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. Author manuscript; available in PMC: 2025 Jun 21.
Published in final edited form as: Osteoarthritis Cartilage. 2025 Feb 18;33(5):590–600. doi: 10.1016/j.joca.2025.02.777

Macrophage phenotypes modulate neoangiogenesis and fibroblast profiles in synovial-like organoid cultures

Qi Gao #, Xiurui Zhang †, Meagan J Makarcyzk †, Laurel Elizabeth Wong #, Madison Sidney Virgil Quig #,†, Issei Shinohara #, Masatoshi Murayama #, Simon Kwoon-Ho Chow #, Bruce A Bunnell ‡, Hang Lin †, Stuart B Goodman #,⁎
PMCID: PMC12182213  NIHMSID: NIHMS2066971  PMID: 39978574

Abstract

Objective:

To investigate the interaction among the cells thought to be foundational to inflammation, fibrosis, and angiogenesis in the synovial membrane.

Method:

We encapsulated fibroblasts, polarized macrophages, and endothelial cells in a 3D culture system. We used this model to determine the cellular transcriptional profiles, cytokine secretion, and vascular formation associated with different macrophage phenotype conditions.

Results:

Neo-angiogenesis reached its maximum level at approximately day 21 in the presence of pro-inflammatory macrophages conditions, but was sustained in the presence of anti-inflammatory macrophages. RNA sequencing revealed an influence of macrophage phenotype on gene expression associated with fibrosis and angiogenesis. Furthermore, by including lipopolysaccharides-coated polyethylene particles (lcPE), an inflammatory stimulus replicating wear debris from joint replacements into our system, insights into the local reaction to byproducts of different biomaterials can be ascertained.

Conclusion:

Chronic inflammation and fibrosis of the synovial membrane are often present in osteoarthritis and post-total joint arthroplasty. Our results suggest that the progression of inflammatory synovial diseases is influenced by macrophage phenotypes.

Keywords: Immunity, 3D culture, Synovitis, Arthroplasty, Particle-induced inflammation

Introduction

Synovium lines the inner surface of the joint capsule and produces fluid that lubricates and nourishes the articular joints. Anatomically, the synovial membrane contains two layers: an inner lining layer that directly encapsulates the articular cavity and an outer sub-lining layer comprising fibroblasts, macrophages, vascular structures, and other sparse infiltrating cells 1,2 (as depicted in Fig. 1A). The synovium responds quickly to joint injuries and holds significant importance in joint immunity.3,4 For example, the presence of byproducts derived from orthopedic implants, such as ultra-high molecular weight polyethylene, activates immune cells that release pro-inflammatory factors, resulting in persistent inflammation, ultimately causing the failure of the joint prosthesis.5,6 Chronic inflammation in synovial joints is sustained by the pro-inflammatory (M1) macrophage phenotype, as opposed to the anti-inflammatory (M2) phenotype that generally resolves inflammation and promotes tissue regeneration. Furthermore, in chronic inflammation, excessive angiogenesis occurs in the synovial sub-lining layer, accompanied by the migration of inflammatory cells from the bone marrow to the joint cavity and synovium.7,8 Chronic synovitis plays a pivotal role in the pathology of many joint diseases, including osteoarthritis (OA).9,10 Therapeutic approaches aimed at restoring synovial homeostasis are an unmet clinical need.

Fig. 1.

Fig. 1

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Illustration of synovial membrane and experimental design. (A) The synovial membrane’s outer sub-lining layer comprises fibroblasts, macrophages, vascular structures, and other sparse infiltrating cells. (B) Fibroblasts, HUVECs, and macrophages were encapsulated in a 3D fibrin-GelMA hydrogel system. The hydrogel was photopolymerized using non-ultraviolet light and used to investigate angiogenesis, fibrosis, and other synovial-related diseases.

Inflammation facilitates neo-angiogenesis, in which the newly formed vessels are thought to contribute to the initial enhancement and subsequent resolution of tissue inflammation.11,12 Indeed, persistent pro-inflammatory processes and the apparent failure of anti-inflammatory processes are the hallmarks of chronic inflammation and the resulting tissue damage.13 Chronic inflammation is associated with the infiltration of macrophages, which in turn secrete cytokines, chemokines, and angiogenic factors to induce the expansion of blood vessels.14,15 In this regard, macrophages are immune modulators that regulate angiogenesis in health and disease.14,16 The balance between pro- and anti-inflammation effects of macrophages is crucial for proper tissue repair and homeostasis.

Synovial fibrosis is associated with the activation of matrix-secreting fibroblasts and extracellular matrix (ECM) reorganization. The mechanistic connection between inflammation and fibrosis highlights the importance of synovial fibroblasts in joint diseases.17 By preventing the recruitment of inflammatory leukocytes together with on-site reprogramming of macrophages, it may be possible to mitigate the effects of uncontrolled fibrosis.

In vitro, human organ-on-a-chip and organoid models recapitulate the microphysiological processes that often cannot be gleaned from animal studies. Here, we establish an in vitro 3D platform that encapsulates macrophages that may polarize to pro- or anti-inflammatory phenotypes, endothelial cells (ECs) that self-assemble into vascular networks, and fibroblasts to model the synovial membrane. The organ chip model can effectively replicate dynamic pathophysiological conditions, providing a valuable method to understand synovial inflammation mechanisms. Our platform has enabled an authentic recapitulation of disease mechanisms using human cells by conducting experiments temporally. In addition, the changes in transcriptional profiles and pathway analysis based on the RNA sequencing analysis were explored in our 3D co-culture model.

Results

Peak angiogenesis was driven by M1 macrophage while sustained by M2 macrophage

We established a vascularized synovial model that faithfully recapitulates clinical tissue to investigate inflammation, angiogenesis, and fibrosis in the synovial membrane. Mesenchymal stem cell (MSC)-derived fibroblasts and Green fluorescent protein (GFP)-labeled HUVECs (human umbilical vein endothelial cells) were encapsulated in a fibrin-GelMA hybrid scaffold (as depicted in Fig. 1B), and the cell-laden scaffold was cultured for 28 days.

A debate persists regarding the contribution of pro-inflammatory or anti-inflammatory macrophage phenotypes to angiogenesis.14,18 To understand more about how macrophages regulate angiogenesis during inflammation and tissue regeneration, we loaded M1 (M1 coculture group) or M2 (M2 coculture group) macrophages, which had been polarized as described in Methods, into our 3D synovial model. Given that short acute inflammation has the potential to progress into chronic inflammation, and considering that this transformational process typically spans over a few weeks,19 we proceeded to culture the scaffolds for 28 days. Dynamic vessel structures were shown in Fig. 2A. There were sporadic distributions of short, thin tubes within the scaffold during the first week. Subsequently, these tubes underwent elongation and branching, forming a hierarchical branching structure by days 21 and 28. Fig. 2B illustrates the vessel branch which is characterized as the new vessel from the main vessel. In the M1 coculture group, a peak phase was observed, with a significant increase in vessel length and volume observed during the initial 21 days, followed by a plateau on day 28. Nevertheless, the length and volume on day 28 remained greater than that on day 7, as shown in Fig. 2C. There was continuous and significant vessel length and volume expansion in the M2 coculture group. A statistical difference was detected on day 21 and day 28 when compared with that on day 7. In addition, vessels in the M2 coculture group revealed a more robust structure. Statistical analysis revealed longer and thicker vessels in the M2 group than in the M1 group after day 14. Taken together, M1 macrophages facilitate angiogenesis within a short period (∼21 days), after which the vessels enter a plateau phase. Conversely, vessel growth persists uninterrupted in the presence of M2 macrophages.

Fig. 2.

Fig. 2

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Macrophage phenotypes modulate angiogenesis. (A) Dynamic vessel formation in the M1 coculture group (top row) and M2 coculture group (bottom row) at different time points. (B) Illustration of branch and branching points in the fluorescent images. (Scale bar = 200 µm.) (C) Statistical analysis of branch length and vessel volume during the culture period in M1 coculture group (D) and M2 coculture group. (E) The upper table summarizes the total branches and branch points observed in the M1 co-culture group, while the lower table summarizes the total branches and branch points observed in the M2 co-culture group. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.).

Macrophage phenotype manages the transcriptional profiles of the synovial membrane

The transcriptional profile of the whole tissue was subsequently compared between the M1 and M2 coculture groups by bulk RNA sequencing. Specific, commonly expressed, and differentially expressed genes (DEGs) were plotted using the Venn diagram approach, as shown in Fig. 3A. Fig. 3B represents the heatmap of the gene expression. Gene Ontology (GO) analysis was performed to highlight biological processes (BPs), as shown in Fig. 3C-F. On day 7, we found that genes related to the regulation of bone remodeling, response to bone morphogenetic protein, and ossification were significantly enriched in the M1 coculture group. In contrast, genes associated with neutrophil activation and leukocyte proliferation were significantly upregulated in the M2 coculture group from day 7 to day 21.

Fig. 3.

Fig. 3

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Transcriptome analysis. (A) Venn diagrams representing the differentially expressed genes specific or common in the M1/M2 coculture group at different time points. (B) The heatmap shows the top differentially expressed genes. (C-F) GO analysis reveals the BPs associated with M1 and M2 coculture groups on day 7, day 14, and day 21. p-value (p.adjust) less than 0.5 are selected. (G-I) The pathway categories of DEGs between the M1 and M2 coculture group on day 7, day 14, and day 21. Orange represents activated pathways, while blue indicates suppressed pathways.

To identify factors driving DEG associated with macrophage phenotypes, we performed upstream regulator analysis and activated/deactivated pathway analysis using QIAGEN Ingenuity Pathway Analysis (IPA).20 As shown in Fig. 3G-I, The IPA analysis identified phagosome formation and pathogen-induced cytokine storm signaling pathway enrichment in the M2 coculture group. The rheumatoid arthritis signaling pathway was activated in the M2 coculture group on day 14 and day 21, in contrast to the M1 coculture group. Specifically, on day 21, hepatic fibrosis signaling pathway, wound healing signaling pathway, OA pathway, role of osteoblasts and chondrocytes in rheumatoid arthritis signaling pathway, and pulmonary fibrosis idiopathic signaling pathway were predicted to be activated in the M2 coculture group. Our data indicates that the presence of M2 macrophages for an extended period increases the risk of developing fibrosis and arthritis, whereas M1 macrophages didn’t show the same effect. Altering the phenotype of macrophages could potentially serve as a therapeutic approach.

Temporal effects on inflammation, angiogenesis, and fibrosis

We next examined the dynamic changes in the transcriptome within the M1 and M2 coculture groups, and the gene expression on day 7 was used as the reference baseline. Fig. 4A-D shows the BPs identified through GO analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis results. In the M1 coculture group, leukocyte migration and chemotaxis were significantly decreased by day 14 compared to day 7, indicating a reduction in immune cell recruitment and inflammatory responses. Furthermore, fibroblast proliferation also showed a decline in activation from day 14 to day 28. BPs related to cell division, including those associated with chromosome segregation, were markedly reduced after day 21. This also supports the result that the plateau phase of vessel formation was accompanied by a reduction in proliferative activity. In contrast, ECM organization was upregulated on day 21, highlighting a shift towards tissue remodeling and structural organization. KEGG analysis further revealed that pathways associated with rheumatoid arthritis, osteoclast differentiation, and phagosome formation were progressively deactivated, reflecting a decrease in inflammatory and immune-related activities as the coculture progressed. IPA analysis was shown in Fig. 4E-G. The results are consistent with GO and KEGG analysis, revealing that cytokine signaling was mainly activated on day 7, aligning with the peak of immune response and inflammation in the M1 coculture group. This temporal shift underscores the transition from an inflammatory phase to a more stable, remodeling phase in the M1 coculture group.

Fig. 4.

Fig. 4

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Dynamic transcriptional profiles of M1 coculture group. (A-B) Comparison of BO across different time points. Downregulated BP terms on days 14, 21, and 28 (A), as well as activated BP terms on days 21 and 28 (B). An analysis of KEGG pathways at various time intervals reveals the downregulation of pathways on days 14, 21, and 28 in comparison to day 7 (C), alongside the upregulation of pathways observed on days 21 and 28 (D). IPA analysis of DEGs comparing day 7 with day 14 (E), day 7 with day 21 (F), and day 7 with day 28 (G), highlighting the pathways and biological processes affected at each time point. The x-axis shows the z-score, while the y-axis shows the corresponding categories of canonical pathways.

GO and KEGG analysis of the M2 cultural group is shown in Fig. 4A-D. Leukocyte activation, particularly in immune response processes, was significantly decreased by day 14, reflecting a reduction in inflammatory activity. The BPs related to cell division, such as chromosome segregation and mitotic sister chromatid separation, were deactivated on day 28, which is later than in the M1 coculture group. In contrast, ECM organization was highly activated between day 14 and 21, suggesting increased tissue remodeling during this period. KEGG analysis further revealed that focal adhesion pathways were upregulated on day 14 compared to day 7, highlighting changes in cellular adhesion and interaction with the ECM. Additionally, deactivated phagosome formation, osteoclast differentiation, and rheumatoid arthritis after day 14 indicated a reduction in inflammatory and immune responses over time. Fig. 4E-G shows that IPA results also support the finding that immune system and cell cycle processes are more active on day 7.

Wear particles differentially modulate angiogenesis in the synovial membrane

We further proceeded to incorporate LPS-coated polyethylene particles (lcPE) in our system to investigate their impact on synovia physiology. In addition, polyethylene particles were used to mimic the wear debris, which contributes to synovitis and OA.21 Identification of the soluble factors extends our understanding of how the immune system regulates angiogenesis. The cytokine levels were examined and compared by collecting the culture medium on day 7 (Fig. 6A). Higher secretion of MIG/CXCL9 (chemokine ligand 9), FLT-3L (Fms-related tyrosine kinase 3 ligand), and IP-10/CXCL10 were significantly higher in the M1 coculture group than the M2 coculture group, which indicates a pro-inflammatory environment. The M1 coculture group also exhibits elevated levels of Interleukin (IL-6) and IL-8 compared to the M2 coculture group, although no significant statistical difference was observed. The M2 coculture group exhibited elevated levels of IL-RA, Tumor Necrosis Factor-alpha (TNFα), IL-10, and Monocyte chemoattractant protein (MCP)-3 secretion, while the expression remained largely unaffected by lcPE stimulation. To our surprise, TNFα was highly expressed in the M2 group but not the M1 group. In addition, we conducted a comparison of cytokine levels with or without lcPE stimulation. Notably, no significant differences were observed, except for a marked increase in IL-17E/IL-25 in the M1 coculture group treated with lcPE compared to the M2 coculture group with lcPE. The lcPE regulators significantly impact angiogenesis in our synovial membrane model but do not affect cytokine secretion.

Fig. 6.

Fig. 6

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Impact of lcPE and macrophage phenotype on cytokine secretion and transcriptome. (A) Level of cytokine expression in the culture supernatant. (B) Impact of lcPE on angiogenesis. Top: comparison of angiogenesis parameters in the M1 coculture group with or without lcPE stimulation. Bottom: comparison of angiogenesis parameters in the M2 coculture group with or without lcPE stimulation. (C) Upregulated BP of in the M1 coculture group with lcPE stimulation on day 28 in comparison to day 7. (D) Downregulated BP of in the M1 coculture group with lcPE stimulation on day 14, 21 and 28 in comparison to day 7. (E) Upregulated BP of in the M2 coculture group with lcPE stimulation on day 28 in comparison to day 7. (F) Downregulated BP of in the M2 coculture group with lcPE stimulation on day 14, 21 and 28 in comparison to day 7. (n=3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

The angiogenesis results are summarized in Fig. 6B. The addition of lcPE to the M1 coculture had no detectable influence on general pattern of time-dependent changes in vessel formation: it increased over the initial 21-day period and then entered a plateau phase through day 28. However, compared to the group without lcPE, there was a more robust increase in vessel length and volume in the lcPE group by day 7. This initial rate of vessel formation was not sustained, as vessel length and volume were greater in the no lcPE group by 21, and the decrease in length and volume was even greater by 28. In the M2 macrophage group, there was no detectable influence of lcPE on angiogenesis in the early phase. However, with time in culture, the increase in vessel volume and length increased more rapidly in the lcPE group.

We further compared the transcriptional difference between groups with or without lcPE stimulation. There were only a few DEGs identified in the M1 coculture group when comparing conditions with and without lcPE on day 21. No differences were observed at other time points. Additionally, no DEGs were observed between the M2 coculture group with and without lcPE, indicating that the presence of lcPE had less impact on gene expression. The findings of this study indicate that the macrophage phenotype plays a crucial role in governing cellular processes, whereas lcPE has a minor effect.

A time series transcriptome analysis was conducted on the M1 coculture group treated with lcPE. From day 7 to day 28, the M1 coculture group with lcPE exhibited a downregulation of several key BPs, including cell division and fibroblast proliferation, as shown in Fig. 6C-D. GO analysis of the M2 coculture group with lcPE is summarized in Fig. 6D-E. When comparing genes expressed on day 7 and day 14, the results showed a significant decrease in leukocyte migration and cellular response to chemokines, suggesting a reduced inflammatory response over time. Interestingly, on day 21, we observed an upregulation of cell-cell adhesion and collagen fibril organization, indicating a shift in cellular behavior towards enhanced structural organization. At the same time, cell division was notably decreased, further emphasizing a decline in cellular proliferation.

Discussion

Inflammation-mediated angiogenesis

Researchers developed 3D coculture systems to replicate the tissue microenvironment, with a primary emphasis on tumor and drug screening in the majority of prevalent clinical scenarios.22 People also create 3D models to investigate joint-related diseases, focusing on areas other than the synovial membrane.23,24 Clinical research indicates that significant cell populations in synovial membranes contain fibroblasts, macrophages, ECs, T cells, B cells, etc. Among them, synovial macrophages are critical in maintaining synovial homeostasis.4 Our work was focused on creating an engineered 3D synovial model that encapsulates fibroblasts, macrophages, and ECs. As demonstrated by the evidence of vessel formation, immune response, and gene expression, our synovial model effectively replicates crucial aspects of human inflammation, angiogenesis, and fibrosis.

Synovial angiogenesis is associated with inflammation and causes chronic synovitis, characterized by increased tissue infiltration of macrophages.25 The non-activated macrophages adopt their phenotypes based on environmental stimulation. M1 macrophages producing pro-inflammatory cytokines are an essential source of post-joint injury. Indeed, M1 macrophages have been found in high concentrations in synovial tissues of OA patients.26 Furthermore, the increased blood flow may stimulate angiogenesis by applying shear stresses on the endothelium and stabilizing new blood vessels. Nevertheless, most investigations suggest a more prominent role of M2 macrophages in angiogenesis by their ability to secrete pro-angiogenic factors such as IL-10 and Transforming growth factor (TGF)β. In addition, M2 macrophages regulate angiogenic Vascular endothelial growth factor (VEGF)-A and VEGF-C expression, which act in a paracrine manner to promote wound repair and neovascularization on local ECs.27 M2 macrophage-derived exosomes promote angiogenesis and increase vascular density.28 Some researchers showed that M2 macrophages have a higher angiogenic potential than M1 macrophages, suggesting the importance of fibroblast growth factor signaling as the working mechanism.29 Others proved that the angiogenic capacity of murine M2 macrophages but not M1 depended on their TIMP-free proMMP (Matrix metalloproteinase)-9.30 Although opposite findings were reported regarding how functional macrophages regulate angiogenesis, we noticed that both pro-inflammatory and anti-inflammatory processes participate in angiogenesis. The anti-inflammatory process leads to critically enhanced angiogenesis.

The secretion of cytokines indicated the specific phenotype of the macrophages. In our system, we have observed two distinct angiogenesis patterns, where peak angiogenesis was initially driven by M1 macrophage and sustained by M2 macrophage. The M2 coculture group with lcPE exhibited the presence of more robust vessels, suggesting a higher potential for blood flow rate. The examination of transcriptional profiles reveals in the M1 coculture group, there was an increase in cell proliferation during the initial stage, a strengthening of the ECM structural component during the intermediate stage, and an immune regulatory feature during the final stage. Angiogenesis showed a comparable pattern when lapel was added to the culture system. lcPE improved the process of angiogenesis in the M1 coculture group during the early phase, whereas it facilitated the self-disassembly process in the later phase. Furthermore, GO analysis indicated a different BP. Abnormal angiogenesis in inflammatory disease may contribute to tissue growth, disordered tissue perfusion, and abnormal ossification. Our research has uncovered the significance of the dynamic transcriptome in controlling angiogenesis and inflammation and aiding in the recovery of proper tissue structure and function.

Fibrosis in inflammation and inflammatory resolution

Fibroblast-like synoviocytes (FLSs) are the main effector cells of knee OA synovial fibrosis. FLS helps to shape and maintain the synovial ECM by producing matrix components (such as fibronectin, collagens, tenascin, proteoglycans, and laminin) and ECM-degrading enzymes (such as proteases, MMPs, and cathepsins. Once activated, FLS is suggested to acquire a myofibroblast (MF)-like phenotype, which promotes fibrogenesis by excessively depositing collagen, precisely types I, III, and V. This excessive collagen deposition results in the disruption of organization and tissue functions and ultimately leads to synovial fibrosis.31 FLSs are known to modulate macrophage gene expression profiles 32 and, therefore, could determine macrophage phenotype in RA. When co-cultured with ECs, FLS from the inflamed joints of patients with advanced RA increases the expression of adhesion molecules on ECs, promoting the adhesion of lymphocytes to the ECs.33

Macrophages play a crucial role in fibrosis, and the studies of macrophage receptors and ligands are investigated as potential targets for anti-fibrotic purposes. During the early inflammatory phase, activated M1 macrophages clear the pathogenic micro-organisms and secrete ECM-degrading MMPs. Subsequently, the development and progression of fibrosis may occur in the tissue repair stage. M2 macrophage-derived microRNAs and extracellular exosomal-miRNAs have also been proposed to promote the progression of pulmonary fibrosis.34 Indeed, multiple studies have linked the pro-fibrogenic signatures of M2 macrophages to the pathogenesis of musculoskeletal disease. It has been suggested that a proportion of anti-inflammatory CD163 positive macrophages tends to be higher in idiopathic pulmonary fibrosis (IPF), while inhibition of M2 macrophage polarization has been shown to inhibit bleomycin-induced IPF in rats.35,36 Additionally, the M2 macrophage contributes to the progression of lung fibrosis. By inhibiting the M2 program, methyl-CpG-binding domain 2 (MBD2) can potentially halt the development of pulmonary fibrosis.37 An excessive TGF response is induced by abnormal polarization of M2 macrophages, consequently fostering pathological tissue fibrosis in musculoskeletal disorders such as scleroderma.38 Researchers further investigate the contribution of M2 macrophage subpopulations. M2b macrophages can mitigate tissue fibrosis by inhibiting cardiac fibroblast proliferation, migration, and differentiation into MFs.39 This suggests that M2b macrophages may be utilized in protective treatments against pathological fibrosis. It has been suggested that Trichostatin A upregulated M2c but inhibited M2a macrophages and, therefore, inhibits the activation of MFs.40 However, others indicated M2c macrophages could promote epithelial–mesenchymal transition if overactivated after an injury, leading to pathological fibrosis.41 Furthermore, reducing the M2c subgroup may improve pulmonary fibrosis.42 Therefore, different subgroups of M2-type macrophages have dual immunomodulatory functions in the fibrosis process. Given that macrophages play a crucial role in fibrosis, novel treatments targeting macrophage polarization to re-establish homeostasis may be beneficial in treating chronic synovitis and thus slowing OA progression.

Conclusion

Macrophage activity is a significant component of the healthy response to infection and injury that consists of tightly regulated early pro-inflammatory activation followed by anti-inflammatory and regenerative activity. Encapsulating fibroblasts, polarized macrophages, ECs, and lcPE in a fibrin-GelMA scaffold create a 3D synovial platform. Using this platform, we found that angiogenesis peaked around day 21 under pro-inflammatory conditions, while it remained consistently sustained in anti-inflammatory circumstances. The transcription profiles changed throughout the 28-day culture period. These profiles provide potential insights into the biological pathways involved in immune modulation, aiming to alleviate chronic inflammation and the resulting synovitis in OA.

Materials and methods

Cell culture and differentiation

Human bone marrow-derived MSCs were maintained in a growth medium containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic-antimycotic. Once 80% confluency was reached, MSCs were detached by 0.25% Trypsin/EDTA (Ethylenediaminetetraacetic acid) and passaged to new flasks. Fibrogenic medium (Advanced DMEM supplemented with 5% FBS, 1% GlutaMAX, 1% antibiotic-antimycotic, and 50 µg/mL Ascorbic acid) was applied to differentiate MSCs to fibroblasts. The differentiation process took four weeks.

GFP-positive HUVECs were obtained from Angio-Proteomie (Boston, MA, USA) and cultured using Endothelial Cell Growth Medium (Promocell C-22011). Medium was changed every 2 or 3 days.

The EasySep™ Human Monocyte Isolation Kit (Stem Cell Technologies) was employed to isolate monocytes from blood of de-identified male donors. Then, the monocytes were derived into naive macrophages by macrophage stimulation medium. (RPMI supplemented with 10% FBS, 1% antibiotic-antimycotic, and 100 ng/mL macrophage colony-stimulating factor). To obtain functional macrophages, 20 ng/mL Interferon gamma (IFNγ) and 10 ng/mL Lipopolysaccharide (LPS) were added to the stimulation medium to polarize the undifferentiated M0 macrophages to the M1 phenotype. Alternatively, 20 ng/mL IL-4 was added to obtain the M2 phenotype.

Set up of the 3D co-culture system

Fibrin-photocrosslinkable methacrylated gelatin (GelMA) hybrid scaffolds were used in our experiments. GelMA was prepared according to a previously reported protocol [39]. 15% GelMA HBSS (w/v) was mixed with 0.15% lithium phenyl-2,4,6-tri-methylbenzoylphosphinate (LAP) as the photoinitiator. Fibrinogen was dissolved in HBSS at 60 mg/mL, and thrombin was prepared at 5 U/mL. Fibrinogen, thrombin, and 15% GelMA were mixed (1:1:4, v/v/v) to obtain the Fibrin-GelMA hybrid gel. Then, the cells were loaded into the hybrid gel and transferred to a cylindrical silicone mold with 5 mm diameter and 2 mm depth. Fibroblasts, HUVECs, and macrophages were seeded at concentrations of 5 million cells/mL, 5 million cells/mL, and 10 million cells/mL, respectively. The cell-seeded hydrogels were photopolymerized using non-ultraviolet light (395 nm) for 2 min. The final concentration of GelMA is 10%. The synovial 3D constructs were cultured in Fibrogenic medium/macrophage stimulation medium/Endothelial Cell Growth Medium (1:1:1 mixture, v/v/v) for 4 weeks.

PE particles were ethanol sterilized and coated with 10 ng/mL LPS. Cells were then cultured with or without lcPE. The final concentration of LPS and cPE in the scaffold were 0.25 ng/mL and 0.125%, respectively.

Live imaging using a confocal microscope and image analysis

The GFP fluorescence visualized the vasculature using a Leica STELLARIS 5 White Light SuperResolution Tau Sense Inverted Confocal (Leica, Wetzlar, Germany). Z-stack images of the HUVEC network were acquired at 10X magnification. Quantitative analysis of the vessel networks was performed using Imiris. A semi-automated approach was used, with manual vessel identification followed by automated calculation of length and diameter. Branches length was calculated as the sum of all edges between two branch points or between a branch point and a terminal point. Diameters were defined as the distance from the center to the branch’s surfaces. Filament length was the sum of all branches’ length within the entire filament graph. One-way ANOVA (Analysis of variance) was applied for comparison.

Luminex

A Human Cytokine Array/Chemokine Array 48-Plex (Eve Technologies, Calgary, Canada) was used to determine the level of cytokine secretion at supernatants. Cytokines and chemokines included in the analysis were as follows: sCD40L, EGF, Eotaxin, Fibroblast growth factor-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1, MCP-3, M-CSF, MDC (CCL22), MIG, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNFα, TNFβ, VEGF-A.

RNA sequencing and functional analysis

TRIzol™ Reagent (Life Technologies, UK) was applied to extract total RNA from the contracts and then treated with chloroform. mRNA was isolated from the aqueous phase by sequentially adding isopropyl alcohol and 75% ethanol. RNA concentration and quality measurements were taken using NanoDrop. Library preparation, quality control and sequencing were performed by Novogene Co., Ltd. RNA-seq data was processed by DESeq2 package to identify DEGs with Log2 (fold change) ≥ 1 and an adjusted p-value ≤ 0.05. Sequence data is available through the GEO database under accession number XXX. The upregulated or downregulated genes were used to complete GO enrichment analysis by ClusterProfiler R package. BPs, molecular functions, and cellular components were identified, and P < 0.05 was regarded as statistically enriched. The raw sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) under the accession number GSE291863.

The IPA system (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) was used for subsequent bioinformatics analysis. Canonical pathway analysis, disease and function, regulator effects, upstream regulators and molecular networks were performed. For canonical pathway analysis, disease and function, the −log (P-value) > 1.3 was taken as the threshold, the Z-score > 2 was defined as the threshold of significant activation, while the Z-score < −2 was defined as the threshold of substantial inhibition. The P-value of overlap < 0.05 was set as the threshold for upstream regulators.

Fig. 5.

Fig. 5

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Dynamic transcriptional profiles of M2 coculture group. (A-B) Comparison of BO across different time points. Downregulated BP terms on days 14, 21, and 28 (A), as well as activated BP terms on days 21 and 28 (B). An analysis of KEGG pathways at various time intervals reveals the downregulation of pathways on days 14, 21, and 28 in comparison to day 7 (C), alongside the upregulation of pathways observed on days 21 and 28 (D). IPA analysis of DEGs comparing day 7 with day 14 (E), day 7 with day 21 (F), and day 7 with day 28 (G).

Acknowledgments

The authors are grateful to other members of the participating laboratories for their contributions and suggestions.

Funding

This work was supported by NIH grants UG3TR002136, R01 AR063713, R01 AR073145, and the Ellenburg Chair in Surgery at Stanford University.

Footnotes

CRediT authorship contribution statement

Q.G.: Methodology, Software, Writing – original draft. X.Z.: Methodology. M.J.M: Methodology. L.E.W.: Methodology. M.S.V.: Methodology. I.S.: Methodology. M.M.: Methodology. S.K.C.: Writing – review & editing. B.A.B.: Writing – review & editing. H.L.: Conceptualization, Writing – review & editing. S.B.G.: Conceptualization, Writing – review & editing.

Conflict of interest

The authors declare no conflict of interest.

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