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Human umbilical cord mesenchymal stem cells improve the survial of flaps by promoting angiogenesis in mice

European Journal of Medical Research volume 30, Article number: 356 (2025) Cite this article

Abstract

Background

Flap necrosis post-operation disturbs surgeons during plastic and reconstructive surgery. This is caused by hypoperfusion and subsequent ischemia–reperfusion injury, where restricted blood flow followed by restored circulation paradoxically exacerbates tissue damage. Mesenchymal stem cells, which show multidirectional differentiation, provide hematopoietic support and are involved in immune regulation and anti-fibrosis, have inspired research on improving the blood supply of flaps.

Methods

Primary human umbilical cord mesenchymal stem cells (HuMSCs), were obtained and subcultured for expansion. The cells of the third generation were incubated in a gelatin sponge. Thirty Kunming mice were randomly divided into three groups, and saline, HuMSCs, and HuMSCs-CM were injected preoperatively into the skin of the back. The vessel density was assessed on the tenth day. Forty-eight Kunming mice were divided into two groups. Group A was subdivided into the saline group, HuMSCs, and HuMSCs-CM groups and pretreated as described above. In Group B, the intervention was changed from injection to subcutaneous embedding. Random flaps were made on the back in both groups on the tenth day after pretreatment. The survival rate of the flap was calculated on the seventh day.

Results

HuMSCs-CM significantly increased the microvessel density on the tenth day after pretreatment. The flap survival rate was higher in the cell and CM groups, rising from approximately 13% to 60% in Group A, and to about 75% in Group B. Moreover, subcutaneous embedding of cell-carrying gelatin sponges improved flap survival compared to other interventions.

Conclusion

Improved cell incubation conditions can enhance its utility. The application of HuMSCs and their conditioned medium promoted the survival of the flap by inducing neovasculogenesis.

Introduction

Flap survival is the focus after transplantation in clinical settings. Flap necrosis is linked to hypoperfusion of local microcirculation in the early stage and free radical damnification and inflammatory lesions induced by ischemia–reperfusion at the late stage. Angiogenesis and vasculogenesis are crucial for the survival of the flap post-transplantation. Currently, strategies such as prefabricated flap [1], skin and soft tissue expansion [2], vascular pressurization [3], ischemic preconditioning [4], and application of platelet-rich plasma [5] and various pro-angiogenic factors [6] have effectively increased the utilization rate of flaps in experimental research and clinical work, enriching studies on the regeneration prefabricated of the flap blood supply.

Mesenchymal stem cells (MSCs) are multipotent stem cells derived from mesoderm with high self-renewal ability. These have the following characteristics: hematopoietic support, immune regulation, and anti-fibrosis. Regulation of various cytokines expression by stem cells with low immunogenicity promotes vascular differentiation and indirectly repairs tissues showing ischemia–reperfusion. In this study, MSCs were mainly derived from bone marrow, fat, and umbilical cord, and their mechanisms of action were through paracrine function. Relevant experiments have confirmed that MSCs promote neovascularization under physiological or pathological conditions by secreting vasoactive growth factors, significantly improving the flap utilization rate. Clinically, human bone marrow-derived mesenchymal stem cells have been used to assist the excessive flap expansion when pre-expanded flaps were used for full-face repair and reconstruction [7]. A more ideal graft was created to reconstruct the large area of facial skin defect. Some scholars have combined the prefabricated flap with regenerative medicine to pre-construct an abdominal island flap with the axis of the rat femoral vessels. Homologous allogeneic adipose MSCs were given during the first stage of the surgery, confirming that adipose-derived stem cells have the same potential to enhance the survival of preconstructed flaps [8]. Compared with bone marrow and adipose stem cells, HuMSCs are relatively valuable for research and clinical settings due to their non-invasive acquisition and isolation methods, low cost, and higher secreted chemokine and growth factor levels. Our experiments have shown that HuMSCs participate in neovascularization by differentiating into vascular endothelial cells and increasing the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), eventually establishing a new microcirculation to promote the survival of the flap [9].

Considering that the mechanisms of MSC action are realized by their secretory function, studies are now exploring their exosomal components, which promote wound healing by inducing the proliferation and migration of vascular endothelial cells, promoting angiogenesis, and reducing the apoptosis of endothelial cells. In the in vitro culture, the cytokine secretion profile and secretion level change owing to the change in the microenvironment of MSCs. Our previous study has shown that MSCs in gelatin sponge stereoscopic culture improve the secretion of various factors to varying degrees, showing good clinical prospects. However, research on the timing of the application of HuMSCs and their products and the changes in local blood supply post-administration is lacking.

Materials and methods

Isolation and culture of HuMSCs

The Ethics Committee of Shantou University Medical College (SUMC, Shantou, China) granted ethics approval for the study. With prior signed informed consent, discarded umbilical cords from healthy pregnant women (excluding infectious diseases such as AIDS, hepatitis, and syphilis) who underwent cesarean section at full-term gestation were obtained for this study. The Wharton’s jelly pieces obtained from umbilical cords were cut into pieces, approximately 2 mm2, under aseptic conditions and evenly plated. To the plates, high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, Inc., USA) containing 2% fetal bovine serum (Gibco, USA), 1% penicillin and streptomycin solution (Beyotime, China), bFGF (Zhuhai Essex Bio-Pharmaceutical Co., Ltd., China), transferrin (Sigma, Sigma-Aldrich, Co., USA), insulin (Sigma, Sigma-Aldrich, Co., USA), and selenium acid (Aldrich, Sigma-Aldrich, Co., USA) was added. The plates were incubated in a humidified CO2 incubator. HuMSCs grew around the tissue block when cultured for 5–7 days, and the medium was regularly changed. The cells were passaged when they reached a confluency of 80–90%. Cells of the third passage were used for the subsequent experiments.

Preparation of gelatin sponge seeded with HuMSCs (GS-MSC) and HuMSCs-CM

Gelatin sponges (Xiang’en, Jiangxi Xiang’en Medical Technology Development Co., Ltd., China) were cut into 20 mm × 20 mm × 10 mm pieces and soaked in 0.75 mg/L PLL solution (Beyotime, China) overnight. After washing thrice with phosphate-buffered saline (PBS), the gelatin sponges were put in six-well plates and pre-wetted with a culture medium before cell seeding. Each gelatin sponge block was seeded with 1.5 mL of HuMSCs suspension (3 × 106 cells/mL) and incubated in an atmosphere with 5% CO2 at 37 °C for 30 min. After incubation for 30 min, gelatin sponges were immersed in 6 mL of prepared culture medium. The medium was changed every two days during the experiment. The conditioned media (CM) of HuCMSCs incubated in the gelatin sponge up to Day 3, Day 7, and Day 14 were collected, centrifuged, and filtered for subsequent experiments.

Evaluation of growth factor levels by enzyme-linked immunosorbent assay (ELISA)

CM was collected from HuCMSCs cultured to day 3 under monolayer culture conditions (Monolayer-CM, Day 3). CM was also collected from HuCMSCs cultured in gelatin sponges up to day 3, day 7, and day 14 (GS-HuMSCs-CM, Day 3, Day 7, Day 14). One day before collecting the supernatant, the medium was changed to a serum-free medium. The supernatant was centrifuged at 3000 rpm for 10 min to remove floating cells and cellular debris and subjected to ELISA. Secreted levels of vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF) and Transforming Growth Factor-β1 (TGF-β1) in the supernatant were quantified using the corresponding ELISA kits (R&D systems, USA) following the manufacturer's protocol. Briefly, microplates precoated with capture antibodies were prepared as per the manufacturer's instructions. Standards and samples were diluted in sample diluent to fall within the assay's linear range. Cell culture supernatants were added to wells, followed by standards, controls, and test samples. Plates were sealed and incubated for 2 h at room temperature, then washed and incubated with covalent chelators. After another wash, substrate chromogenic solution was added, and plates were incubated in the dark for 20–30 min. At the end of incubation, 50 μL of termination solution was added to terminate the reaction, and absorbance was measured at 560 nm using a microplate reader. Data from three independent and repeated assays were analyzed and growth factor levels were calculated from four-parameter logistic curves.

Experimental flap model

The experimental subjects comprised Kunming mice, adhering to the specific pathogen-free (SPF) standard. These mice, aged 4–5 weeks and weighing 30–40 g, were procured from the animal laboratory of Shantou University Medical College. The experimental protocols involving mice followed the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments), and approved by the medical animal care & welfare committee of Shantou University Medical College under approval ID (SUMC2022-603). Mice were sacrificed by cervical dislocation following administration of anesthesia by inhalation of isoflurane.

Thirty Kunming mice were randomly divided into three groups (Fig. 1). The back was disinfected, and hair was removed after anesthetizing the animals. Subsequently, HuMSCs, saline, and HuMSCs-CM were intradermally injected into the back once every 2 days for a total of three times. The injection points were located in the pre-formed skin flap area, arranged in 2 × 5 rows, with 10 points and 0.1 ml per point. On the tenth day after pretreatment, mice in each group were dissected to observe the vascular distribution of the dorsal skin and five areas were selected for anastomotic branch counting (measured by 2 blinded judges) and histological observation according to the route of the main blood vessel trunks.

Fig. 1
figure 1

Schematic diagram of animal experiment. a Injection Pretreatment. b The injection pretreatment group underwent flap surgery on day 7. c Embedded carrier group underwent flap surgery on day 7. d Model of the random flap

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Forty-eight Kunming mice aged 4–5 weeks and weighing 30–40 g were randomly divided into two groups. Group A was subgrouped and pretreated as described above, and animals in group B were pretreated by subcutaneous embedding, in which a small transverse incision was made on the back at a distance of 2 cm from the tail. Gelatin sponges with stem cells, saline, or HuMSCs-CM were placed subcutaneously. All mice were anesthetized and disinfected, and hair was removed on the tenth day. A random flap measuring 1 × 4 cm was designed along the long axis in the middle of the back, reaching the deep fascia layer and sutured back to the original position. After the operation, the mice were wrapped in a dressing. All mice were dissected seven days postoperatively to observe the gross necrosis of the flaps and vascular distribution. The flap survival rate (%) was calculated as (viable area/total flap area) × 100%. Necrotic regions of the flap were visually identified by their dark purple or black color, dry and hard texture, and absence of capillary response, while viable areas exhibited normal skin elasticity and a healthy pink color. Necrotic regions and the actual extent of the contracted flap were marked with a felt-tip pen. The entire flap was photographed (EOS 6D, Canon, Japan) under standardized conditions with a fixed light source and distance. The proportion of viable and necrotic areas was quantified using the ImageJ image analysis system. Skin tissue sections from the proximal, middle, and distal regions of the flap were subjected to staining. The experiments have been reported according to the ARRIVE guidelines 2.0.

Histology

On the tenth day of dorsal skin pretreatment and the seventh day postoperation, animals were euthanized and dorsal skin and flap specimens were harvested as described previously. Specimens were placed into 4% paraformaldehyde followed by gradient dehydration in ethanol. These were subjected to paraffin-embedding and sectioned into 5 μm-thick slices. Tissue segments were placed on slides and subjected to hematoxylin and eosin (HE) staining (Beyotime, China). The CD31 immunohistochemical staining procedure includes deparaffinization with xylene, rehydration through graded ethanol, antigen retrieval in preheated citrate buffer, blocking endogenous peroxidase activity with 3% hydrogen peroxide, and nonspecific binding with 5% goat serum. Subsequently, sections were incubated with primary anti-CD31 antibody (Beyotime, China) overnight at 4 °C, followed by biotinylated secondary antibodies and streptavidin-HRP. 3,3'-diaminobenzidine (DAB) was used as a chromogenic agent for 5 min. The sections were counterstained with hematoxylin and sealed after gradient dehydration and transparency.

Statistical analysis

All data are presented as the mean ± standard deviation (SD), and comparisons between groups were analyzed by one-way analysis of variance (ANOVA). The comparisons among growth factor levels are presented. A p < 0.05 indicated a considered statistically significant difference.

Results

Identification of HuMSCs

The primary culture of HuMSCs was an adherent culture (Fig. 2). After 5–7 days, cells gradually migrated outward from the surrounding of Wharton jelly tissue. Simultaneously, the cells, with polygonal star and spindle shape, continued to replicate and proliferate and finally showed mutual fusion.

Fig. 2
figure 2

Primary and subculture results of HuMSCs (original magnification: 40 ×). a HuMSCs migrated outward from the tissue block when cultured for 5–7 days, with spindle-shaped and polygonal star shape. b HuMSCs'proliferation reached 80%−90% fusion when primarily cultured for 14 days. c The second generation cells reached 90% confluence. d The third-generation cells reached 90% confluence

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Change in HuMSC-secreted growth factors in the GS-HuMSC group

Supernatants from monolayer cultures and GS-HuMSCs were collected and growth factors were measured (Fig. 3). GS-HuMSCs released significantly higher levels of factors on day 3 than the monolayer cultured group (p < 0.001). VEGF secretion was the highest among the four growth factors, and its secretion increased successively over one week. TGF-β1 and HGF secretion levels peaked on day 3, and that of KGF peaked on day 7 but remained at a higher level compared to the monolayer group.

Fig. 3
figure 3

Growth factor assessment of HuMSCs100% confluence group, monolayer group (day 3), and GS-HuMSCs-CM groups (day 3). A-D showed the secretion trend of VEGF、KGF、HGF、TGF-β1(**p < 0.01. ***p < 0.001. ****p < 0.0001.)

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Dorsal vascular network on day 10 after pretreatment

There are four dorsal dermal vascular trunks in mice, which extend centripetally from the base of both shoulders and hind limbs to the center of the back, and branch out into small branches, some of which anastomose with the adjacent trunk branches forming a dorsal vascular network. On day 10, thickening of the dorsal vessel diameter, the most obvious increase in branching, and increased mutual anastomosis of the branches was observed in the GS-HuMSCs-CM group (Fig. 4). Anastomotic branches refer to collateral branches formed between adjacent structures within the same area. Five visual fields (1 × 1 cm/view) were randomly selected for counting anastomotic branches. The mean anastomoses in the HuMSCs group and the GS-HuMSCs-CM group were 6.13 ± 0.83/cm2 and 6.88 ± 1.76/cm2, respectively, which were significantly higher compared with the control group (4.73 ± 0.82/cm2) (P < 0.01). The results in the HuMSC group compared with the GS-HuMSCs-CM group showed no statistically significant difference (P > 0.05).

Fig. 4
figure 4

Comparison of dorsal vascular network of skin in each group after 10 days’ pretreatment. a General observation of vascular network. a, b and c were the results of normal saline pretreatment on day 4, day 7 and day 10; d, e and f were the results of GS-HuMSCs-CM pretreatment on day 4, day 7 and day 10; g, h and i were the results of HuMSCs pretreatment on day 4, day 7 and day 10. The red circle in the figure shows one of the main blood vessels in the back of mice. It can be observed that HuMSCs group and GS-HuMSCs-CM group had the most obvious increase in blood vessel branches on the 10 th day and more anastomosis with each other. b Anastomotic branches on day 10. (*P < 0.05; **P < 0.01)

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Survival of flaps

The distal part of the skin flap in both groups darkened gradually and the surface was covered with a crust, starting on the third postoperative day. On the seventh day, necrosis no longer extended, and the necrotic part was blackish brown, covered with a crust, and poorly elastic. In Group A (Fig. 5), excluding the obvious error factors caused by clamp damage during operation, six mice in each subgroup were included. The flap survival rates in the HuMSC group and the GS-HuMSCs-CM group were 64.85 ± 8.09% and 61.93 ± 12.03%, respectively, significantly higher than those in the control group (13.70 ± 5.55%) (P < 0.01). There was no statistically significant difference between the HuMSC and GS-HuMSCs-CM groups (P > 0.05). Different degrees of flap retraction were observed on postoperative day 7. In Group B (Fig. 6), six mice in each group were included. The flap survival rates in the GS-HuMSCs group and the GS-GSHuMSCs-CM group were 79.11 ± 9.96% and 75.98 ± 15.94%, respectively, significantly higher than those in the GS-control group (13.98 ± 9.65%) (P < 0.01).

Fig. 5
figure 5

Comparison of flaps in Group A on day 7 post-operation. a General observation. b Measurement results. (**P < 0.01; ***P < 0.001)

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Fig. 6
figure 6

Comparison of flaps in Group B on day 7 post-operation. a General observation. b Measurement results. (**P < 0.01; ***P < 0.001)

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Histopathological analysis and Immunohistochemistry

Staining of the back skin after 10 days of pretreatment

After 10 days of pretreatment, the skin of five regions of the back was randomly selected for staining based on the vascular trunk alignment (Fig. 7). Histological results microvessel density (MVD) of HuMSC and GS-HuMSCs-CM groups at 6.24 ± 1.37 and 5.32 ± 0.48, respectively, significantly higher than that of the control group (2.53 ± 0.71) (P < 0.01). Two-by-two comparisons confirmed no significant difference between HuMSC and GS-HuMSCs-CM groups (P > 0.05). The total thickness of the skin and subcutaneous layer in the HuMSCs group and GS-HuMSCs-CM group was 508.06 ± 74.6 μm and 542.91 ± 33.4 μm, respectively, significantly higher than that of the control group (438.31 ± 28.0 μm) (P < 0.05).

Fig. 7
figure 7

Comparison of staining results of dorsal skin in each group after 10 days’ pretreatment. a Results of HE staining and CD31 immunohistochemical staining. a, d and g are the results of HE staining under a microscope with a 40 ×; b, e and h are the results of HE staining under a microscope with a 200 ×. c, f and i are the results of CD31 immunohistochemical staining under a microscope with a 200 ×. Blood vessels are shown by the red arrows in the figure. It can be observed that the HuMSCs group and GS-HuMSCs-CM group have more blood vessels. b Counting results. A is the thickness of the skin and subcutaneous layer under a 40 × microscope, B and C are the comparison of total microvascular diameter and MVD count under 200 × microscope (*P < 0.05; **P < 0.01; ***P < 0.001)

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HE staining of the flap tissues

Histological results showed that the distal part of the three groups was more fragmented than the proximal ones. There were different degrees of inflammatory infiltration in the proximal and distal ends. Distal microvessels in the HuMSC and the GS-HuMSCs-CM groups were easier to identify than those in the control group, regardless of groups A or B. Microvessel density in tissue sections of three regions of the whole flap was assessed. In group A (Fig. 8), the MVD of the HuMSC group and GS-HuMSCs-CM group was 7.86 ± 1.34/field of view and 7.66 ± 0.42/field of view, respectively, significantly higher than that of the control group (4.22 ± 1.20/field of view) (P < 0.01). There was no statistically significant difference in the results between the HuMSC and the GS-HuMSCs-CM groups (P > 0.05). In group B (Fig. 9), the MVD of the GS-HuMSC and GS-GSHuMSCs-CM groups were 8.00 ± 0.94/field of view and 7.81 ± 0.65/field of view, respectively, significantly higher than that of the control group (3.66 ± 1.22/field of view) (P < 0.01). There was no statistically significant difference between the GS-HuMSC and GS-GSHuMSCs-CM groups (P > 0.05).

Fig. 8
figure 8

Comparison of staining results of flap in group A. a HE staining results. a, c, e, g, i, k, m, o and q are the results under a microscope with a 40 ×, b, d, f, h, j, l, n, p and r are the results under a microscope with a 200 ×. Blood vessels are shown by the arrows in the figure. It can be observed that the HuMSCs group and GS-HuMSCs-CM group have more blood vessels. b Measurement results. (*P < 0.01)

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Fig. 9
figure 9

Comparison of staining results of flap in group B. a HE staining results. a, c, e, g, i, k, m, o and q are the results under a microscope with a 40 ×, b, d, f, h, j, l, n, p and r are the results under a microscope with a 200 ×. Blood vessels are shown by the arrows in the figure. It can be observed that the GS-HuMSCs group and GS-GSHuMSCs-CM group have more blood vessels. b Measurement results. (*P < 0.01)

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Discussion

Flap necrosis involves several mechanisms. Vascular closure occurs when the perfusion pressure of the flap is lower than the critical closure pressure of the small arteries, and ischemia of distal tissue and flap necrosis ensues. Subsequently, rebound vasodilation associated with sympathetic mechanisms causes local reperfusion. The massive release of reactive oxygen species in the early stages of reperfusion stimulates the opening of the mitochondrial permeable conversion holes, leading to the depolarization of the membrane potential and rupture of the outer mitochondrial membrane. Apoptosis molecules are released into the cytoplasm, activating the apoptosis cascade. The damage to tissue follows [10]. Accumulation of neutrophils post reperfusion blocks microcirculation and the release of chemotrophic factors, further promoting leukocyte accumulation in the ischemic tissue. Such accumulation induces inflammatory cascades to damage endothelial cells and destroy the integrity of blood vessels. Toxic products, such as oxygen-free radicals released by necrotic neutrophils aggravate tissue damage [11,12,13]. Relevant mechanisms have provided insights into the prophylactic treatment of flap necrosis, such as attenuating free radical damage [14], ischemic pre-adaptation [15], up-regulation of angiogenesis-related factors [6], modulation of diastolic vascular factors [16], prefabricated flaps [1], vasodilatation [2], and inhibition of inflammatory responses [13]. Among them, studies related to improving microcirculatory perfusion are notable.

Neovascularisation is a complex process but involves two main prepossess: angiogenesis and vasculogenesis [17]. The former refers to the generation of new microvessels from pre-existing capillary networks. The latter refers to the process by which endothelial progenitor cells reorganize to form new capillaries and dock with the original vascular network. Various angiogenic factors play an indispensable regulatory role in this process. VEGF serves as the master regulator of angiogenesis. Its secretion peak reflects stem cells'heightened responsiveness to pro-angiogenic demands under hypoxia, inflammation, or mechanical stress. It promotes the proliferation, migration of endothelial cells and enhances vascular permeability, providing the initial signal for vascular sprouting. KGF primarily targets epithelial cells, promoting the repair of surrounding tissues and indirectly stabilizing the microenvironment of nascent vasculature. HGF demonstrates stem cells'regulatory capacity in orchestrating complex tissue regeneration processes, including vascular branching and extracellular matrix remodeling. VEGF initiates signaling mainly by binding to VEGFR-2, the main functional receptor, which undergoes dimerization and autophosphorylation upon activation, thereby initiating the PI3 K/Akt pathway, the MAPK/ERK pathway, etc. VEGF activates PI3 K, which generates the second messenger, PIP3, thereby activating Akt [18]. Akt promotes endothelial cell survival by suppressing pro-apoptotic proteins (e.g., BAD and Caspase-9) [19], and enhances NO synthase activity [20], further dilating blood vessels to improve local perfusion of the flap. In addition, VEGFR-2 phosphorylation activated the Ras-Raf-MEK-ERK cascade reaction to promote endothelial cell proliferation and migration [21]. Furthermore, the hypoxic microenvironment post-flap transplantation stabilizes HIF-1α, a transcription factor that directly binds to the hypoxia-responsive element (HRE) within the VEGF promoter, upregulating VEGF transcription. A previous study confirmed that MSCs promoted angiogenesis by regulating the hypoxia-inducible factor-1α/VEGF pathway [22]. The effectiveness of stem cells applied to various ischemic disease models has now been demonstrated. A study confirmed that HuMSCs improved limb blood supply in the hindlimb ischemia model, and ERK and PI3 K-Akt pathway activation may underlie the mechanism by which they promote angiogenesis [23]. Some scholars transplanted VEGF gene-transfected HuMSCs into a diabetic rat model of limb ischemia, corroborating the finding that stem cells stimulate angiogenesis and increase perfusion [24]. Although existing studies have demonstrated the limited ability of pro-angiogenic factors to promote vascular neogenesis in organisms under physiological conditions due to the lack of stimulation by ischaemic and hypoxic conditions [25], the number of anastomotic branches and MVD in the back skin of the mice pre-treated with HuMSCs and GS-HuMSCs-CM in this study was higher than that in the control group. This may be attributed to the paracrine supplementation of the stem cells with a high content of growth factors absorbed into capillaries and induced neovascularisation by stimulation of local intradermal injection. MSCs promote neovascularisation by secreting many vasoactive growth factors [26]. MSCs differentiate into vascular endothelial cells and smooth muscle cells [27], directly generating new blood vessels. These are stabilized by binding to pericytes or smooth muscle cells [28]. Furthermore, stem cells may exert their anti-inflammatory effects by reducing the mRNA levels of tumor necrosis factor-α(TNF-α), macrophage inflammatory protein-α, and vascular cell-adhesion molecule-1 (VCAM-1) [29], thereby improving the flap survival rate.

In the histological staining of the flaps, we also observed varying degrees of inflammatory infiltration in the proximal and distal ends. Inflammatory infiltration phases following flap surgery can be divided into distinct phases. During the acute inflammatory phase, neutrophils and M1 macrophages massively infiltrate the tissue, releasing pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [30]. Concurrently, post-reperfusion neutrophil aggregation triggers an inflammatory cascade that exacerbates tissue damage. Neutrophil clusters obstruct capillary lumina, leading to microcirculatory occlusion [31]. Chemotactic factors released by neutrophils induce continuous WBC migration to ischemic tissue, resulting in endothelial cell injury and loss of vascular integrity. Infiltrating neutrophils further damage the flap, while necrotic neutrophils release toxic products such as oxygen free radicals and proteases, which intensify tissue injury. During this phase, inflammatory cytokines (e.g., SDF-1, IL-8) guide the rapid recruitment of HuMSCs to the ischemic area of the flap [32]. These cells secrete fators that effectively mitigate excessive inflammatory responses. In the subacute phase, HuMSCs secrete TGF-β and IL-10 to enhance M2 macrophage expansion while suppressing M1-associated factors such as iNOS [33]. In the remodeling phase, inflammatory infiltration in the flap markedly diminishes, with M2 macrophages dominating the microenvironment and regulatory T cell (Treg) proportions increasing alongside enhanced collagen deposition.

In addition to the application of stem cells in pro-angiogenesis, stem cell-related derivatives have been studied. MSC-derived exosomes produce hypoxia-inducible factor-1α during the induction of VEGF expression, which is crucial in activating the process involved in angiogenesis [34]. The conditioned medium of MSCs contains various types of pro-vascular growth factors and exosomes carrying various biomolecules. Their exocytosis levels change with the change in culture conditions. Stem cells obtained from traditional wall culture have relatively lower exocytosis and biological activities. The 3-D culture system is consistent with the growth environment of the cells, which can more effectively activate the functions of the stem cells. 3-D culture methods include scaffold-free and scaffolded models. The former [35] takes advantage of the aggregation tendency of adherent cells to construct cell spheres by providing a low-adhesion or non-adhesion culture environment. The latter employs various biomaterials, such as gelatine microspheres [36] and platelet-poor plasma gel [37]. In our previous study, pro-angiogenic cytokine content increased in HuMSCs cultured in 3-D systems [38]. In this study, two primary administration methods were investigated through experimental models: subcutaneous injection and subcutaneous embedded drug delivery systems. The former method involves injecting drugs into the subcutaneous connective tissue (located between the dermis and muscle layer). Drugs administered through this route are slowly absorbed by local capillaries or lymphatic vessels, resulting in absorption rates that depend on the drug's physicochemical properties and local blood flow characteristics. The latter way encapsulates the drugs in biocompatible materials, such as gelatin sponges, polylactic-co-glycolic acid (PLGA) microspheres [39], which are then implanted subcutaneously. These carrier systems enable controlled drug release through two main mechanisms: gradual diffusion via the material's micropores or sustained release during carrier degradation. In clinical practice, the choice of drug delivery methods often needs to be combined with the half-life of the drug, the treatment cycle of the disease and other factors for comprehensive consideration. The enrichment of the vascular network may be related to high pro-angiogenic cytokine content. However, there was no significant difference in MVD of the back skin pretreated with HuMSCs and GS-HuMSCs-CM in this study, which may be because: 1. the non-ischemic stereoscopic environment in vivo is more conducive to activating the function of stem cells after HuMSC administration. Thus, the effect of promoting angiogenesis was similar to that of the CM. 2. Half-life of cytokines prevented GS-HuMSCs-CM from showing a superior outcome to administration. Accordingly, we subsequently supplemented the results on improving local circulation by embedding a slow-release model in the flap experiment.

Although most experiments have focused more on the postoperative delivery of angiogenesis-related substances to improve flap survival, some have shown that delivery of endothelial progenitor cells immediately after surgery may limit the ability to prevent flap necrosis [40]. Since significant improvements in neovascularization were observed only 14 days after injection, EPC delivery before inducing ischemia may be more effective. In our study, intradermal injections were administered (intraventricular injections were not chosen due to the possibility of poor local effects due to long-distance migration and organ filtration [41]) to cells and CM to the pre-formed flap site. Flap survival was significantly higher in the HuMSC and GS-HuMSCs-CM groups compared with the control group, which was presumed to be associated with an increase in the total area of the capillary bed and an increase in perfusion per unit area due to the action of cell-secreted associated factors, such as VEGF and b-FGF. In addition, the mechanism by which stem cells improve flap survival relies on their involvement in vascular network construction and immunomodulatory responses. The inflammatory environment may play a benign mediating role in stem cell function. The flap survival rate of the HuMSCs group in group A was slightly higher than that of the GS-HuMSCs-CM group; however, this statistical difference was not significant, presumably related to factors such as cells’ survival being susceptible to the surrounding environment, low survival rate in ischemic tissue, vulnerable to negative feedback regulation, uneven distribution of secreted factors in the treatment area, and easy retention in the local area. In the gelatin sponge-loaded slow-release model (Group B), the 3-D stereoscopic environment locally provides a good space for the cells to release high levels of exocytosis factors continuously, and the gelatin sponge carrying the cells shows better application than the gelatin sponge simply soaked in CM.

In addition, on day 7, the flap retraction rate tended to be lower in the HuMSC and GS-HuMSCs-CM groups. Flap retraction can be categorized into immediate retraction and late retraction. Immediate retraction is mainly due to elastic fiber retraction; late retraction may be related to the structure and content of collagen fibers and elastic fibers, the latter of which was observed in this experiment. Among the numerous paracrine factors secreted by HuMSCs, TGF-β1 is a fibrosis-promoting growth factor involved in immune regulation. TGF-β1 [42] increases extracellular matrix mRNA expression and protein synthesis through related signal transduction cascades, resulting in an abundance of collagen fibers and overexpression of the extracellular matrix. Factors such as b-FGF act directly on cells in tissues to stimulate cell division and multiplication. It was therefore hypothesized that the skin structure was remodeled due to the intervention of associated factors secreted by stem cells, manifested as an increase in skin thickness in HuMSCs and GS-HuMSCs-CM preconditioned groups. This result might further explain the difference in flap retraction rates among the groups.

In this study, exocytosis cytokines from stem cells were used to intervene in the skin blood supply and improve the survival rate of the flap, providing a novel research direction for regenerative therapy in the field of wound repair and reconstruction. Although the model showed results aligning with our initial preconception, some limitations of the study warrant further consideration: 1. a comparison of the effects between the preconditioned and postoperative interventions is lacking. 2. The effects of subcutaneous embedding and injection on the subcutaneous vascular network were not explored. 3. Considering that markers may interfere with the early proliferative capacity of cells [43], we did not label the activity of stem cells in vivo after transplantation. It is possible to analyze the local microenvironmental conditions of cells delivered in vivo to analyze the recruitment and activity of stem cells at the treatment site. 4. Perhaps modulating the local microenvironment, such as local pH or oxygen concentration, may optimize the release of exosome components and enhance factor activity. 5. Integrating carrier properties into the optimization of drug delivery systems may be more effective in sustaining the activity of cytokines. These issues merit further investigation. Our study may provide ideas for the application of regenerative medicine in flap research and its clinical translation: Firstly, optimizing drug delivery systems was explored by utilizing gelatin sponges as carriers to deliver stem cells and their derivatives to the flap bed. Future efforts could focus on developing innovative biomaterial carriers or modifying existing carrier models using targeted delivery techniques, thereby reducing intervention frequency and enhancing patient compliance. Secondly, optimizing flap design was addressed by clarifying the vascular distribution of murine dorsal skin through anatomical studies, which offers insights for clinical flap design. For example, perforator flap design could be personalized using imaging techniques such as color Doppler ultrasound, combined with interventions to optimize vascular bed conditions and improve flap survival. Thirdly, integrating visualization techniques with tools like micro-CT or laser Doppler flowmetry could enable real-time microcirculatory assessment, guiding timely interventions to prevent flap necrosis.

Conclusion

Improved cell incubation conditions can effectively enhance the utility of cells. The application of HuMSCs and their CM can promote the survival rate of the flap by inducing neovasculogenesis.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

bFGF:

Basic fibroblast growth factor

CM:

Conditioned medium

ELISA:

Enzyme-linked immunosorbent assay

GS:

Gelatin sponge

GS-HuMSC:

Gelatin sponge seeded with HuMSCs

GS-HuMSCs-CM:

Conditioned medium from HuCMSCs cultured in gelatin sponges

GS-GSHuMSCs-CM:

Gelatin sponge soaked with conditioned medium from HuCMSCs cultured in gelatin sponges

HGF:

Hepatocyte growth factor

HuMSCs:

Human umbilical cord mesenchymal stem cells

KGF:

Keratinocyte growth factor

MSCs:

Mesenchymal stem cells

MVD:

Microvessel density

TGF-β1:

Transforming growth factor-β1

TNF-α:

Tumor necrosis factor-α

VCAM:

Vascular cell-adhesion molecule

VEGF:

Vascular endothelial growth factor

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Acknowledgements

We would like to thank the Department of Obstetrics and Gynecology, the Second Affiliated Hospital of SUMC, for their assistance in obtaining umbilical cords.

Funding

This study was supported by Shantou Medical Science and Technology Planning Project, 2022-169-66 and 2022-81-41.

Author information

Authors and Affiliations

  1. Department of Plastic Surgery and Burns Center, Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, China

    Siyi Ma, Jintao Ni, Yuping Kuang, Zhixia Wang & Lujun Yang

  2. Research Center for Translational Medicine, Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, China

    Danyan Ye & Lujun Yang

Authors
  1. Siyi Ma
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  2. Jintao Ni
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  3. Danyan Ye
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  4. Yuping Kuang
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  5. Zhixia Wang
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  6. Lujun Yang
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Contributions

Siyi Ma was responsible for study conception, experimental design, histological analysis, data interpretation, manuscript preparation, and final validation. Jintao Ni and Danyan Ye conducted most of the experiments. Yuping Kuang and Zhixia Wang helped perform the histological analysis. Lujun Yang assisted in the design of the experiment and provided financial support. All authors discussed the results and contributed to the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Lujun Yang.

Ethics declarations

Ethics approval and consent to participate

Protocols for sampling human umbilical cord tissue were approved by the Institutional Review Board of Shantou University Medical College. All participants provided their written consent to participate in this study, and procedures were performed following our institutional guidelines and the Declaration of Helsinki. All animal work was carried out following protocols approved by the Institutional Review Board of Shantou University Medical College (Title of the ethics approval project “The role of human umbilical cord mesenchymal stem cells and their exoproducts in the preconstruction of blood supply in skin flap,” approval number, SUMC2022-603 and date of approval, Oct. 27, 2022). Mice were monitored daily by laboratory members and by animal health technicians.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Ma, S., Ni, J., Ye, D. et al. Human umbilical cord mesenchymal stem cells improve the survial of flaps by promoting angiogenesis in mice. Eur J Med Res 30, 356 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02602-7

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02602-7

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European Journal of Medical Research

ISSN: 2047-783X

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