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Therapeutic effect of three-dimensional hanging drop cultured human umbilical cord mesenchymal stem cells on osteoarthritis in rabbits

Stem Cell Research & Therapy volume 15, Article number: 311 (2024) Cite this article

Abstract

Background

Mesenchymal stem cells (MSCs) have shown a positive effect on Osteoarthritis (OA), but the efficacy is still not significant in clinical. Conventional two-dimensional (2D) monolayer culture method is prone to cause MSCs undergoing replication senescence, which may affect the functions of MSCs. Three-dimensional (3D) culture strategy can sustain cell proliferative capacity and multi-differentiation potential. This study aimed to investigate the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured by 3D hanging drop method on OA.

Methods

hUC-MSCs were isolated from umbilical cord and cultured by 3D hanging drop method for 48 h. Scanning electron microscopy (SEM) was used to observe gross morphology 2D and 3D hUC-MSCs. Transcriptome comparison of gene expression differences between 2D and 3D hUC-MSCs. GO enrichment analysis, KEGG pathway enrichment analysis and GSEA enrichment analysis were used to analyze the impact of 3D hanging drop culture on the biological functions of hUC-MSCs. Female New Zealand rabbits (n = 12) were divided into 4 groups: Normal group, Model group, 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group. After 8 weeks, the gross and histological appearance of the cartilage was evaluated by safranin O-fast green staining and Mankin scoring system. The expression of type I collagen and type II collagen was detected by immunohistochemistry. The levels of IL-6, IL-7, TNFα, TGFβ1 and IL-10 in the knee joint fluid were tested by ELISA.

Results

3D hanging drop culture changed cell morphology but did not affect phenotype. The MSCs transcriptome profiles showed that 3D hanging drop culture method enhanced cell-cell contact, improved cell responsiveness to external stimuli and immunomodulatory function. The animal experiment results showed that hUC-MSCs could promote cartilage regeneration compared with Model group. 3D hUC-MSCs treatment group had a higher histological score and significantly increased type II collagen secretion. In addition, 3D hUC-MSCs treatment group increased the expression of anti-inflammatory factors TGFβ1 and IL-10.

Conclusion

The above experimental results illustrated that 3D hanging drop culture method could enhance the therapeutic effect of hUC-MSCs, and showed a good clinical application prospect in the treatment of OA.

Introduction

Osteoarthritis (OA) is a degenerative joint disease with a high incidence in middle-aged and elderly people. In China, 10–17% of people over 40 years old suffer from OA, and the number of patients with OA continues to increase as population aging situation aggravating [1]. OA is currently considered a complex multifactorial disease of joints, rather than being simply viewed as a classic degenerative disease caused by wear and tear. Increasing evidence supports that low-grade chronic inflammation plays an important role in OA [2]. Immune cells infiltrate joint tissue in the early stages of inflammation, various joint cells release cytokines and chemokines, then the complement system is activated. Subsequently, cartilage degradation factors such as matrix metalloprotein (MMP) and prostaglandin E2 (PGE2) are released, resulting in extracellular matrix (ECM) metabolic imbalance and irreversible cartilage degradation [3].

Current treatment strategies for OA mainly include pharmaceuticals and joint replacement surgery. Non-steroidal anti-inflammatory drugs (such as ibuprofen, opioid analgesics) and immunosuppressants (such as methotrexate) can suppress inflammation and relieve some symptoms, but their ability to promote cartilage repair is not sufficient. Besides that, the fibrocartilage is easily formed after joint replacement surgery, its mechanical properties are inferior to those of articular cartilage [4, 5]. With the development of tissue engineering and cell regeneration technology, stem cell-based therapy shows encouraging results in repairing damaged cartilage [6,7,8].

Mesenchymal stem cells (MSCs) can be obtained from bone marrow, adipose tissue, umbilical cord blood, umbilical cord stroma (Wharton’s jelly) and other tissues. Since their excellent differentiation ability and immunomodulatory function, MSCs are widely used for the treatment of a variety of diseases in clinical, including OA [9,10,11,12]. Many in vitro studies have shown that multiplication and differentiation properties of MSCs are related to the tissue source, health status and the physiological status of the donor [13]. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are more suitable for clinical application because the advantages of higher expansion potential, lower immunogenicity and higher paracrine potential than other tissue-derived MSCs [14]. In addition to the therapeutic efficacy, the lower cost of cell preparation is also an important factor for the wider application of hUC-MSCs. Therefore, treatment of OA with hUC-MSCs may be an affordable, safe and effective treatment option [15].

Existing studies show that MSCs can promote chondrocyte regeneration and inhibit extracellular matrix degradation in OA treatment. The most important thing is that MSCs can attenuate the inflammatory microenvironment induced by macrophages [16]. However, conventional two-dimensional (2D) culture method often fails to maintain the stability of MSCs, leading to cellular aging and decline in cell functions. It ultimately affected the clinical efficacy of MSCs in treating OA [17, 18]. Three-dimensional (3D) cell culture system can simulate conditions similar to the microenvironment in vivo, and the 3D cultured cells exhibit a variety of in vivo-like characteristics in terms of morphology, behavior, metabolism, cell heterogeneity, and cell-cell or cell-matrix interactions [19, 20]. The hanging drop method is often used as a 3D culture method due to its simple operation and does not require expensive or specific tools. 3D hanging drop culture method can keep MSCs in an undifferentiated state and maintain their high migration properties, it has been shown to enhance the anti-inflammatory effect of MSCs [21].

In this study, the transcriptome profiles of 2D and 3D hUC-MSCs were compared to investigate the impact of 3D hanging drop culture method on cell therapy efficacy. Furthermore, knee joint patellar cartilage defects were constructed in rabbit model. 2D hUC-MSCs and 3D hUC-MSCs were injected into joint cavity respectively for comparison of cartilage repair ability. After 8 weeks, the statuses of the cartilage repair in different groups were assessed by gross and histological appearance observations, modified Mankin scoring system was used to quantify the degree of OA. The production of type I collagen and type II collagen was detected by immunohistochemical staining. For evaluating the immunomodulatory effect of hUC-MSCs, the expression levels of inflammatory cytokines in joint fluid were also detected by ELISA.

Materials and methods

2D culture of hUC-MSCs

The primary hUC-MSCs isolated from human Wharton’s Jelly were donated by Department of Respiratory Medicine and Neonatology of the Children’s Hospital of Chongqing Medical University. Cells were cultured in DMEM/F12 (Dakewe Biotech Co., China) supplemented with 5% human platelet lysate (Elitecell, USA) at 37 °C and 5% CO2. Cells were sub-cultured onto plastic flasks when the density reached about 80%. The third passage (p3) was used for identification and follow-up experiments.

p3 hUC-MSCs were harvested by trypsin/EDTA (0.05%/0.02%), adjusted the cell concentration to 106/mL with saline, and filtered through a 100 μm sieve. 100 µL of cell suspension was incubated with CD34-PE, CD44-FITC, CD45-FITC, CD73-PE, CD90-PE, CD105-PE, HLA-DR-PE and isotype control, respectively. All antibodies were purchased from Biolegend (California, USA). Washed three times with PBS and analyzed using flow cytometer (Novocyte 2040 R, Agilent, USA).

3D hanging drop culture of hUC-MSCs

3D hanging drop culture method was performed as previously described [22]. Briefly, After the density of hUC-MSCs reached 90%, cells were trypsinized and centrifuged at 300 g for 10 min. Then cells were resuspended in culture medium, dropped cell suspension onto the culture dish at 35 µL/drop (2 × 105 cells/drop). Inverted the culture dish, placed it in a tray containing PBS to prevent evaporation at 37 °C and 5% CO2. After culturing for 48 h, the hanging spheroids were collected, and cells were dispersed by trypsin/EDTA.

Scanning electron microscopy (SEM)

Harvested 2D hUC-MSCs and 3D hUC-MSCs by trypsin/EDTA respectively, washed with PBS. Fixed with Karnovsky’s fixative (2% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer) for 30 min at room temperature in the dark, washed with PBS. After 1% osmium tetroxide post-fixation and ethanol dehydration (from 30 to 100%), treated the sample with a mixture of ethanol and isoamyl acetate (V/V = 1/1) for 30 min. Treated with pure isoamyl acetate for 1 h and dried by supercritical CO2 method, immobilized to coverslips via a one-component cyanoacrylate adhesive, and mounted onto aluminum holders using conductive tabs. Finally sputter coated with a gold layer. Analysis was performed using a field-emission scanning electron microscope (SU8010, Hitachi, Japan). SEM images were recorded with an acceleration voltage of 10 kV.

Transcriptome sequencing of 2D and 3D hUC-MSCs

Harvested 2D hUC-MSCs and 3D hUC-MSCs, RNA was extracted using the Trizol reagent (Life Technologies, Carlsbad, California, USA) following manufacturer’s instructions, synthesized cDNA using RNA as template. The quality of cDNA library was evaluated by the Agilent Bioanalyzer 2100 system. Paired-end sequencing was carried out on HiSeq 2500 (Illumina). Raw reads were quality checked with FastQC and then mapped against the Human Reference Genome GRCh38 (Ensembl) using HISAT2. Differentially expressed genes (DEGs) were defined considering a false discovery rate (FDR) with a significance threshold of log2FoldChange > 1 and p < 0.05. Functional annotation of DEGs, GO enrichment and KEGG enrichment analyses were performed in R.

Subsequently, gene set enrichment analysis (GSEA) was employed to perform the enrichment analysis. The normalized read counts were imported into the GSEA (v4.3.2) platform from Broad Institute with two gene sets being tested, including gene ontology and KEGG pathway. An enrichment score (ES) for each gene set was calculated. This ES reflected the degree to which a gene set was overrepresented at the top or bottom of a ranked list of genes. Tested gene sets were considered to be statistically enriched when p < 0.01 and FDR < 0.25.

Ethical considerations

The female New Zealand rabbits, weighing (2.5 ± 0.3) kg, were purchased from Suzhou Xishan Biotechnology Co., Ltd. (Suzhou, China). The Institutional Ethics Committee of Chongqing Perfect Cell Biotechnology Co., Ltd. approved all experimental protocols. All surgical procedures were performed in accordance with Guidelines for the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35,892–2018 (Issued 6 February 2018 Effective from 1 September 2018). All experimental facilities had a national authorization to perform animal experiments (approval ID: SYXK 2020-0017). This study had been reported in line with the ARRIVE guidelines 2.0.

All animals were placed in separate cages and allowed to move freely. Administered intramuscularly ampicillin sodium (20 mg/kg) twice daily to prevent infection for seven days postoperatively [23]. Observed daily for their general health condition, local infection and mobility.

Sample size

The sample size was calculated by using the resource equation approach. Considering the error degrees of freedom (DFs), the DFs in the following formulas were replaced by 20 [24]. This study was aimed to compare four groups with three repeated measurements. Therefore, the numbers of animals per group required were:

n = 20 / kr + 1 = 20 / (4 × 3) + 1 = 2.67 =  rounded up to 3 animals/group.

The total numbers of animals required were:

$$\text{N} = {\text{n}}\times\text{k}\text{ =}\text{ 3}{\:\times\:4=}\text{ 12 }\text{ animals}.$$

Where N = total number of subjects, k = number of groups, n = number of subjects per group, and r = number of repeated measurements. According to the calculation results, 12 rabbits were finally used for subsequent experiments.

Cartilage defect models and cell implantation experiments

Rabbits were randomly divided into four groups: Normal group (without surgery and treatment), Model group (animals undergoing knee cartilage defect surgery and receiving saline injection through the knee joint cavity), 2D hUC-MSCs treatment group (animals undergoing knee cartilage defect surgery and receiving 2D cultured hUC-MSCs injection through the knee joint cavity) and 3D hUC-MSCs treatment group (animals undergoing knee cartilage defect surgery and receiving 3D hanging drop cultured hUC-MSCs injection through the knee joint cavity). The rabbits were anesthetized by intravenous injection of pentobarbital sodium (40 mg/kg) into the marginal ear vein, combined with intramuscular injection of xylazine hydrochloride solution (30 mg/kg) for analgesia. Both knee joints were disinfected by iodophor and opened via a medial parapatellar approach, the patella was dislocated laterally. Full-thickness osteochondral defects (4 mm in diameter, 5 mm in depth) were created in trochlear grooves with a hand drill. Removed the cartilage and bone debris, washed defected site with saline. Relocated the patella, closed soft tissue in layers [25, 26].

Doses used in preclinical studies of MSCs to treat knee arthritis varied by species and body size, with doses injected into rabbit models ranging from 106 to 17 × 106 cells [27]. In this study, for 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group, resuspend 2 × 106 cells in 1 mL of saline and injected them into the joint cavity, respectively. Model group was treated with 1 mL saline. The animals were euthanized by intravenous injection of 100 mg/kg sodium pentobarbital (Sigma Aldrich, USA) at 8 weeks post-surgery, the knee joints and joint fluid were collected for evaluation, and the carcasses were bagged and frozen.

Safranin O-fast green staining and Mankin score

Briefly, the knee joints of four groups were fixed in 4% paraformaldehyde for 48 h and washed with PBS. Decalcification for two weeks then embedded in paraffin. Cut into 5 μm sections with a microtome. For safranin O-fast green staining, samples were stained with fresh Weigert’s staining solution for 4 min, followed by 5 min with Fast Green and 5 min with Safranin O solutions. Sealed the slides with resin for observing. Image J was used to quantify the percentage of positive areas stained by safranin O-fast green as previous described [28]. Briefly, red colour intensity (RCI) was calculated in the entire cross-sectional slice area by obtaining red, green, and blue (RGB) image planes in a scale of 256 values (black = 0). The fraction of red (RF) was defined as the ratio of the R component to the sum of the R, G, and B components: RF = R/(R + G + B) and expressed as a percentage. Severity of OA was assessed according to the modified Mankin scoring system, which ranges from 0 to 14 points including four characteristics: structure (0–6 points), cellular abnormalities (0–3 points), matrix staining (0–4 points), and tidemark integrity (0–1 point) [29].

Immunohistochemistry staining

Slices were dewaxed in xylene and hydrated in ethanol aqueous solution. Heated at 95 °C in a 0.01 M sodium citrate buffer (pH = 6.0) to expose the surface antigens. Cooled at room temperature, then the sections were incubated with 3% H2O2 for 10 min to quench endogenous peroxidase activity, washed with PBS. Blocked in goat serum at 37 °C for 15 min, incubated with the primary antibody dilution solution directed against type I collagen (1:1000, Novus, USA) and type II collagen (1:500, Novus, USA) overnight at 4 °C. Subsequently, washed with PBS and incubated with biotinylated secondary antibody working solution at 37 °C for 20 min. Washed with PBS and incubated with streptavidin-HRP working solution at 37 °C for 20 min. Counterstained slices with DAB, and sealed with a glass slide by resin. The images were analyzed using Image J and quantified by assessing the average optical density (AOD) in each sample.

Enzyme-linked immunosorbent assay

Centrifuged joint fluids of four groups at 1000 g for 20 min, collected the supernatant. ELISA kits of rabbit IL-6 (ml027345), rabbit IL-7 (ml036818), rabbit TNF-α (ml027766), rabbit TGF-β1 (ml028093) and rabbit IL-10 (ml027158) were purchased from Shanghai Mlbio Biotechnology Co. (Shanghai, China), the levels of cytokines were detected according to the manufacturer’s instructions. Measured the OD value of each well at a wavelength of 450 nm, repeated three times for each group. Drew a standard curve according to the measured OD values of different concentrations of standard substances, obtained a linear regression equation. Substituted the OD values of the samples into the equation to calculate the concentration of the samples.

Statistical analysis

All experimental data were expressed as mean ± standard deviation. The results were analyzed by one-way analysis of variance (ANOVA) using Origin 2018. Statistical significance of differences between means was determined using Tukey’s double test, p-values less than 0.05 (p < 0.05) were considered statistically significant (* represented p < 0.05, ** represented p < 0.01, *** represented p < 0.001, ns represented no significance).

Results

Identification of hUC-MSCs

As presented in Fig. 1a, cells began to migrate from the edge of tissue after culturing for 7 days. Cells displayed fibroblast-like spindle-shaped morphology. After reaching high confluence, cells were subcultured. Flow cytometric data of p3 hUC-MSCs (Fig. 1c) demonstrated that cells were positive for CD44, CD73, CD90 and CD105, but negative for CD45, CD34 and HLA-DR. Specifically, the positive rate of cell count was 100% for CD44, 99.98% for CD73, 100% for CD90 and 99.93% for CD105, respectively. These results indicated the successful isolation of hUC-MSCs.

Fig. 1
figure 1

Identification of hUC-MSCs. (a) Morphology of 2D hUC-MSCs (scale bar represented 200 μm) and scanning electron microscopy (SEM) image of 2D hUC-MSCs (scale bar represented 5 μm); (b) 3D hanging drop culture method of hUC-MSCs and observation of 3D sphere after 48 h (scale bar represented 200 μm) and SEM image of 3D hUC-MSCs (scale bar represented 5 μm); (c) Flow-cytometric analysis of surface markers expressed on 2D hUC-MSCs; (d) Flow-cytometric analysis of surface markers expressed on 3D hUC-MSCs

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Identification of 3D hanging drop cultured hUC-MSCs

After culturing hUC-MSCs using 3D hanging drop method for 48 h, the cells aggregated into 3D spheres as shown in Fig. 1b. Flow cytometric data demonstrated the positive rate of cell count was 99.32% for CD44, 99.92% for CD73, 100% for CD90 and 96.27% for CD105, respectively. Besides that, cells were still negative for CD34, CD45 and HLA-DR.

Morphological comparison of 2D and 3D hUC-MSCs

SEM observed the differences of gross morphology between 2D and 3D hUC-MSCs. The surface of cells cultured in 2D was smooth except for protruding proteins, while 3D hUC-MSCs were slightly smaller and rougher. In particular, there were many microvilli-like structures on the cell surface (Fig. 1b), which was beneficial to cell interaction and intercellular communication [30].

Identification of differentially expressed genes between 2D and 3D hUC-MSCs

The analysis of differential gene expression tests was shown in Fig. 2. As shown in Fig. 2b, a total of 5910 genes were differentially expressed in the two groups of cells, of which 3235 genes were up-regulated, 2675 genes were down-regulated. 10,656 genes had no significant difference (|log2FoldChange| > 1 and p < 0.05). All significantly differentially expressed genes were shown in Supplementary Material 1.

Fig. 2
figure 2

Transcriptome analysis results. (a) The volcano plot of DEGs in 2D hUC-MSCs vs. 3D hUC-MSCs; (b) Venn diagram of DEGs in 2D hUC-MSCs vs. 3D hUC-MSCs; (c) The heatmap of DEGs in 2D hUC-MSCs vs. 3D hUC-MSCs; (d) GO analysis of up-regulated genes in 3D hUC-MSCs compared with 2D hUC-MSCs; (e) KEGG analysis of up-regulated genes in 3D hUC-MSCs compared with 2D hUC-MSCs

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GO enrichment analysis showed that compared to the 2D monolayer cultures, the formation of 3D aggregates led to a multitude of changes in mechanical stress, cell–cell and cell–ECM contacts, these factors in concert regulate MSCs properties. These up-regulated genes in 3D hUC-MSCs were enriched in biological processes such as response to stimulus (GO:0050896), cell communication (GO:0007154), and multicellular organism development (GO:0007275).

GO terms related to replicative senescence (GO:0090399), regulation of angiogenesis (GO:0045765), regulation of immune response (GO:0050776), biological adhesion (GO:0022610), cell migration (GO:0016477), extracellular exosome biogenesis (GO:0097734) were screened out, and significantly regulated genes were listed as shown in the Table 1. These biological functions affected the repair effect of MSCs after implantation into the knee joint. DEGs results indicated replicative senescence-related genes such as Tp53 and Serpine1were significantly downregulated in 3D hUC-MSCs. TP53 was a pro-apoptosis component of the DNA damage response and activated caspases. Serpine1 was known as “Plasminogen activator inhibitor-1” (PAI-1), which was transcriptionally activated by TP53 and played a role in senescence. Serpine1 also negatively regulated transplanted MSCs survival and adhesiveness via promoting anoikis [31].

From the perspective of secreting chemokines and cytokines, 3D hUC-MSCs significantly upregulated the expression of genes for pro-angiogenic factors (such as VEGF and FGF2) and inflammatory regulatory factors (such as IDO1, IL-1β, TSG-6, STC-1). TSG-6 could inhibit extravasation of leukocytes (mainly neutrophils and macrophages) at the site of inflammation, which played important role in OA. STC-1 secreted by MSCs might have both anti-inflammatory and anti-apoptotic effects by upregulating the expression of mitochondrial uncoupling protein 2 [32]. Although angiogenesis was associated with pain and OA progression, clinical results showed that increased VEGF level caused by injection of MSCs did not cause pain [33]. VEGF was a key survival factor for chondrocytes. In addition, the pro-angiogenic factor FGF2 could also exert anti-apoptotic effect. 3D hUC-MSCs also upregulated the expression of ICAM-1 and VCAM-1, which could enhance the immunosuppressive effect by improving adhesion to T cells [34].

For exosome synthesis, SDC4 and RAB27A/B were significantly expressed in 3D hUC-MSCs, these genes were involved in endosomal membrane budding to form multivesicular bodies (MVBs) and MVB distribution [35]. MSCs-derived exosomes were found to exert beneficial therapeutic effects on OA [36]. These results showed that 3D hanging drops method contributed to inhibit senescence caused by replication in vitro and improved cell biology functions, including migration, adhesion, vascular repair, immunomodulation and release of exosomes.

All significantly up-regulated genes were projected to 328 KEGG pathways (Supplementary Material 3). The top 20 positively enriched pathways were shown in Fig. 2e. The upregulated genes in 3D hUC-MSCs involved signaling pathways such as cancer pathway, TNF signaling pathway, MAPK signaling pathway, cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, and rheumatoid arthritis. TNF and TNF receptor (TNFR) superfamilies members regulated cell differentiation, survival and programmed death, but their most critical functions were related to the immune system [37]. It was found that various dysfunctional cross-talking intracellular signaling pathways played an important role in the pathogenesis of rheumatoid arthritis, such as JAK-STAT and NF-κB. These signaling pathways were involved in cell survival and immune regulation. Pathogenesis of OA was similar with rheumatoid arthritis, they were both characterized by polyarthritis with progressive joint damage, disability, immunologic abnormalities and inflammation [38]. The results showed that these upregulated genes in 3D hUC-MSCs were mainly involved in immune-related signaling pathways.

Table 1 List of significantly regulated genes in 3D hUC-MSCs compared with 2D hUC-MSCs
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GSEA analysis

GSEA analysis was employed for a deeper enrichment investigation, results showed gene sets related to cytokine responses were significantly overrepresented at the top of the ranked list (Tables 2 and 3). It further implied that 3D hanging drop culture could improve the sensitivity of cells to the external environment. Indeed, Acute phase response (ES = 0.74; FDR q-value = 0.00) gene set was ranked 6th in the list (Supplementary Material 2: Table S1), leading edge genes were shown in the Fig. 3a, these up-regulated genes played an important role in immunomodulation.

Fig. 3
figure 3

GSEA enrichment plots of 3D hUC-MSCs. (a) GSEA enrichment plot of acute phase response with leading edge gene subsets; (b) GSEA enrichment plot of rheumatoid arthritis with leading edge gene subsets

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GSEA analysis of KEGG pathway showed most of the positive enriched gene sets were related to immune-related signaling pathways, including Rheumatoid arthritis pathway. Leading edge genes were shown in the Fig. 3b, these upregulated genes were mainly related to monocyte-macrophage system migration. GSEA results were consistent with the KEGG results, indicating that 3D hanging drop culture method mainly up-regulated genes related to immune regulation, and could promote hUC-MSCs to secrete chemokines and cytokines for recruiting monocytes.

Table 2 List of top 5 positive enriched gene sets (GO terms) in 3D hUC-MSCs
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Table 3 List of top 5 positive enriched gene sets (KEGG pathway) in 3D hUC-MSCs
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Histological evaluation of rabbit knee articular defected cartilage

After 8 weeks, the incision of the operative site was healed well. All animals survived successfully with no adverse events observed. The cartilage defect in the 2D hUC-MSCs treatment group significantly decreased and had a distinct boundary. For the 3D hUC-MSCs treatment group, the regenerated tissue was well integrated with the surrounding tissue with no obvious boundary. However, the model group had larger defect area on the surface, with degenerate and hyperplasia (Fig. 4c).

Fig. 4
figure 4

Gross appearance observation at 8 weeks post-surgery. (a) Full-thickness osteochondral defects (4 mm in diameter, 5 mm in depth) in trochlear grooves; (b) The Mankin scores of cartilages in four groups. * indicated p < 0.05, ** indicated p < 0.01; (c) Observation of gross appearance of the cartilages in four groups

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Safranin O-fast green staining was used to observe the formation of new cartilage matrix microscopically. It was observed from Fig. 5a that the surface of the articular cartilage at the defect site was irregular, disordered arrangement of cells with slightly shallow cytoplasm staining, and no obvious collagen fiber formation in the model group. The defects of the 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group were filled with neocartilage, and there were a large number of cartilage-like cells tightly arranged, accompanied by the formation of cartilage lacuna. Quantitative result of safranin O staining showed that both 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group significantly promoted cartilage matrix formation (p < 0.05). The content of collagen fibers in the 3D hUC-MSCs treatment group was higher than that in the 2D hUC-MSCs treatment group.

Fig. 5
figure 5

Histological staining evaluation. (a) Safranin O-fast green staining, type I collagen and type II collagen immunohistochemical staining. The scale bar represented 100 μm; (b) Quantify the percentage of positive areas stained by safranin O. * indicated p < 0.05, ns indicated no significance; (c) Quantify the percentage of positive areas stained by type I collagen. * indicated p < 0.05, ns indicated no significance; (d) Quantify the percentage of positive areas stained by type II collagen. * indicated p < 0.05, ** indicated p < 0.01

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Histopathological gradation of the severity of OA were quantified according to modified Mankin scoring system. As shown in Fig. 4b, the score of the normal group was 0, and the overall scores of the model group were 6–9 due to irregular notched surface and severe reduction in the matrix staining. The surface of the 2D hUC-MSCs treatment group was slightly uneven with an overall score of 4–6 points (p < 0.05). While the cartilage defects in 3D hUC-MSCs treatment group were mostly repaired, with an overall score of 3–4 points (p < 0.01). According to these results, hUC-MSCs significantly promoted the secretion of extracellular matrix to repair defected cartilage, and the repair effect of 3D hUC-MSCs treatment group was better than that of the 2D hUC-MSCs treatment group.

Expression of type I and type II collagen in defected cartilage

The expression levels of type I collagen and type II collagen in four groups were examined using immunohistochemical analysis after 8 weeks. In the model group, type I collagen was expressed but type II collagen was barely expressed, indicating the regenerated cartilage was composed of fibrocartilage. Both the 2D hUC-MSCs treatment group and the 3D hUC-MSCs treatment group expressed type I and type II collagen, which showed that the neocartilage was composed of hyaline cartilage and fibrocartilage. Quantification results showed the positive rate of type II collagen in 3D hUC-MSCs treatment group was significantly higher than 2D hUC-MSCs treatment group (p < 0.05). In addition, a large number of cells appeared in the cartilage layer after treating with 3D hUC-MSCs.

Expression of inflammatory cytokines in knee joint fluid

To evaluate the immunomodulatory function of hUC-MSCs, inflammatory cytokines in knee joint fluids were detected by ELISA at 8 weeks post-surgery. Compared with the model group, the levels of pro-inflammatory cytokines decreased significantly in the 2D hUC-MSCs treatment group, including TNF-α (p < 0.05), IL-6 (p < 0.05) and IL-7 (p < 0.001). But the expression levels of anti-inflammatory cytokines did not change significantly. For 3D hUC-MSCs treatment group, the expression level of pro-inflammatory cytokine TNF-α was reduced (p < 0.05), at the same time anti-inflammatory cytokines TGF-β1 and IL-10 were significantly increased (p < 0.05) (Fig. 6). These results indicated that the immunomodulatory effect of hUC-MSCs cultured by 3D hanging drop method was significantly better than that of 2D plate cultured hUC-MSCs.

Fig. 6
figure 6

The levels of cytokines in knee joint fluid. * indicated p < 0.05, *** indicated p < 0.001

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Discussion

Damaged cartilage had limited self-repair ability since lacked blood vessels, nerves, and lymphoid tissue [39,40,41]. MSCs therapy had become the most promising strategy for treating OA. MSCs created a regenerative microenvironment in joints by producing immunomodulatory and nutritional factors. However, many clinical experiments had shown that these factors were not sufficient to completely regenerate cartilage defects. The reasons might be related to poor cell quality before injection and a significant decrease in cell viability after injection [42].

In order to obtain enough MSCs for clinical treatment, 2D monolayer culture method was usually applied for rapid expansion. However, continuous 2D subculture induced cell replicative senescence, affecting the function and quality of cells [43]. Cellular senescence was a permanent cell cycle arrest characterized by persistent DNA damage response activation. After DNA damage, CHK1 and CHK2 were activated to transmit the damage signal to the downstream participating effector protein, Tp53 [44, 45]. In this study, hUC-MSCs were formed into 3D spheroids using the hanging drop culture method. The results of flow cytometry showed that short-term 3D hanging drop culture did not affect the expression levels of surface markers of hUC-MSCs. DEGs results showed that CHK1, CHK2, and Tp53 were all down-regulated.

In 2023, Krasnova et al. found the loss of cellular actin fiber tension in 3D MSCs spheroids, MSCs underwent a morphological transition from spindle-shaped cells to small, rounded cells. Besides changes in cell morphology, autophagy occurred in cells after 3D hanging drop culturing, which might be related to regulation of aging [46]. On the other hand, an oxygen gradient was formed in the 3D MSCs spheroids, which was characterized by a high oxygen concentration on the surface and a mild hypoxic state in the center of the aggregates. Lower oxygen tension improved cell lifespan and protected them from DNA damage [47]. As shown in Supplementary Material3, autophagy and hypoxia-related signaling pathways were involved in KEGG pathway enrichment results. REDD1 (DDIT4) was a stress-responsive protein induced by hypoxia. Elevated levels of REDD1 desensitized cells to apoptotic stimuli. REDD1 inhibited mTORC1 via regulation of TSC2 activity by two mechanisms [48, 49]. mTOR inhibition resulted in downregulation of CHK1 and CHK2, while activating autophagy [50, 51]. DEGs results showed REDD1 and autophagy initiating kinase ULK1 were highly expressed in 3D hUC-MSCs, 3D hanging drop method might inhibit cell replicative senescence by maintaining a lower oxygen tension state.

Homing and adhesion of MSCs in the local environment of damaged tissue determined their therapeutic function. CXCR4 encoded a well-known cognate receptor for CXCL12, the CXCL12/CXCR4 axis mediated injected MSCs homing to sites of injury, and MSCs could effectively adhere to articular cartilage [52, 53]. Previous studies had shown that cell homing and cell adhesion abilities of 2D cultured MSCs were weakened, which affected cell colonization after implantation in the knee joint [54]. During MSCs spheroids formation, dispersed cells aggregated due to long-chain ECM fibers consisting of RGD motifs that allowed to bind cell-surface integrin, and this led to upregulated cadherin expression. Cadherin accumulated on the surface of cell membrane, and the hemophilic cadherin–cadherin binding between neighboring cells allowed to tighten connections between cells [55]. High expression of CXCR4 was also associated with lower oxygen tension [56]. Overall, 3D hUC-MSCs had stronger tropism and adhesion, making it easier to reach and interact with inflammatory areas in the knee joint.

Numerous preclinical and clinical studies had demonstrated the MSCs possess immunomodulatory and regenerative properties and can be used to treat a range of immune system-related diseases and pathologies. Even during the global pandemic of SARSCoV-2, MSCs therapy had been put forward as promising therapeutic regimens for severe SARSCoV-2 patients [57]. MSCs promoted cartilage repair through the production of growth factors, cytokines, chemokines and exosomes, as well as the inhibition of the production of pro-inflammatory factors, rather than direct differentiation and cell replacement [58]. The loss of cell-cell contact resulting from 2D culture could reduce the secretion of trophic factors by MSCs. More importantly, MSCs might face harsh microenvironments after transplantation such as hypoxia, oxidative stress, damage signals, inflammation, and immune responses. This environment impaired cell vitality and function [59]. Therefore, 2D cultured MSCs did not show an obvious effect on OA.

Bartosh et al. found 3D assembly of MSCs drove transcriptome changes with activation of immune responsive pathways including chemokines and IL-1 signaling. These pro-inflammatory agents within the spheroid microenvironment could further prime MSCs to secrete anti-inflammatory factors, such as TSG-6 and STC-1 [60]. In this study, 3D hanging drop cultured hUC-MSCs also upregulated the expression of TSG-6 and STC-1. DEGs results showed that IL1A, IL1B, IL1R1, IL1R2 were highly expressed in 3D hUC-MSCs as seen in Supplementary Material 1, which were consistent with previous results. But IL-1 signaling pathway was not involved in the KEGG pathway enrichment results. It had been reported that the expression of IL-1β and TNF-α could be augmented by exposure to hypoxia [61]. In this study, most of the up-regulated genes were related to TNF signaling pathway as shown in Supplementary Material3. GSEA analysis results also showed that TNF signaling pathway (ES = 0.62; FDR q-value = 0.00) gene set was ranked 6th in the list as seen in Table S2 (Supplementary Material 2). DEGs results indicated that TNF and TNFR2 (TNFRSF1B) were highly expressed in 3D hUC-MSCs as seen in Supplementary Material 1. TNFα/TNFR2 signaling pathway was responsible for immunosuppression, MSCs produced higher levels of immunosuppressive molecules (TSG6 and other growth factors such as HGF, IGF1, and VEGF) through the TNFα/TNFR2 signaling pathway [62]. These results were also consistent with studies done by Dorronsoro et al., which revealed the importance of TNF-mediated activation of NF-κB for priming the immunosuppressive function in MSCs [63]. Furthermore, expression of TNFR2 had been shown to correlate with increased IL-10 and TGFβ production [64, 65].

Several of the pro-inflammatory including IL-family cytokines and TNFα, activated JAK/STAT signaling pathway in both direct and indirect ways [66]. Moreover, “cross-talk” between JAK/STAT signaling, MAPK and PI3K pathway had been demonstrated, these signaling pathways were involved in the regulation of cell proliferation, survival, development, and apoptosis [67]. IDO1 secretion had been reported to be dependent on the JAK/STAT signaling pathway [68]. A study by Wang et al. showed regulation by IDO1 through its metabolite kynurenic acid which activated the aryl hydrocarbon receptor (AhR) that bound to the promoter of TSG6 and enhanced its expression [69]. Therefore, transcriptome results showed that the immune regulatory factors and growth factors secreted by 3D hUC-MSCs were cooperatively regulated by multiple signaling pathways, among which the TNFα/TNFR2 signaling pathway might play a key role. It could be speculated that MSCs spheroids created a pro-inflammatory and hypoxia microenvironment, MSCs used autocrined proinflammatory cytokines as molecular switches of their anti-inflammatory properties. But the immune regulatory mechanism initiated by 3D culture might be different from the immune regulatory mechanism initiated by pro-inflammation and hypoxia. Latest research showed that the anti-inflammatory effects of MSCs primed by pro-inflammatory and hypoxia gradually declined over time, but the anti-inflammatory properties were fully retained after 3D culturing [70]. Spheroids formed by 3D hanging drop culture method induced changes in cell-matrix and cell-cell interactions, resulting in significant rearrangements of the physical forces acting on each cell. Ultimately, cell polarization, cytoskeletal organization, and morphology would change. MSCs in 3D aggregates experienced a much softer environment than those grown in 2D monolayer materials. The stiffness and elasticity of the matrix significantly affected the paracrine properties of MSCs [71]. In 2016, the importance of elasticity-related signaling in MSCs spheroids was pointed out by Cesarz et al., who showed that soft elasticity-induced mechanical signaling increased BMP2 expression and IL1 signaling upstream of pro- and anti-inflammatory gene expression [72]. It was unknown whether multiple factors (pro-inflammatory, hypoxia and mechanical signaling) synergistically affected the immunoregulatory ability of MSCs. The specific mechanisms and signaling pathways involved were required to be further explored and verified.

For verifying the therapeutic effects of 2D and 3D hUC-MSCs on OA, rabbit knee joint patellar cartilage defects model was constructed and treated with 2D hUC-MSCs and 3D hUC-MSCs respectively. The inflammatory environment created by cartilage damage was critical for progression of OA [73, 74]. Neutrophils were first recruited to secrete proinflammatory mediators and activation of helper T1 (Th1) cells, polarizing infiltrating macrophages into M1 macrophages. Next, M1 macrophages secreted pro-inflammatory factors (TNF-α and IL-6) that not only induced chondrocyte destruction, but also inhibited the synthesis of Col II and GAGs by promoting the production of proteolytic enzymes [75, 76]. IL-7 produced by damaged chondrocytes promoted the degradation of extracellular matrix proteins and cooperated with other cytokines to accelerate cartilage destruction [77]. The results of safranin O-fast green staining and immunohistochemistry confirmed that hUC-MSCs could promote the production of GAGs and type II collagen to form new cartilage matrix. Compared with 2D hUC-MSCs treatment group, 3D hUC-MSCs treatment group promoted the proliferation of chondrocytes and collagen deposition, significantly improved the morphologic features and histologic characteristics of the defected cartilages after 8 weeks.

According to the results of ELISA of knee joint fluid, 2D hUC-MSCs reduced the expression of inflammatory cytokines IL-6, IL-7 and TNF-α, but the expressions of anti-inflammatory cytokines IL-10 and TGF-β1 were not affected. 3D hUC-MSCs significantly down-regulated the expression of TNF-α while up-regulated the expressions of IL-10 and TGF-β1. 2D hUC-MSCs did not play a very good immunoregulatory role due to insufficient secretion of cytokines. Combined with the transcriptome results, in an inflammatory environment, 3D hUC-MSCs might secrete more factors such as TSG-6, STC-1 and IDO1 by activating the TNFα/TNFR2 signaling pathway and JAK/STAT signaling pathway. STC-1 inhibited MMP3/13 to prevent matrix degradation and inhibit the proliferation of OA fibroblast-like synoviocytes [78]. TSG6 was an important factor of MSCs’ immunomodulatory effects. TSG6 enhanced the expansion of regulatory T cells as well as inhibited neutrophil recruitment by directly modulating neutrophil adhesion to the endothelium [79, 80]. TSG6 could also enhance the inhibitory effect on MMPs [81]. Furthermore, TSG6 bound to fragments of hyaluronan, therefore diminishing pro-inflammatory effect [82]. IDO1 were responsible for the transformation of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype [83]. The M2 phenotype secreted anti-inflammatory cytokines IL-10 and TGF-β1, which suppressed inflammation and promoted tissue remodeling [84]. IL-10 had been shown to have chondroprotective effects, and it might act as a stimulator of chondrocyte proliferation [85]. Upregulation of TGF-β increased the proteoglycan content of articular cartilage and induced an increase in the expression level of type II collagen mRNA, with a corresponding increase in the ratio of type II collagen to type I collagen [86]. Therefore, 3D hUC-MSCs created a better regenerative microenvironment for OA and activated cartilage repair by secreting more cytokines.

Conclusion

Immune regulation for treating OA had become a new strategy. MSCs therapy was the most promising treatment method. However, 2D culture method weakened the biological functions of MSCs, especially the immune regulatory ability. 3D hanging drop culture method down-regulated the cell replication senescence-related gene TP53, up-regulated the cell homing-related gene CXCR4 and various adhesion-related genes. More importantly, 3D hanging drop culture method significantly improved the genes expression of anti-inflammatory genes (such as TSG6, STC-1, IDO1). In the rabbit cartilage defect model, 3D hUC-MSCs showed better effects in cartilage regeneration and immune regulation. Therefore, MSCs cultured by 3D hanging drop method might be more practical for treating OA.

Data availability

We have submitted the raw of RNA sequencing data to the Sequence Read Archive (SRA) of NCBI (accession number PRJNA1077984), which is publicly available at https://www.ncbi.nlm.nih.gov/sra/PRJNA1077984.

Abbreviations

AOD:

Average optical density

CCR 7:

C-C chemokine receptor 7

CCR 9:

C-C chemokine receptor 9

CCR 10:

C-C chemokine receptor 10

CHEK 1:

Checkpoint Kinase 1

CHEK 2:

Checkpoint Kinase 2

Col II:

Type II collagen

CXCL12:

CXC motif chemokine ligand 12

CXCR4:

C-X-C motif chemokine receptor 4

CXCR2:

C-X-C motif chemokine receptor 2

DEGs:

Differentially expressed genes

ECM:

Extracellular Matrix

ELISA:

Enzyme-linked immunosorbent assay

ES:

Enrichment score

FDR:

False discovery rate

FGF2:

Fibroblast growth factor 2

GAGs:

Glycosaminoglycans

GSEA:

Gene set enrichment analysis

GO:

Gene Ontology

hUC-MSCs:

Human umbilical cord-derived mesenchymal stem cells

ICAM 1:

Intercellular cell adhesion molecule-1

IDO1:

Indoleamine 2,3-Dioxygenase 1

IL-1β:

Interleukin-1β

IL1 RA:

Interleukin 1 Receptor Antagonist

IL-6:

Interleukin-6

IL-7:

Interleukin-7

IL-10:

Interleukin-10

ITGA1:

Integrinα1

ITGA2:

Integrinα2

ITGA7:

Integrinα7

ITGA10:

Integrinα10

KEGG:

Kyoto Encyclopedia of Genes and Genomes

MAPK:

Mitogen-activated protein kinase

MSCs:

Mesenchymal stem cells

MVBs:

Multivesicular bodies

OA:

Osteoarthritis

OD:

Optical density

PAI-1:

Plasminogen activator inhibitor-1

PBS:

Phosphate Buffered Saline

PDGF:

βPlatelet-Derived Growth Factorβ

PTGS 2:

Prostaglandin-endoperoxide synthase 2

RAB27A:

Ras-related protein 27 A

RAB27 B:

Ras-related protein 27B

RCI:

Red colour intensity

RGB:

Red, green, and blue

RF:

Fraction of red

SDC-4:

Syndecan-4

SDCBP:

Syndecan binding protein

SEM:

Scanning electron microscopy

SMPD3:

Sphingomyelin phosphodiesterase 3

STC-1:

Stanniocalcin-1

TGF β1:

Transforming growth factor β1

Th1 cells:

T helper 1 cells

TNFα:

Tumor Necrosis Factor Alpha

TP53:

Tumor protein 53

TSG101:

Tumor susceptibility gene 101

TSG-6:

Tumor necrosis factor α stimulated gene-6

VCAM 1:

Vascular cell adhesion molecule 1

VEGF:

Vascular endothelial growth factor

2D:

Two-dimensional

3D:

Three-dimensional

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Acknowledgements

This work received technical support and scientific advice from the public [Department of Respiratory Medicine and Neonatology of the Children’s Hospital of Chongqing Medical University] and the public [The animal laboratory of Qilu Hospital of Shandong University]. The authors also thank Hangzhou Yanqu Information Technology Co., Ltd. for providing scanning electron microscopy and transcriptome sequencing technical support.

Funding

The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Author notes

  1. Xiaoyu Dai and Yuanshan Gan equally supervising authors.

Authors and Affiliations

  1. Chongqing Perfect Cell Biotechnology Co. LTD, Chongqing, 400700, P.R. China

    Qiang Fu, Mei Han, Xiaoyu Dai, Ruian Lu, Enjie Deng, Xuemei Shen, Feng Ou, Yongguang Pu, Xueqin Xie, Kang Liu & Yuanshan Gan

  2. Stem Cell and Regenerative Medicine Research Center, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, P.R. China

    Dong Li

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  1. Qiang Fu
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Contributions

QF and DL equally conceived and designed the study. QF, MH, XD, RL, ED, XS, FO, YP, XX, KL and YG performed the experiment, analyzed the data, performed the statistical analysis, and wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Dong Li.

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Ethics approval and consent to participate

Department of Respiratory Medicine and Neonatology of the Children’s Hospital of Chongqing Medical University had confirmed that there was ethical approval for the collection of umbilical cord tissue and human cells (Approval Number: 2022 − 347; Approval Date: October 09, 2022), and the donors’guardians had signed informed consent. All research protocols involving hUC-MSCs had been reviewed by the Institutional Ethics Committee of Chongqing Perfect Cell Biotechnology Co., Ltd. (Approval Number: KYLL-202302-04; Approval Date: February 25, 2023). All participants consented to be part of this study. This study was approved by the Institutional Ethics Committee of Chongqing Perfect Cell Biotechnology Co., Ltd. (Title: Animal experimental study on the treatment of osteoarthritis by three-dimensional hanging drop cultured umbilical cord mesenchymal stem cells; Approval Number: DWLL202303; Approval Date: September 1, 2023). All surgical procedures were performed in accordance with Guidelines for the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35892–2018 (Issued 6 February 2018 Effective from 1 September 2018). All experimental facilities had a national authorization to perform animal experiments (approval ID: SYXK 2020-0017). This study has been reported in line with the ARRIVE guidelines 2.0.

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The authors declare that there is no competing interests.

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Fu, Q., Han, M., Dai, X. et al. Therapeutic effect of three-dimensional hanging drop cultured human umbilical cord mesenchymal stem cells on osteoarthritis in rabbits. Stem Cell Res Ther 15, 311 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-03905-y

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512