, appropriate non-parametric tests were used.

Results

Characterization of hydrogel physical properties

In this study, we evaluated the gelation properties of CS/GP hydrogels and composite hydrogels containing different concentrations of HEC at 25 °C (room temperature) and 37 °C. Under the condition of 37 °C, except for the pre-gel solution with a 3% GP concentration and without HEC, which did not form a gel, all other hydrogel samples remained liquid at room temperature and rapidly transitioned to a gel state at 37 °C, demonstrating their excellent temperature sensitivity (Fig. 1A). The addition of HEC significantly shortened the gelation time of the hydrogel, and this gelation time decreased with increasing HEC concentration, showing a negative correlation (Fig. 1B). The water content of all hydrogels exceeded 90%, and with the increase of HEC concentration, the water content increased, suggesting the high water-retention property of CS/HEC/GP (Fig. 1C). We observed that the addition of HEC slowed down the degradation rate of the hydrogel, showing a negative correlation between degradation rate and HEC concentration. Nevertheless, CS/HEC/GP still exhibited good degradation ability compared to CS/GP hydrogels (Fig. 1D).

Fig. 1
figure 1

Characterization of the physical properties of hydrogel with different formulations: A Schematic representation of hydrogel gelation at 25 °C and 37 °C (CS/GP(5%) group: Containing 2%CS and 5% GP; CS/GP(3%) group: Containing 2%CS and 3% GP; + 0.2%HEC group: Containing 2%CS, 3% GP and 0.2% HEC; + 0.4%HEC group: Containing 2%CS, 3% GP and 0.4% HEC; + 0.6%HEC group: Containing 2%CS, 3% GP and 0.6% HEC); B Statistical chart of gelation time of hydrogel at 37 °C; C Statistical chart of water content in hydrogel; D Statistical chart of mass retention rate of hydrogel

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Characterization of CB-MNCs

The CB-MNCs used in this experiment were identified by flow cytometry and met the CB-MNCs identification criteria [23]. The CB-MNCs demonstrated excellent viability post-resuscitation, with a cell survival rate exceeding 90% (Fig. 2A, B). The cell diameters distributed mostly in the range of 7–14 µm (Fig. 2C). The endotoxin test performed using the gel clot method showed an endotoxin level of < 0.25 EU/mL (See Table S1), indicating that the CB-MNCs were of high purity and free from significant endotoxin contamination, ensuring the safety and reliability of the cells for experimental use. Phenotypic analysis of the cells (Fig. 2D–H) showed that CB-MNCs consisted of monocytes (CD45+/CD14+), lymphocytes (CD45+/CD3+, CD45+/CD19+, CD45+/CD56+), and a small number of stem cells (CD34+). Figure 2I showed that the majority of cells were CD3+CD4+ helper T cells, accounting for the largest proportion, followed by monocytes (CD14+), with the majority being M1 type and a smaller proportion of M2 type. Other significant populations included CD3−CD16+CD56+ NK cells, CD3+CD8+ cytotoxic T cells, and CD3−CD19+ B cells. Smaller proportions of double-positive T cells (CD3+CD4+CD8+), hematopoietic stem cells (CD34+), and endothelial progenitor cells (CD34+CD133+CD309+) were also present.

Fig. 2
figure 2

Characterization of CB-MNCs: A Ordinary optical microscopy image of CB-MNCs; B Survival rate of CB-MNCs; C Diameter distribution chart of CB-MNCs (horizontal axis represents the cell diameter (μm), and the vertical axis represents the number of cells); D–H Results of surface antigen identification for CB-MNCs; I Chart of the cellular composition ratio of CB-MNCs

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Biocompatibility tests for hydrogel

After 1 day of cultivation within the hydrogel, except for the CS/GP group, the cell viability in all other groups was above 90%, while the cell viability in the CS/GP group was less than 60%, indicating that CS/HEC/GP were conducive to the survival of CB-MNCs within them. As the cultivation time extended, the cell viability decreased in all groups. However, even after cultivation for 14 days, the cell viability in all CS/HEC/GP groups remained above 80%. Interestingly, in the CS/HEC/GP group containing 0.4% HEC, the cell viability was significantly higher than that in the group containing 0.2% HEC (p < 0.001), as shown in Fig. 3A, B. We found that the CS/HEC/GP did not significantly affect the migration rate of CB-MNCs. At the detection points of 3 h, 6 h, 12 h, and 24 h, although the number of migrating cells in the CB-MNCs group was slightly higher than that in the CB-MNCs@CS/HEC/GP group, this difference was not statistically significant (Fig. 3C, D). Toxicity analysis of the extracts from CS/HEC/GP containing different concentrations of HEC was conducted before transplantation (Fig. 3E). The results showed no significant difference between the extracts of CS/HEC/GP with varying HEC concentrations and the control group(RPMI 1640 medium), indicating that the introduction of HEC did not affect the biological safety of the hydrogel system.

Fig. 3
figure 3

Biocompatibility tests for hydrogel: A Live/Dead fluorescence staining image, scale line 250 µm (CS/GP(5%) group: Containing 2%CS and 5% GP; + 0.2%HEC group: Containing 2%CS, 3% GP and 0.2% HEC; + 0.4%HEC group: Containing 2%CS, 3% GP and 0.4% HEC; + 0.6%HEC group: Containing 2%CS, 3% GP and 0.6% HEC); B Statistical chart of the proportion of live cells in a single field of view, ***p < 0.001 vs. CS/GP(5%) group, †p < 0.05 vs. + 0.2% HEC group, †††p < 0.001 vs. + 0.2% HEC group; C Transwell assay image, scale line 250 µm; D Quantification of migrated cells in (A); E Cell viability of CB-MNCs after 1 day of culture in 100% CS/HEC/GP extract medium

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Establishment of PUs model in aged mice

We established an aged model by treating male C57BL mice with D-gal and analyzed the expression levels of aging-related proteins P53 and GLB1. Western blot results (Fig. 4A, B) showed a significant increase in the protein levels of P53 and GLB1 in the D-gal treated aged group compared to the young control group (PBS-treated) (p < 0.001). By placing 2 ceramic magnets on each side of the mice dorsal skin fold and intermittently applying pressure (Fig. 4C), ulcer reactions were observed in the skin after three ischemia–reperfusion (IR) cycles and 7 days of rest, resulting in the formation of four PUs wounds. These PU wounds exhibited high consistency in size (Fig. 4D).

Fig. 4
figure 4

Establishment of pressure Injury model in aged mice: A Western blot analysis of P53 and GLB1 protein expression, N = 5 per group (Full-length blots are presented in Supplementary Fig. S1); B Statistical results of (A); C Four ceramic magnets applying constant pressure to the skin; D, Schematic diagram of a PU wound

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CB-MNCs@CS/HEC/GP therapy accelerates PUs closure

At days 3, 7, and 14 post-treatment, both in young and aged murine models, the CB-MNCs and CB-MNCs@CS/HEC/GP groups demonstrated a significantly higher wound healing rate compared to the CS/HEC/GP and PBS groups. Specifically, the CB-MNCs@CS/HEC/GP group showed a significantly higher wound healing rate than the CS/HEC/GP group (p < 0.05), the CB-MNCs group (p < 0.01), and the PBS group (p < 0.01) (Fig. 5A, B).

Fig. 5
figure 5

CB-MNCs@CS/HEC/GP therapy accelerated the healing of PUs: A Representative images showing the effects of different treatments on wound healing in young and aged mice, scale = 2.5 mm; B Comparison of wound healing rates; C Statistical area under the healing curve from (B). Data are mean ± SD of (n = 5 per group) *p < 0.05, **p < 0.01, ***p < 0.001 CB-MNCs@CS/HEC/GP vs. CS/HEC/GP group. †p < 0.05, ††p < 0.01, †††p < 0.001 CB-MNCs@CS/HEC/GP vs. CB-MNCs group, #p < 0.05, ##p < 0.01, ###p < 0.001 CB-MNCs@CS/HEC/GP vs. PBS group

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To further evaluate the combined effects of cell therapy, hydrogel application, and age on wound healing, we performed a three-factor ANOVA on the Area Under the Healing Curve (AUHC). In the young murine group, both the CB-MNCs (p < 0.05), CS/HEC/GP (p = 0.021), and CB-MNCs@CS/HEC/GP (p < 0.001) treatments significantly increased the AUHC compared to the control group (CB-MNCs- Hydrogel-), with the CB-MNCs@CS/HEC/GP group showing a significantly higher AUHC than the CB-MNCs group (p = 0.009). In the aged group, three treatments also significantly increased the AUHC compared to the control group (CB-MNCs, p = 0.007; CS/HEC/GP, p < 0.001; CB-MNCs@CS/HEC/GP, p < 0.001), and the AUHC was significantly higher in the CB-MNCs@CS/HEC/GP group compared to the CS/HEC/GP group (p = 0.021) and the CB-MNCs group (p < 0.001) (Fig. 5C). Notably, after treatment with CB-MNCs@CS/HEC/GP, there was no statistically significant difference in AUHC between the aged and young groups. The three-way ANOVA revealed no significant interaction among the three factors (cells, hydrogel, and age). However, there was a significant two-factor interaction between CB-MNCs and hydrogel, indicating a synergistic effect in increasing the AUHC (p = 0.012) (See Table S2).

CB-MNCs@CS/HEC/GP therapy inhibits inflammatory response

Immunofluorescence analysis was used to evaluate the expression levels of CD11c (M1-like macrophage marker) and CD206 (M2-like macrophage marker) (Fig. 6A). On day 7, CB-MNCs@CS/HEC/GP treatment in the young group significantly decreased CD11c and increased CD206 expression, reducing the M1/M2-like macrophage ratio compared to the control group (CB-MNCs- Hydrogel-) (p = 0.007). In the aged group, both CB-MNCs (p = 0.006) and CB-MNCs@CS/HEC/GP (p = 0.025) treatments significantly lowered the M1/M2-like ratio compared to the control group (CB-MNCs- Hydrogel-). Across both age groups, the M1/M2-like ratio was lower with CB-MNCs@CS/HEC/GP than with CS/HEC/GP or CB-MNCs alone. No significant difference in M1/M2-like ratio was observed between the aged and young groups after CB-MNCs@CS/HEC/GP treatment (Fig. 6B). Three-factor ANOVA revealed significant interactions between cells, CS/HEC/GP, and age (p = 0.045), showing a synergistic effect between CB-MNCs and CS/HEC/GP, though moderated by age (See Table S2).

Fig. 6
figure 6

CB-MNCs@CS/HEC/GP therapy reduced the M1/M2-like Mφ ratios: A IF staining of CD11c and CD206 to assess the number of M1-like Mφ and M2-like Mφ in wound tissue, scale line = 50 µm; B Statistical analysis of the M1/M2-like Mφ ratios in wound tissue from (A). Data are mean ± SD of (n = 5 per group)

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To further explore the anti-inflammatory effects of CB-MNCs@CS/HEC/GP, we measured TNF-α (pro-inflammatory) and IL-10 (anti-inflammatory) levels by immunohistochemistry (Fig. 7A, B). In both young and aged groups, CB-MNCs@CS/HEC/GP significantly reduced TNF-α and increased IL-10 levels compared to the control group (CB-MNCs- Hydrogel-) (all p < 0.001). Across both age groups, TNF-α was significantly lower and IL-10 higher with CB-MNCs@CS/HEC/GP than with CS/HEC/GP or CB-MNCs alone. No significant difference was observed in TNF-α or IL-10 levels between the aged and young groups after CB-MNCs@CS/HEC/GP treatment (Fig. 7C, D). Three-way ANOVA showed a synergistic effect of CB-MNCs and CS/HEC/GP in reducing TNF-α (p = 0.007) and increasing IL-10 (p = 0.031), without significant three-way interactions among cells, hydrogel, and age (See Table S2).

Fig. 7
figure 7

CB-MNCs@CS/HEC/GP therapy decreased TNF-α levels and increased IL-10 levels: A IHC staining for TNF-α to assess pro-inflammatory levels in wound tissue, scale line = 200 µm; B IHC staining for IL-10 to assess anti-inflammatory levels in wound tissue, scale line = 200 µm; C Statistical analysis of pro-inflammatory levels in wound tissue from (A); D Statistical analysis of anti-inflammatory levels in wound tissue from (B). Data are mean ± SD of (n = 5 per group)

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CB-MNCs@CS/HEC/GP therapy promotes cell proliferation and angiogenesis

To assess angiogenesis and cell proliferation, Ki67 and CD31 co-immunostaining was conducted on day 3 of treatment (Fig. 8A). In both young and aged mice, CB-MNCs@CS/HEC/GP treatment significantly increased Ki67 expression, indicating enhanced cell proliferation, compared to the control (CB-MNCs-Hydrogel-) (both p < 0.001). Ki67 levels were also significantly higher in the CB-MNCs@CS/HEC/GP group than in the CS/HEC/GP or CB-MNCs groups (all p < 0.001). No significant difference in Ki67 expression was observed between young and aged groups treated with CB-MNCs@CS/HEC/GP (Fig. 8B). A significant interaction between CB-MNCs and CS/HEC/GP (p = 0.022) suggested a synergistic effect in promoting Ki67 expression (See Table S2).

Fig. 8
figure 8

CB-MNCs@CS/HEC/GP therapy promoted cell proliferation, angiogenesis, and re-epithelialization: A IF staining of Ki67 to assess the proliferative capacity of cells in wound tissue, scale line = 50 µm; B Statistical analysis of the proliferative capacity of wound tissue from (A); C IF staining of CD31 to assess the level of angiogenesis in wound tissue, scale line = 50 µm; D Statistical analysis of angiogenesis in wound tissue from (C). Data are mean ± SD of (n = 5 per group)

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CD31 staining was conducted to assess angiogenesis. On day 3, revealing that CB-MNCs@CS/HEC/GP treatment significantly increased new blood vessel formation in both young and aged mice compared to the control (CB-MNCs- Hydrogel-) (both p < 0.001) (Fig. 8C). The CB-MNCs@CS/HEC/GP group also exhibited a significantly greater number of blood vessels than either the CS/HEC/GP (young: p < 0.001; aged: p = 0.017) or CB-MNCs group alone (young: p = 0.002; aged: p < 0.001). Similar results were observed on day 7, with no statistically significant difference in angiogenesis between young and aged groups treated with CB-MNCs@CS/HEC/GP (Fig. 8D). Three-factor ANOVA indicated a significant interaction between CB-MNCs and CS/HEC/GP (p = 0.023), confirming a synergistic effect in promoting angiogenesis (See Table S2).

CB-MNCs@CS/HEC/GP therapy Increased collagen deposition

Immunofluorescence analysis on day 7 post-treatment was conducted to evaluate Vimentin (fibroblast marker) and Vimentin/α-SMA (myofibroblast markers) expression (Fig. 9A). CB-MNCs@CS/HEC/GP treatment significantly increased Vimentin+ fibroblasts in both young and aged groups compared to the control (CB-MNCs-Hydrogel-) (p < 0.001). For Vimentin+/SMA+ myofibroblasts, no significant changes were seen in the young group, but both CB-MNCs (p < 0.05) and CB-MNCs@CS/HEC/GP (p < 0.001) treatments increased expression in the aged group compared to control. Vimentin+/SMA+ myofibroblast levels after CB-MNCs@CS/HEC/GP treatment in aged mice were similar to those in young mice (Fig. 9B, C). Three-way ANOVA showed no significant three-way interaction, but a two-way interaction between cells and hydrogel was significant (p = 0.04), indicating a synergistic effect (See Table S2).

Fig. 9
figure 9

CB-MNCs@CS/HEC/GP therapy increased collagen deposition: A IF staining for Vimentin and α-SMA to assess the number of fibroblasts and myofibroblasts in wound tissue on day 7 post-treatment, scale line = 50 µm; B Statistical analysis of fibroblast counts from (A); C Statistical analysis of myofibroblast counts from (A); D Comparison of collagen deposition in wound tissue on days 3 and 7 of treatment, scale line = 200 µm; E, Statistical analysis of collagen deposition in wound tissue from (D). Data are mean ± SD of (n = 5 per group)

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Masson’s trichrome staining on day 7 revealed increased collagen deposition in both young and aged groups after CB-MNCs@CS/HEC/GP treatment compared to control (CB-MNCs-Hydrogel-) (p < 0.001) (Fig. 9D). Collagen levels were higher in CB-MNCs@CS/HEC/GP than in CS/HEC/GP (young: p = 0.057; aged: p = 0.017) and CB-MNCs groups (young: p = 0.009; aged: p < 0.001). Collagen deposition was similar between aged and young groups following CB-MNCs@CS/HEC/GP treatment (Fig. 9E). Three-way ANOVA indicated an interaction between cells and hydrogel on days 3 (p = 0.026) and 7 (p = 0.019), suggesting a synergistic effect in promoting collagen deposition (See Table S2).

CB-MNCs@CS/HEC/GP promotes healing of PUs by promoting changes in cell types within the PUs wound

Histological results showed that, regardless of age group, the dermal structure was more intact after CB-MNCs@CS/HEC/GP treatment compared to the CS/HEC/GP group, CB-MNCs group, and PBS group. Additionally, the CB-MNCs@CS/HEC/GP group induced a greater number of follicles around the wound site compared to the other three groups, and the healing effect was histologically closer to the control group (normal skin group) (Fig. 10A).

Fig. 10
figure 10

CB-MNCs@CS/HEC/GP promotes healing of PUs by promoting changes in cell types within the PUs wound: A HE staining on day 14 of treatment, scale line = 500 µm; B IF detection of CB-MNCs-induced cell type changes, scale line = 50 µm; C Western Blot analysis of Collagen1, CD31, Keratin5, and α-SMA protein expression levels under different treatment conditions (Full-length blots are presented in Fig. S1); C–F Statistical analysis of Collagen1, CD31, Keratin5, and α-SMA protein levels. Data are mean ± SD of (n = 5 per group)

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To investigate the mechanism of action of CB-MNCs@CS/HEC/GP in PUs treatment, we used Nucleoli as a specific marker for human cell nuclei and performed immunofluorescence staining for Vimentin, CD31, keratin5, and α-SMA to assess the potential of CB-MNCs to promote changes in cell types, specifically fibroblasts, endothelial cells, keratinocytes, and smooth muscle cells. The results indicated that CB-MNCs@CS/HEC/GP treatment effectively induced the presence of these cell types (Fig. 10B).

Western blot analysis of COL1, CD31, keratin5, and α-SMA protein levels was performed to further investigate the role of CB-MNCs@CS/HEC/GP in PUs treatment (Fig. 10C). The results showed that, in the young group, compared to the control group (CB-MNCs- Hydrogel-), CB-MNCs@CS/HEC/GP treatment significantly promoted the expression of Collagen1, CD31, Keratin5, and α-SMA proteins (Collagen1, p < 0.01; CD31, p < 0.001; Keratin5, p < 0.01; α-SMA, p < 0.01), all significantly higher than the CS/HEC/GP and CB-MNCs treatment groups. Similar results were observed in the aged group after the same treatment (Collagen1, p < 0.05; CD31, p < 0.001; Keratin5, p < 0.001; α-SMA, p < 0.01). Three-factor ANOVA determined that there was no significant three-factor interaction among cells, hydrogel, and age for Collagen1, CD31, Keratin5, and α-SMA protein expression levels, but there was a significant two-factor interaction between cells and hydrogel (Collagen1, p = 0.023; CD31, p = 0.020; Keratin5, p = 0.025; α-SMA, p = 0.045), indicating a synergistic effect of CB-MNCs and CS/HEC/GP in increasing the expression of these proteins (Fig. 10E–G) (See Table S2).

Discussion

Due to age-related changes in skin tissue, PUs in the older population often exhibit poor wound healing [24, 25]. Among numerous emerging therapeutic approaches, CB-MNCs has shown great potential [5, 6]. In this study, we developed a composite of CB-MNCs and CS/GP/HEC, which was applied to PU wounds in a D-gal-induced aged murine model. We found that the CS/GP/HEC maintained the cell viability of CB-MNCs and significantly promoted wound healing in aged mice when combined with CB-MNCs.

Hydrogels are hydrophilic three-dimensional networked polymer materials that exhibit outstanding performance in fluid absorption, surface cooling, and pain control at the wound bed [26]. Hydrogels intended for wound therapy must meet several key criteria. Firstly, high water content is essential, as it not only helps to maintain wound moisture and promote new cell growth but also significantly reduces the risk of infection. Secondly, hydrogels need to possess appropriate adhesion to ensure stability on the wound while ensuring good biocompatibility and non-toxicity to prevent potential adverse reactions. Lastly, ideal hydrogels should be gradually absorbed or degraded as the wound healing process progresses, thereby reducing the risk of secondary surgical removal of the material [27]. Previous studies have shown that various hydrogels can promote the healing of chronic wounds [28,29,30]. Among them, CS/GP hydrogels have certain advantages in filling wound defects and providing a moist environment for wounds due to their excellent biocompatibility and unique thermosensitivity [31]. However, the high concentration of GP exerts a certain toxicity to the body and cells, limiting its widespread application. Therefore, we optimized the formulation of CS/GP hydrogel to reduce the amount of GP used while enhancing the overall performance of the gel, making it safer and more effective for the treatment of chronic wounds.

In this experiment, we added the long-chain macromolecule substance HEC to the CS/GP hydrogel, which entangled with the CS molecular chain, and the hydroxyl groups of HEC could also form hydrogen bonds with the amino groups on CS and GP [32], accelerating the gel formation and shortening the required gelation time. The optimized CS/GP/HEC can remain in a liquid state for a long time at room temperature and quickly form a gel at 37 °C, meaning it can adapt to the irregular space of PU wounds. Additionally, the addition of HEC increases the overall water content of the CS/GP hydrogel, resulting in better wound cleaning effects. The CS/GP/HEC degrades by approximately 50% after soaking in PBS at 37 °C for 14 days. After the addition of HEC, there are no chemical reactions between the components of the hydrogel, and only weak intermolecular forces are present, thus maintaining the stability and degradability of the CS/GP/HEC. Compared to the CS/GP hydrogel, although the degradation rate of the CS/GP/HEC is slightly slower, it still exhibits good stability and degradability.

To efficiently load the cells, hydrogels should exhibit good biocompatibility and non-toxicity to prevent immune reactions and inflammation, as well as possess appropriate porous structures to facilitate cell attachment and nutrient exchange [27]. We analyzed the cytotoxicity of CS/GP/HEC and found that the CS/GP/HEC extract had no significant impact on the growth status of CB-MNCs cells. Previous studies had suggested that high concentrations of GP couldhave negative effects on cells due to the high osmotic pressure it induces [16, 17], while HEC, through hydrogen bonding with CS-GP solution, reduced the amount of GP used, thereby enhancing the overall biocompatibility and safety of the hydrogel [18]. However, due to the fact that HEC contains the crosslinking agent glutaraldehyde [33], high concentrations of glutaraldehyde are natural toxic by-products of late-stage glycation [34], thus excessive HEC may actually impact cellular activity. Testing of the CS/GP/HEC co-cultured with CB-MNCs cells showed that the hydrogel with 5% GP concentration exhibited poor biocompatibility, with a significant amount of cell death within 1 day, while the hydrogel with 3% GP concentration and CS/GP/0.4% HEC group demonstrated good biocompatibility, with the latter showing superior performance. Furthermore, we analyzed the effect of the CS/GP/HEC on the migration rate of CB-MNCs cells using a Transwell assay, which showed no significant impact on cell migration. This may be attributed to the interconnected porous network structure inside the hydrogel, which provides cell adhesion support and migration space [31].

After confirming the biocompatibility of the CS/GP/HEC, we established an aged murine model with PUs induced by D-gal treatment in C57BL male mice. Subsequently, PUs were created on the back of each mouse, and CB-MNCs cells encapsulated in CS/GP/HEC were locally applied to the PUs wounds in the aged murine model induced by D-gal treatment, following previous reports [21, 22]. We found that CS/HEC/GP, CB-MNCs, and CB-MNCs@CS/HEC/GP all promoted wound healing in both young and aged mice post-treatment, with the CB-MNCs@CS/HEC/GP therapy significantly outperforming other treatments. The CS/HEC/GP as a carrier played an important synergistic role in CB-MNCs-mediated therapy, particularly evident in the treatment of aged mice. The CS/HEC/GP provided a moist environment for wound healing and served as a barrier against harmful substances, resulting in better healing performance compared to the control group. CB-MNCs cells may play a crucial role in wound healing, as in the absence of CS/HEC/GP, leakage of cell suspension and damage to the wound microenvironment lead to low cell concentration and activity, affecting therapeutic outcomes. In the case of CS/HEC/GP loaded with CB-MNCs, CS/HEC/GP serves as a scaffold for CB-MNCs, providing a stable three-dimensional microenvironment for CB-MNCs at the PU wound site, thereby increasing engraftment and prolonging cell viability. CB-MNCs are continuously released, resulting in accelerated wound healing.

Wound healing is a complex biological process that can be divided into three stages: inflammation, proliferation, and remodeling [35]. We investigated the effects of CB-MNCs@CS/HEC/GP on these three stages of wound healing. Firstly, the inflammatory phase involves the activation of lymphocytes, monocytes, and macrophages, which release inflammatory cytokines (such as TNF-α, IL-6) to participate in the process of clearing wound bacteria and foreign substances. During wound healing, the number of pro-inflammatory macrophages (M1-like Mφ) gradually decreases and transitions to anti-inflammatory macrophages (M2-like Mφ), which secrete anti-inflammatory factors (such as IL-10) and promote cell proliferation and new blood vessel formation [36]. Although short-term inflammation is beneficial for wound repair, sustained expression of inflammatory mediators at the wound site can interfere with the proliferation process [37]. Therefore, changes in the M1/M2-like Mφ ratios and the expression levels of TNF-α and IL-10 are crucial for wound healing. CB-MNCs are rich in lymphocytes and monocytes, which can secrete immunomodulatory factors such as interleukin-10 to regulate immunity, reduce inflammation, and promote tissue regeneration [5]. Our study confirmed that CB-MNCs@CS/HEC/GP significantly alleviated wound inflammation by downregulating the expression of M1-type macrophages and pro-inflammatory cytokine TNF-α, and upregulating the expression of M2-type macrophages and anti-inflammatory cytokine IL-10, creating favorable conditions for promoting wound healing. Although the interaction suggested a synergistic effect between CB-MNCs and CS/HEC/GP in reducing the M1/M2-like Mφ ratios, age-related factors may partially offset this synergy. However, the degree of decrease in the M1/M2-like Mφ ratios after CB-MNCs@CS/HEC/GP treatment in the aged group was comparable to that in the young group, indicating that the treatment modality of CB-MNCs@CS/HEC/GP benefited more from inflammation regulation in aged individuals.

The second stage of wound healing is proliferation, characterized by cell proliferation and angiogenesis. Our study found that regardless of whether in young or aged mice, the application of CB-MNCs increased the expression of Ki67 in skin wound tissues, while CB-MNCs@CS/HEC/GP treatment showed even higher Ki67 expression, especially in aged mice, where this effect was more pronounced. The increase in Ki67 expression suggests potential acceleration of the cell division and proliferation process [38], indicating that CB-MNCs itself has a certain promoting effect on cell proliferation, while the CS/HEC/GP's facilitation of CB-MNCs transplantation and survival further enhances this effect, which is more pronounced in aged individuals. Subsequently, we detected the number of newly formed blood vessels in skin wound tissues and found that, compared to the other three groups, regardless of whether in young or aged mice, the CB-MNCs@CS/HEC/GP group exhibited a significant increase in the number of newly formed blood vessels in skin wound tissues.

The third stage of wound healing is tissue remodeling, where fibroblasts and myofibroblasts play crucial roles by synthesizing the extracellular matrix (ECM) and mediating wound contraction, with collagen being the major ECM component [39]. Collagen deposition plays a vital role in wound repair by providing an appropriate three-dimensional microenvironment to support cell proliferation, migration, and differentiation [40]. In our study, we found that CB-MNCs could promote collagen deposition by increasing the number of fibroblasts and myofibroblasts, and the CS/HEC/GP's enhancement of CB-MNCs's promoting effect on transplantation and survival further augmented this effect, with the effect being particularly significant in the aged murine group.

The integrity and regeneration of skin appendages during wound healing not only contribute to restoring the normal structure and function of the skin but also help reduce scar formation and improve the quality of wound healing [41, 42]. In our study, compared to the other three groups, the CB-MNCs@CS/HEC/GP group exhibited a greater number of hair follicles induced around the wound, and the healing effect was histologically closer to normal skin. To further explore the potential mechanism by which CB-MNCs@CS/HEC/GP promoted PUs healing, we used human Nucleoli as a specific marker for human cell nuclei and observed the ability of CB-MNCs to promote changes in cell types, specifically fibroblasts, endothelial cells, keratinocytes, and smooth muscle cells, in PU wounds. This finding is consistent with previous literature, indicating that CB-MNCs can promote the presence of various cell types in the appropriate microenvironment to restore tissue function [43, 44]. Western blot analysis further validated the significant impact of CB-MNCs on the expression levels of the above key cellular markers. These results suggested that CB-MNCs@CS/HEC/GP therapy could promote wound closure by contributing to the development of required cell types in PU wounds, facilitating hair follicle regeneration, and improving healing quality.

In summary, the use of CS/HEC/GP loaded with CB-MNCs may enable more CB-MNCs to be transplanted in situ at PU wounds, and enhance the survival and vitality of CB-MNCs in the inflammatory environment of PU wounds. Over time, CB-MNCs begin to act on skin wound tissues, exerting immunomodulatory effects, reducing inflammation, and by contributing to the development of required cell types to replace damaged cells, they promote cell proliferation, angiogenesis, and collagen deposition in skin wound tissues, ultimately shortening the healing time of PUs in aged mice and improving healing quality.

The limitation of this study lies in its reliance on an animal model; future research should validate these findings through clinical trials. Additionally, although we observed the promoting effect of CB-MNCs@CS/HEC/GP on specific cellular markers, the precise mechanism of action requires further investigation. Understanding the molecular interactions between CB-MNCs and PU wounds, as well as their functional roles in different stages of wound healing, will be crucial for developing more effective PUs treatment strategies.

Conclusion

In this study, we successfully developed a composite of CB-MNCs and CS/GP/HEC for the treatment of aged pressure injury wounds. The CS/GP/HEC is suitable for carrying and sustained release of CB-MNCs, maintaining the cellular activity of CB-MNCs, alleviating inflammation at the wound site, promoting cellular proliferation, angiogenesis, collagen remodeling, re-epithelialization, and regeneration of skin appendages at the wound site, thereby accelerating the wound healing process. Our research suggests that local transplantation of CB-MNCs@CS/HEC/GP is a promising new strategy for wound healing.

Data availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Abbreviations

CB-MNCs:

Cord blood mononuclear cells

CS/HEC/GP:

Chitosan/hydroxyethyl cellulose/glycerophosphate

PUs:

Pressure ulcers

ANOVA:

Analysis of variance

AUHC:

Area under the healing curve

Mφs:

Macrophages

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Acknowledgements

I extend my gratitude to my colleagues in the Laboratory Group 7-8. Their enthusiastic support and altruistic aid have allowed me to overcome the various obstacles and challenges that arose during my research. We acknowledge the use of artificial intelligence in this research. Specifically, we used ChatGPT for English grammar correction.

Funding

This work was funded by the research grants from Taishan Scholars (No. tsqn202103146), the Natural Science Foundation of Shandong Province (ZR2023MH075), the National Natural Science Foundation of China (82070392, 81702194, 81801953), Key research and development program of Shandong Province (2019GSF108041).

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Authors and Affiliations

  1. Department of Geriatric Medicine & Laboratory of Gerontology and Anti-Aging Research, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China

    Zhi-cheng Yang, He Lin, Hui Pan & Zhi-hao Wang

  2. Shandong Qilu Stem Cell Engineering Co., Ltd, Jinan, 250012, Shandong, China

    Guo-jun Liu, Feng Gao & Zhong Wang

  3. School of Nursing and Rehabilitation, Shandong University, Jinan, 250012, Shandong, China

    Zhi-cheng Yang, Jun-lu Zhu & Xiao-hong Zhang

Authors
  1. Zhi-cheng Yang
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  2. He Lin
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  3. Guo-jun Liu
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  4. Hui Pan
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  5. Jun-lu Zhu
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  7. Feng Gao
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  8. Zhong Wang
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  9. Zhi-hao Wang
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Contributions

Zhi-cheng Yang and He Lin contributed to the performance of the study, analyzed and interpreted the data, and wrote the manuscript (Please note that Zhi-cheng Yang and He Lin are co-first authors who contributed equally to this study). Guo-jun Liu, Feng Gao and Zhong Wang provided technical support. Hui Pan and Xiao-hong Zhang contributed to the study's performance and data interpretation; Jun-lu Zhu contributed to manuscript revision.

Corresponding author

Correspondence to Zhi-hao Wang.

Ethics declarations

Ethics approval and consent to participate

The study was conducted under the approved project "Experimental Study on Hydrogel-Loaded Human Umbilical Cord Blood Mononuclear Cells Promoting Pressure Ulcers Healing in Mice." This project was reviewed and approved by the Ethics Committee of the School of Nursing and Rehabilitation, Shandong University. The approval number is No: 2023-D-013, and the approval date is September 15, 2023. The human umbilical cord blood mononuclear cells used in this study were provided by Shandong Qilu Stem Cell Engineering Co., Ltd, which has obtained ethical approval for the collection and use of these cells from the Shandong Provincial Health Department. The approval number for this is Lu Wei Yi Zi [2011] No. 158, and the approval date is December 31, 2011. Written informed consent was obtained from all patients or their guardians for the use of the samples in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that during the study, our institution received an in-kind contribution of cord blood mononuclear cells (total number of cells, 1.6 × 109) from Shandong Qilu Stem Cells Engineering Co. There are no further conflicts of interest to declare.

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Yang, Zc., Lin, H., Liu, Gj. et al. CB-MNCs@ CS/HEC/GP promote wound healing in aged murine pressure ulcer model. Stem Cell Res Ther 16, 52 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04177-w

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04177-w

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512