Human umbilical cord-derived mesenchymal stem cells and auto-crosslinked hyaluronic acid gel complex for treatment of intrauterine adhesion

hUC-MSCs preparation

Frozen hUC-MSCs from passages (P) 4 to P8 were thawed and cultivated in DMEM with 1% penicillin/streptomycin (Gibco, USA) and 10% (v/v) fetal bovine serum (Gibco, USA) under conditions suitable for cell growth (37° C and 5% CO₂). After 48 hours, upon reaching 80-90% confluence, cells were harvested by stem cell digestive juice (Gibco, USA) treatment for subculturing. Experiments utilized cells from both the fourth and eighth passages.

hUC-MSCs culture and recovery on HA-gel

hUC-MSCs (1×105 cell per well) and 300 ul of HA-gel [Con., 5 mg/ml; Bioregen, Co., Ltd., China, approved by CFDA as a medical device (no. 20153641542)] were gently mixed evenly with sterile syringes in 24-well plates and then added DMEM (Gibco, USA), including 10% (v/v) FBS (FBS, Gibco, USA),1% penicillin/streptomycin (Gibco, USA). HA-gel was digested using hyaluronidase following a 48-hour co-culture and released hUC-MSCs, which were recovered and cultured for 48 hours at 37° C and 5% CO₂ incubator. Afterwards, the cells were harvested using pancreatin digestion for use in future experiments.

Phenotype characterization and identification of hUC-MSCs on an HA-gel

The morphological parameters of hUC-MSCs, mixed both with or without HA-gel, were observed at 0, 24, and 48 hours of culture. Meanwhile, Cell morphology was observed after culturing for 48 hours when hUC-MSCs (1×105 cells per well), released from HA-gel, were plated in 6-well plates. The representative pictures were captured using the Olympus CKX4 microscope (Olympus, Japan) after 24 and 48 hours post-culture. To assess the phenotype of the recovered hUC-MSCs, cell surface markers (CD90, CD105, CD34, CD45) were detected with fluorescence-activated cell sorting (FACS). To identify the differentiation potential of hUC-MSCs on HA-gel, the mesenchymal stem cells’ capabilities to promote osteogenesis, adipogenesis, and chondrogenesis were evaluated. Briefly, recovered hUC-MSCs (4×104 cells per well) were plated in 6-well plates and treated with specific induction media. Specifically, osteogenic induction medium (Invitrogen, USA) and adipogenic differentiation medium (Invitrogen, USA). Alizarin Red S (Invitrogen, USA) staining was used to confirm the positive induction of osteogenic differentiation after 21 days, and oil red O (Invitrogen, USA) staining was used to confirm the positive induction of adipogenic differentiation after 14 days. Chondrogenic induction was performed using the pellet method, and after 14 days of induction, Alcian Blue (Invitrogen, USA) and Safranin O (Invitrogen, USA) staining were applied for identification. This comprehensive strategy enabled a thorough evaluation of the hUC-MSCs’ multipotent differentiation capabilities.

hUC-MSCs safety assessment on an HA-gel

To investigate the safety of hUC-MSCs on the HA-gel and to evaluate the viability of hUC-MSCs, trypan blue staining was performed. This approach enabled accurate counting of the live cell following isolation hUC-MSCs from HA-gel. The experiment was carried out in three replications to ensure reliability and consistency in results.

Cell proliferation was tested on hUC-MSCs and released hUC-MSCs from HA-gel. plates with 96 wells with DMEM and 1× 4 cells were sown per well, and cells were cultivated for 4 hours. The manufacturer’s directions assessed stem cell proliferation using the CCK-8 assay (Abcam, UK). The microplate reader measured optical density at 450 nm after 24, 48, and 72 hours. Eight complex holes were present in each group, and statistics were gathered for the average optical densities.

An Annexin V Apoptosis Detection Kit from Invitrogen in the United States was used to identify cell apoptosis. At 48 hours after co-culture, hUC-MSCs were collected and followed by hyaluronidase digestion. For control, hUC-MSCs were cultivated in DMEM with 10% FBS as the negative control and in water as a positive control. Cells were put in into Eppendorf tubes to undergo the process of centrifugation, for 5 minutes at 4° C while being given two PBS washes collecting 1~5× cells. In the following step, 5 μl of FITC-labeled annexin V in 100 μl of binding buffer, 15 minutes at room temperature, and 10 μl of PI were added.400 μl of binding buffer was added stirred and put on the ice following the anti-dark reaction. Data analysis was done using FACS.

Scratch assay was used to gauge the capacity of the cells to migrate. DMEM without FBS was used to seed cells on six-well plates, and they were allowed to develop until 90% of them were confluent. After cell adherence, the plates were incubated with serum-free medium in an incubator with 5% CO₂ at 37° C as a step after being scratched with a sterile pipette tip to make straight wounds. The cells were examined using an Olympus (Japan) microscope at 0 and 16 hours.

hUC-MSCs cytogenetic evaluation on an HA-gel

Telomerase activity of hUC-MSCs on an HA-gel was examined using a modified gel-telomerase repeated amplification technique (TRAP) test. The positive control was hUC-MSCs cultured with a regular medium, while as the negative control Hela cells and human skin fibroblasts were used, and the baseline was set to a TSR8 control. Using the TRAPeze telomerase detection kit in accordance with the manufacturer’s instructions, cells were screened for telomerase activity. The following step included the utilization of cells to be tested for telomerase activity using TRAPese telomerase detection kit, in accordance with the manufacturer’s instructions.

To evaluate the chromosome stability of the hUC-MSCs on an HA-gel, karyotype analysis was used to recover hUC-MSCs from HA-gel compared to hUC-MSCs undergoing similar analysis. Colchicine was briefly added to the culture 2 hours prior to the end of the process at a final concentration of 20 μg/ml. The cells were then collected and centrifuged for 10 minutes at 2000 rpm after being digested for 8 minutes with 2 cc of pancreatic enzymes and EDTA solution. In 8 ml of a hypotonic solution containing 0.075 M KCl, cell pellets were then resuspended, incubated for 30 min at 37° C in a water incubator, and then centrifuged for 10 minutes at 2,000 rpm after being fixed in 2 ml of a 3:1 methanol to the acetic solution. After further centrifugation, the supernatant was taken out, and 8 ml of ice-cold fixative solution was added. After being put onto a cold slide, Giemsa staining was applied after the cells had been trypsinized and treated at 75° C for three hours. Metaphase status image was captured using a Leica fully automated scanner (Leica, Germany), and karyotyping was analyzed with CytoVision software (Leica, Germany).

Establishment of the IUA-induced rat model

Female Sprague-Dawley rats weighing 250±20g were purchased from the Guangdong Medical Experimental Animal Center with license number SCXK (Yue) 2018-0002. The rats were kept in a controlled environment in which we have maintained sterility, by ensuring the absence of contaminants. In addition, the rats were provided with unrestricted availability of food and water. The ethical utilization of animals in this study was approved by the Ethics Committee of Guangzhou Women and Children’s Medical Center.

To minimize individual differences and ensure reliable data, we used the random assignment method for grouping. The 40 female SD rats were numbered individually, and then the rats were randomly divided into 5 groups (8 rats each), including control (CON) group, IUA group, HA-gel group, hUC-MSCs group, and combined treatment (HA-gel+hUC-MSCs) group. Among 8 rats in each group, 1 was used for pathology testing, 3 for protein molecular testing, and 4 for in vivo imaging and fertility experiments.

The IUA model was established using mechanical injury and lipopolysaccharide (LPS) induction [28]. After administering chloral hydrate anesthesia, all rats were put on the operation table. A surgical procedure involved creating a 3 cm longitudinal cut along the central axis of the abdominal region to gain access to the uterus. Subsequently, a small incision of approximately 3 mm was made in the lower 1/3 portion of the uterus. A 2-3 mm diameter scraping spoon gently scraped and damaged the endometrium until the uterine wall became rough and semitransparent. Vascular forceps were employed to secure the ends of the uterus, while an LPS solution measuring 0.5 ml in volume was introduced into the uterine cavity. The solution was withdrawn after 5 minutes. Lastly, the uterus was sutured after LPS-soaked cotton thread was introduced into the uterine cavity. Following the cleansing of the abdominal cavity with a normal saline solution, before closure. After 24 hours of post-operation, the cotton thread was removed.

In the IUA group, no additional treatment was administered after modeling. Conversely, in the hUC-MSCs group, after modeling, 1 ml of normal saline solution containing 3×10^5 hUC-MSCs was injected into one side of the uterine horn. In the HA-gel group, an experimental treatment consisted of administering 1 mL of uterine protective gel. This was injected into one side of the uterine horn after the modeling procedure. In the HA-gel+hUC-MSCs group, 1 ml of uterine protective gel containing 3×10^5 hUC-MSCs was injected into one side of the uterine horn after modeling. During the treatment period, all groups except for the IUA group received oral administration of 0.026 mg estrogen daily as an adjuvant therapy. Rats were euthanized after 4 weeks of therapy, and endometrial tissues were excised and subsequently immersed in a 10% formaldehyde solution for fixation in preparation for subsequent analysis.

Functional assessment of the uteri

7 days post-surgery and treatment injections, a subgroup of rats (n = 4) was transferred to separate cages, where they were paired with male rats for 96 hours to enable mating. After 10 days post-mating, the female rats were euthanized, and the number of fetuses on both uterine sides was counted.

Hematoxylin-eosin staining

Rat endometrial tissues were dried in a graded series of alcohol, cleaned in xylene, and fixed in paraffin at 60° C to observe the histopathological changes. The pieces of the paraffin blocks were then flattened by floating them in a 42° C water bath after being sectioned (3 μm thick). The sections were then mounted on glass slides and incubated at 60° C overnight. The paraffin sections were successfully deparaffinized for 10 minutes in xylene I and II. Following deparaffinization, the sections were immersed for 2 minutes in 100%, 95%, and 75% ethanol, respectively. The specimens were subjected to a hematoxylin staining process for 3 minutes. This was followed by differentiation in a solution of 0.5% hydrochloric acid alcohol for 3 seconds, after which the specimens were washed with tap water for 10 seconds. The counterstaining procedure was performed using eosin for 5 minutes. Following this the sample was rinsed with tap water to remove any remaining dye and was dehydrated for two minutes in ethanol concentrations of 85%, 95%, and 100%. The sections were then immersed in xylene I and xylene II for 3 minutes each. To finalize the preparation, neutral resin was used to mount the sections, which were dried and stored. Three fields of view from each sample of uterine tissue were chosen for high-power microscopy examination.

The width of the uterine cavity was measured using ImageJ software, and the number of endometrial glands was recorded.

Masson staining

To access the collagen deposition in rat endometrium, paraffin-embedded sections were first deparaffinized and hydrated. They were stained with Masson’s trichrome stain, rinsed quickly with a 2% acetic acid solution, then differentiated for 5 minutes with 5% phosphotungstic acid, soaked in a 0.2% acetic acid solution and stained for 5 minutes with a bright green staining solution, three times with 0.2% acetic acid solution, twice with 100% ethanol to dehydrate, and once with xylene to clear for 5 minutes, and finally sealed with neutral gum. Using the high-power microscope, 3 fields of view for each uterine tissue sample were examined. The ratio of collagen deposition within endometrial tissue was calculated using ImageJ software.

Immunofluorescence assay

Tissues were fixed for 20 minutes in 95% alcohol. Following this, the sections were subsequently subjected to treatment with a solution of 0.5% Triton X-100 (Sigma, USA) at ambient temperature for a duration of 20 minutes. The samples were incubated for a total of one hour at a temperature of 37° C, in the presence of 10% normal goat serum obtained from Gibco, United States. Subsequently, the slides underwent treatment with primary antibodies targeting the human nuclear antigen (Abcam, USA) and vimentin (Abcam, USA) for an extended period at 4° C, following the initial blocking step. After that, the sections were treated with Alexa Fluor-conjugated secondary antibodies from the American company Thermo Fisher Scientific for an additional hour at room temperature. 4,6-diamidino-2-phenylindole (Life Technologies, USA) staining was performed to visualize the DNA. Finally, fluorescence microscopy images were captured using a Leica DMI6000B microscope completing the immunofluorescence procedure.

Western blot

The bicinchoninic acid (BCA) assay was used to extract and quantify total protein lysates. Following this, a total of 50 μg of protein was applied onto a sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis. After the electrophoresis, the proteins were then transferred onto polyvinylidene fluoride membranes. To prevent non-specific binding, these membranes were incubated at room temperature for an hour in a solution containing 5% bovine serum albumin (BSA). Once the blocking step was completed, we conducted a two-hour incubation with primary antibodies against SDF-1 (1:300, Santa Cruz, USA), CXCR4 (1:500, Santa Cruz, USA), TAZ (1:300, Abcam, USA), and p-transcriptional co-activator with PDZ-binding motif, referred to as p-TAZ (1:800, CST, USA). The membranes were then incubated with a secondary antibody (1:1,000, Beyotime, China) that was conjugated to horse radish peroxidase. As a loading control, glyceraldehyde-3-phosphate dehydrogenase was utilized. Using the Tanon-5200 gel imaging system, the bands were visualized.

Statistical analysis

Statistical analyses were conducted using SPSS software (version 22.0 IBM, USA). The mean ± standard deviation for continuous variables was presented. A one-way analysis of variance (ANOVA) was employed to compare group means, followed by the LSD test for between-group comparisons. Analyzing the outcomes of fertility tests was done using the 2. One, two, and three asterisks, respectively, indicate that the significance levels of P< 0.05, P < 0.01, and P < 0.001, respectively. A significance threshold of P < 0.05 was employed.

Data availability

Upon reasonable request, the data will be made available to interested parties.

Consent for publication

All authors have granted their permission for the publication of identifiable information, including the text, figures, and other materials, contained in this manuscript.

Phenotype characterization and verification of hUC-MSCs on HA-gel

In the group treated with 50%HA-gel most of the cells were able to migrate out of the gel, and the migrated cells were able to adhere and grow well. In contrast, after 48 hours, in the 80%HA-gel group, the cells remained trapped within the gel forming a spherical form (Figure 1A). In addition, FACS results indicated that CD90 (99.80%) and CD105 (95.20%) were positive makers; CD34 (0.39%) and CD45 (0.10%) were the negative makers (Figure 1B). After being cultured in an enriching environment, the positively identified cells differentiated into osteoblasts, which initiated bone matrix formation, chondroblasts, which formed chondrogenic pellets within 21 days of induction, and adipocytes, which developed lipid droplets within 14 days of induction, as shown in Figure 1C.

Figure 1. hUC-MSCs characteristics and differentiation potential. (A) The effect of HA-gel on the biological behavior of hUC-MSCs; (B) Surface markers characteristic of hUC-MSCs measured by flow cytometry; (C) The morphologic and intrinsic characterization of hUC-MSCs.

Safety assessment of hUC-MSCs on an HA-gel

The hUC-MSCs in the 50%HA-gel group were able to migrate out of the gel and exhibit average adherent growth. Hence there is no need to conduct further tests on their adhesive capabilities in this group. In the 80% HA-gel group, the condition of the cells was observed after culturing for 48h, and the gel was enzymatically digested to recover the cells for seeding into a 6-well plate. After that, the cells were cultured for 24 and 48 hours, and their morphology was observed to assess whether the gel had any impact on the adherent growth of MSCs. After 48 hours, the hUC-MSCs derived from the 80% HA-gel group still exhibited typical morphology characteristics of mesenchymal stem cells. They appeared spindle-shaped and showed adherent growth (Figure 2A). The flow cytometry study results revealed that compared to the PBS control group, the cells from the 50%HA-gel group did not exhibit a significant increase in necrotic, early, and late apoptotic cells (Figure 2B). The CCK-8 assay results indicated that after 48 hours of culture with 50% and 80% HA-gel, the hUC-MSCs did not significantly impact their growth and proliferation capability when returned to normal culture conditions (Figure 2C). The scratch assay results showed that both the 50% and 80% HA-gel did not significantly affect the migration ability of hUC-MSCs cells (Figure 2D).

Figure 2. Safety assessment and verification of hUC-MSCs on an autocrosslinked HA-gel. (A) The influence of HA-gel on the morphology and adhesion of hUC-MSCs; (B) The influence of HA-gel on apoptosis of hUC-MSCs by FACS; (C) The effect of HA-gel on the proliferation ability of hUC-MSCs evaluated using CCK-8 assay; (D) The effect of HA-gel on the migration ability of hUC-MSCs evaluated by scratch assay.

The effects of HA-gel on the genetic behavior of hUC-MSCs

Figure 3A demonstrates that Hela cells exhibit significant telomerase activity. In comparison to Hela cells, the telomerase activity of hUC-MSCs cultured with 50% and 80% GA-GEL for 48 hours has shown markedly lower levels. This indicates that the telomerase activity of MSCs is not abnormally upregulated in the presence of HA-gel, which furthermore enhances that HA-gel-treated hUC-MSCs do not pose a risk of tumor formation. Additionally, the chromosomal structure’s specific morphology is exhibited with clarity in Figure 3B. After 48 hours of culture with 80% HA-gel, there is no apparent occurrence of deletions, duplications, inversions, or other abnormalities in the chromosomes of hUC-MSCs.

Figure 3. The effects of HA-gel on the genetic behavior of hUC-MSCs. (A) Telomerase activity detection; (B) Chromosomal karyotype analysis.

The effect of HA-gel on hUC-MSCs in the IUA model

A total of 40 rats are divided into 5 experimental groups: ① Control group (CON), ② IUA group, ③ HA-gel group, ④ hUC-MSCs group, ⑤ HA-gel + hUC-MSCs group. Each group consists of 8 rats. When compared to untreated hUC-MSCs, the morphological differences in hUC-MSCs treated with HA-gel were merely insignificant, and the cell growth status was good in both groups. The effectiveness of cell membrane labeling was evaluated using PKH26 dye, and subsequent observation under a fluorescence microscope revealed that cells from both groups showed significant staining. As shown in Figure 4A there was no significant difference in the number of stained cells between the groups. In vivo, imaging was used to observe the residence time of hUC-MSCs in the rat uterine endometrium and evaluate HA-gel’s supporting and fixing effect on hUC-MSCs at the uterine site. HA-gel could prolong the residence time of hUC-MSCs in the rat uterine endometrium (Figure 4B).

Figure 4. The effect of HA-gel on hUC-MSCs in IUA model. (A) The morphological differences; (B) The residence time of hUC-MSCs in the rat uterine endometrium.

Endometrial morphological structure and extracellular matrix deposition

HE staining results showed that in the CON group, the rat uterine endometrium consisted of neatly arranged single-layer columnar epithelial cells and stroma with abundant small blood vessels. Oval or circular endometrial glands were distributed in the submucosa and basal layers, and the uterine cavity was in an open state. Compared to the CON group, the IUA group exhibited a narrower uterine cavity (P<0.01), thinner endometrium, disorganized arrangement of epithelial cells, partial glandular atrophy, and decreased glandular quantity (P<0.05). The HA-gel group demonstrated a considerably bigger uterine cavity than the IUA group (P <0.001), with no significant changes in endometrial thickness, partial glandular atrophy, or glandular quantity (P>0.05). The hUC-MSCs group on the other hand exhibited a significantly wider uterine cavity (P<0.01), thicker endometrium, partial glandular atrophy, and increased glandular quantity (P<0.05). In the HA-gel+hUC-MSCs group, there was a notable enlargement of the uterine cavity (P<0.05), alongside with a thicker endometrium. The glands were predominantly oval or circular, and there was a significant increase in glandular quantity (P<0.01). These results indicate a remarkable improvement in tissue structure (Figure 5A).

Figure 5. Effect of different treatments on endometrial regeneration and collagen remodeling. (A) HE stains shows the width of uterine cavity and number of endometrial glands; (B) Masson staining indicates the degree of endometrial fibrosis.

Results from Masson staining revealed that the IUA group of rats had much more collagen deposition than the CON group(P<0.001) and severe fibrosis of the uterine endometrium. Compared to the IUA group, the HA-gel group showed some improvement in uterine endometrial tissue fibrosis, but the difference was insignificant (P>0.05). Rats in both the hUC-MSCs group and the HA-gel+hUC-MSCs group exhibited a significant decrease in collagen deposition in the uterine endometrial tissue(P<0.001), while the collagen deposition in HA-gel+hUC-MSCs group was less than that in the hUC-MSCs group (P<0.001) (Figure 5B).

Effects of different treatments therapy on endometrial proliferation, fibrosis and estrogen regulation

We further investigated the effects of HA-gel+hUC-MSCs therapy on fibrotic cytokines. Integrin β1, ER, TGF-1, Ki67, Vimentin, and VEGF were significantly upregulated, while MMP-9 was considerably downregulated in the IUA group compared to the CON group (Figure 6A). While the expression of the Integrin β1, ER, TGF-β1, Ki67, Vimentin, and VEGF was dramatically increased, MMP-9 was decreased in the HA-gel+hUC-MSCs group compared with the IUA group (Figure 6A). The HA-gel + hUC-MSCs group exhibited notable alterations in the expression of genes associated with fibrosis, estrogen, and cellular differentiation. Moreover, the IHC analyses revealed that the expression of levels of Integrin β1, ER, and TGF-β1 expression levels were consistent with those of Western blot (Figure 6B).

Figure 6. Effects of different treatments on endometrial proliferation, fibrosis, and estrogen regulation. (A) The expression levels of Integrin β1, ER, TGF-β1, Ki67, Vimentin, VEGF, and MMP-9 were determined by Western blot; (B) Integrin β1, ER, and TGF-β1 were confirmed by immunohistochemical analyses. RE, Estrogen Receptor; TGF, Transforming Growth Factor; VEGF, Vascular Endothelial Growth Factor; MMP, Matrix Metalloproteinase.

Effects of different treatments on fertility restoration

To evaluate the effect of HA-gel+hUC-MSCs treatment on fertility restoration, pregnant uteri and the associated embryos were collected with the outcomes shown in Figure 7. The IUA group had a considerably decreased pregnancy rate and a reduced quantity of embryos compared to the CON group. However, the HA-gel+hUC-MSCs group exhibited improved fertility compared with the other groups. The findings suggest that the utilization of HA-gel + hUC-MSCs treatment has the potential to effectively regenerate a functional endometrium, therefore facilitating the effective implantation of embryos and increasing fertility restoration prospects.

Figure 7. Effects of different treatments on fertility restoration.