Category: 3. Business

This preliminary study is the first to assess the use of HCR wrapping for patient-specific guides in VT scar ablation.

Innovative solutions are needed to precisely identify and target complex epicardial substrate during concomitant open-chest surgery—enabling accurate localization on the arrested heart—and in stand-alone procedures, where prolonged surgery and the need for an EP team in the room present additional challenges. Epicardial guides would allow the surgeon to improve navigation, accuracy, safety, and therapeutic efficacy in difficult-to-treat ablation patients.

Herein, we aimed to develop a feasible, cost-effective, and rapid method for creating epicardial surgical guides, focusing on early feasibility and technical viability. Compared to traditional 3D printing methods, HCR molding offers an efficient and affordable alternative that yields promising potential.

Rationale behind silicone and molding

The emergence of 3D printing technologies in the last decade has created abundant application opportunities in the field of precision medicine and surgical education [10]. However, 3D printing resins face challenges, such as brittleness, poor dimensional accuracy and low mechanical strength, especially after post-processing and sterilization, as well as limited material options, high costs, and lack of standardization [11,12,13]. In addition, uncured monomers or leachates may release cytotoxic byproducts, further complicating their use in surgical environments [14]. To overcome the limitations of traditional 3D printing resins, we chose a silicone-based solution—an elastomer widely used in the medical field [15]. Some silicones are approved for long-term implantation and have a proven track record in dynamic (cardiovascular) surgical environments. With its flexibility, durability, stability, versatility, and resistance to extreme temperatures, silicone can be fully customized to meet the specific needs of this epicardial application.

Although silicone modeling has been used to create customized molds [16,17,18], no studies have fabricated silicone specifically as an epicardial guide, posing unique challenges in identifying a material that meets all necessary criteria. Material choice is a critical factor to the success of a patient-specific medical device. Properties such as silicone hardness and flexibility help the guide maintain shape and function despite the heart’s constant motion, which prevents gaps that could lead to uneven lesions.

We also considered liquid silicone rubber molding, a widely used technique in medical device manufacturing and studies. However, this method requires creating an inverted mold with several components (multi-part mold system, vent channels, and silicone channels) that must be precisely aligned and securely connected using interlocks to prevent movement. This makes the process significantly more labor-intensive and less suitable for a streamlined, efficient, and rapid customized workflow for ablation treatments. Another barrier is that the liquid silicone for our required durometer would be too viscous at room temperature, making injection into the mold difficult and prone to air entrapment. We also considered direct 3D silicone printing, a more novel approach. However, it was not our preferred choice due to potential challenges with support structures, layer adhesion, and viscosity control, which could compromise integrity and make the process tedious.

An advantage of silicone wrapping is that sculpting results in a smoother inside surface due to the natural smoothness of milled silicone; unlike 3D printing, which can create rougher surfaces. This technique also allows customization using different silicones, curing times, and pigments to meet tailored requirements.

Thermal insulation and procedural safety

The guide manufacturer is responsible for ensuring compliance with design requirements (form, fit, and function) throughout the entire device’s life cycle [19]. A critical consideration in the development of surgical guides is their thermal insulation performance under ablative stress. These properties are vital for two primary reasons: ensuring procedural safety during targeted ablation and protecting adjacent, non-target anatomy from thermal injury. The material’s electrical non-conductivity reduces the risk of unintended heating when ablation instruments come into accidental contact with the guide’s edges—a scenario that can result in localized temperatures as high as 70–90 °C during radiofrequency ablation or below 0 °C during cryo-ablation, potentially causing collateral tissue damage. Due to the lack of conductivity data in the material datasheet, preliminary tests were conducted simulating accidental instrument-guide contact. These experiments confirmed that the guide effectively prevented thermal injury to underlying tissue. However, further comprehensive thermal and mechanical characterization—particularly post-processing and sterilization—remains necessary to validate the material’s suitability for clinical application.

Design considerations

Guide stability on the epicardial surface is of critical importance to ensure precise lesion placement. Material testing indicated that silicone with a 70 Shore A hardness and 2–3 mm thickness provided an optimal balance between flexibility and intraoperative stability on the beating heart. In addition, preliminary foldability tests were conducted to assess suitability for minimally invasive approaches, revealing that the current guide designs are too bulky for efficient trocar introduction, largely due to excess material at the apex base. However, this limitation is expected to be less significant in smaller, target-specific guide designs. Future work should explore alternative silicones and ensure uniform thickness without excess material. Moreover, to facilitate insertion and removal of the guide through a trocar, incorporating a secure locking system with small tabs on the lateral sides that click when folded might prove useful.

Finally, an important challenge in epicardial guide use for minimally invasive stand-alone ablation is achieving stable anchoring of the model on a beating heart. Potential solutions we considered include micro-suction ports, adhesive surfaces, or specialized gripping materials.

Broader clinical applicability and translational pathway

While the guide in this feasibility study was designed to cover a broad scar region, the end objective is to use preprocedural input from the EP to incorporate predefined ablation targets in form of ‘windows’ in the mask, directly providing the surgeon with an ablation roadmap or lesion set. By providing intra-operative guidance, it could enhance precision and reduce reliance on full real-time mapping and a complete EP team, since lesions are identified pre-operatively. However, the customization of these ablation openings requires further finetuning as they depend on patient pathology, surgical approach (open-chest or minimally invasive), and the type and size of the catheter used.

A crucial early step in clinical translation is assessing biocompatibility and characterizing the sterilized silicone material to confirm its suitability, thermal stability, mechanical integrity, and overall safety for clinical use. Preclinical validation is essential to rule out risks, such as loose fragments or allergic reactions, and to assess the guide’s fit, stability, efficacy and precise electro-anatomic alignment. Swine models are commonly used due to physiological similarity to humans and comparable heart rates, beginning with open-chest studies and advancing to minimally invasive approaches [20]. For these experiments, subject-specific guides are fabricated from pre-operative cardiac CT. While device alignment partially relies on visible anatomical landmarks (aorta, pulmonary artery ring, and left ventricular apex)—which are accessible via both open-chest and thoracoscopic approaches—epicardial electro-anatomic mapping is still needed during early translational phases. Alignment accuracy can be quantified by measuring the percentage overlap between pre-operative targets (scar and mapping) and post-ablation areas confirmed by mapping, and post-explant TTC staining and histology.

The expected benefits—allowing concomitant VT ablation, improved consistency, shorter procedures, fewer repeat ablations—translate into both clinical and economic value. Though regulatory and compliance efforts for clinical translation are significant, they are minor compared to the costs of repeat ablations, prolonged procedures, and hospital stay/visits—especially once integrated into routine practice. While the training and integration of the device into the surgical workflow may initially add time to the procedure, it is expected to reduce overall operative time over the long term—particularly if intraoperative mapping can be reduced, potentially eliminating the need for an EP team in the operating room.

This silicone-based approach can serve as a basis for the engineering and development of other customizable guides. The potential for further applications is vast, as this can be adapted to meet specific mapping and procedural requirements. For example, our group previously developed an epicardial guide for Brugada syndrome ablation, highlighting the arrhythmogenic substrate in the right ventricular outflow tract, based on electro-anatomical mapping data [21]. In addition, we have created guides for coronary artery mapping and optimal bypass target placement during coronary artery bypass grafting [22], and sinus node protection for inappropriate sinus tachycardia ablation. Furthermore, this approach could extend to other (non-)cardiac procedures for anatomical modeling, pre-operative planning, and surgical training.

Limitations and future considerations

Several limitations should be considered. As this was an early proof-of-concept study, the 3D-printed mold was fabricated from non-biocompatible PLA. Thus, biocompatibility testing, including in vitro cytotoxicity assessments as per ISO 10993–5:2009 (https://www.iso.org/standard/36406.html), as well as post-sterilization material characterization assessments are warranted for future research. Following, in vivo porcine experiments are needed on the beating heart to assess stability, fit, ablation efficacy, and electro-anatomic alignment more in-depth.

Landscape of single cell nuclei of bladder cancers

Nine patients diagnosed with NMIBC were enrolled in this study. All patients were treated with adjuvant BCG after endoscopic resection. Three patients experienced disease progression. They were classified as “progressors”. The remaining six patients who did not experience disease progression were classified as “non-progressors.” Detailed clinicopathological information of these patients is provided in Fig. 1A.

To characterize cell types in the tumor microenvironment (TME) of NMIBC, a total of 14 specimens (P-2, P-8 and P-9 specimens per patients including three post-progression specimens, 1 cell-free specimen) were subjected to snRNA-seq using resection specimens before BCG treatment. A total of 73,979 nuclei were obtained after removing cell doublets (median of 5,804 nuclei, 1,000–8,720 nuclei per patient) and profiled for mRNA quantification. Detailed information on snRNA-seq is available in Supplementary Table S1. Clustering of snRNA-seq revealed six major cell types: 61,837 epithelial cells, 898 endothelial cells, 5,973 T cells, 1,907 B cells, 1,712 myeloid cells, and 1,652 fibroblasts.

A t-distributed stochastic neighbor embedding (tSNE) plot was used to illustrate cell distribution as shown in Fig. 1B. Notably, epithelial cells constituted a significant fraction of cells, indicative of high tumor purity across samples. Moreover, heterogeneity between Progression and NP samples was observed as shown in pairwise tSNE plots (P-2, P-8, and P-9), Primary and progression samples formed well-separated clusters, indicating distinct transcriptional differences (Fig. 1C). The expression of marker genes for each major cell type is depicted in Fig. 1D, including CDH1 [18] for epithelial cells, VWF [19] for endothelial cells, COL1A1 [20] for fibroblasts, CSF1R [21] for myeloid cells, CD2 [22] for T cells, and MS4A1 [18] for B cells. Additionally, cluster-specific gene expression of selected markers is presented in Fig. 1E.

The TGF-β signaling pathway is activated in malignant cells with disease progression

Epithelial cells were further subject to fine-clustering. tSNE plots of epithelial cells revealed that the clustering of epithelial cells was largely determined with respect to individual patients, indicative of a substantial level of inter-patient heterogeneity of malignant cells (Fig. 2A). We then performed inferCNV to determine copy number alterations (CNA) based on gene expression. Using inferCNV analysis, we identified significant interpatient heterogeneity in inferred CNA profiles across nine bladder cancer malignant cells (Fig. 2B). Notable arm-level gains were observed in chromosomes 1q, 5p, and 20p, while frequent losses were detected in 9p, 11p, 17p, and 19p. Hurst et al. [23] have reported that gains of 1q and 5q and losses of 9p, 11p, 17p, and 19p are associated with disease progression of bladder cancers. In addition, Bellmunt et al. [24] have found that gains in 20p are linked to disease recurrence, while López et al. [25] have identified a connection between gains in 5q and bladder cancer recurrence.

We analyzed malignant cells using longitudinal specimens obtained before treatment and after disease progression. Functional gene sets were identified using ssGSEA with representing sample-specific disease progression using Hallmark pathway. This analysis identified molecular functions enriched in the difference of NES values between Progression and Primary specimens. It highlighted tumor progression-related functions, such as WNT–β-catenin signaling and TGF-β signaling, which are consistently enriched across malignant cells during disease progression (Fig. 2C). This result suggests that TGF-β might play a key progression-related molecular function in disease progression after BCG (Figs. 2D–2F). Pseudo-temporal analysis representing the transition from treatment-naïve to progression samples indicated that each paired sample of malignant cells passed through smooth one-dimensional curves. Specifically, cells from treatment-naïve samples were ordered at early pseudotime points, followed by cells from progression samples at later pseudotime points. This indicates a clear separation in differentiation trajectories of treatment-naïve and progression cells. Results of the ssGSEA analysis of TGF-β signaling along the pseudotime trajectory showed that TGF-β signaling enrichment scores increased progressively from treatment-naïve to progression samples, consistent with the trajectory analysis.

TME cell subtypes associated with disease progression

Sub-clustering of macrophages (Fig. 3A; proliferating, M2 and SPP1 + macrophages) revealed three distinct subtypes. Pseudotime trajectory analysis showed bifurcation into two major branches, with proliferating and some subtypes located in earlier stages, M2 macrophages diverging into another branch, and SPP1 + macrophages appearing in the later phase (Fig. 3B). M2 macrophages were relatively more abundant in the Progression group than in the Progression-Before BCG group. Proliferating myeloid cells showed a gradual increase from NP to Progression-Before BCG and remained elevated in the Progression group, whereas SPP1 + macrophages exhibited a consistent decrease across groups, with the lowest proportion observed in the Progression group (Fig. 3C). The top 10 marker genes of macrophage subtypes are shown in Fig. 3D. The LYZ gene was identified as a marker gene for proliferating myeloid cells [26, 27]. In previous studies on bladder cancer, LYZ has been used as a myeloid cell marker. Moreover, Gu et al. have demonstrated a significant association between these cells and proliferation. Regarding the TTC7B gene, He et al. have found notable correlations between its expression and infiltration levels of various immune cells, including M2 macrophages [28].

Sub-clustering of dendritic cells (Fig. 3E; DC1, DC2, and DC3) demonstrated sequential differentiation. The pseudotime trajectory (Fig. 3F) followed an order from DC3 (LAMP3 +) at the earliest stage, through DC2 (CD1A +), and finally to DC1 (XCR1 +) at the latest stage, highlighting dynamic changes in cell states. For dendritic subtypes, CD1A (DC2) cells showed only a modest and variable increase in the Progression group compared with NP and Progression-Before BCG, without a consistent trend across patients (Fig. 3G). Dendritic cells are known for their roles in antigen presentation and immune response orchestration. They exhibited CADM1 expression, particularly in XCR1 (DC1), as highlighted by our DEG results where CADM1 emerged as a marker (Fig. 3H). To understand CADM1’s function [29], future research needs to be conducted on tumor progression where CADM1’s role has been extensively studied. Kang et al. have found that the ADGRG5 gene in CD1A (DC2) is associated with dendritic cells, showing a stronger correlation with ADGRG5 expression than in other types of immune cells tested [30]. In our study, ADGRG5 was identified as a marker in DC2. In DCs, CCR7-dependent migration from peripheral tissues to lymphoid tissues plays a critical role in host defense against pathogens and immune tolerance maintenance [31]. This migratory process is governed by complex intracellular signaling pathways that can regulate both DC movement and inflammatory responses. Our analysis identified CCR7 as a marker gene specifically in LAMP3-expressing DCs (DC3), as shown in our differential gene expression data (Fig. 3H).

Fibroblast subtypes (Fig. 3I; myoCAF, iCAF, and SMC) also showed distinct developmental patterns. Pseudotime analysis (Fig. 3J) indicated that myoCAF and iCAF dominated the early phase, while SMC and iCAF became predominant later, with the trajectory culminating in iCAF and SMC states. Results revealed a higher proportion of myoCAF cells in the Progression Before BCG group compared with the Progression group. Additionally, SMC cells showed a consistent proportion across all groups. Notably, iCAF cells maintained the highest proportion in all groups, although they were slightly reduced in the Progression group compared with NP and Progression Before BCG groups (Fig. 3K). Both iCAF and myoCAF showed similar cell proportions, although iCAF demonstrated a stronger correlation with reduced survival rates. The top 10 marker genes of fibroblast subtypes are displayed in Fig. 3L.

Sub-clustering of endothelial cells (Fig. 3M; venous, tip-like, arterial, and lymphatic) revealed further heterogeneity. Pseudotime trajectory analysis (Fig. 3N) indicated that venous and tip-like cells appeared in early stages, arterial and lymphatic cells arose but later disappeared, leaving tip-like cells predominant at the end. The distribution of endothelial cell subtypes within each group is illustrated in Fig. 3O. In the NP group, venous cells constituted the highest proportion, followed by tip-like cells. Arterial and lymphatic cells were present in relatively smaller proportions. In the Progression Before BCG group, tip-like cells dominated almost entirely, with a small presence of venous cells. Arterial and lymphatic cells were nearly absent. In the Progression group, tip-like cells also constituted the highest proportion, although the proportion of venous cells was significantly reduced compared with levels in NP and Progression-Before BCG groups. Additionally, proportions of arterial and lymphatic cells slightly increased. The top 10 marker genes of endothelial subtypes are shown in Fig. 3P. In humans, loss-of-function mutations in VEGFC or VEGFR3 can lead to lymphedema, while the application of recombinant VEGFC can stimulate robust lymphangiogenesis in adults, suggesting its therapeutic potential for lymphedema and tissue repair.

To distinguish the function of each subtype, we performed hallmark pathway analysis using ssGSEA and selected the top 10 hallmark pathways. As shown in Supplementary Fig. 1A, Proliferating Macrophage subtype was enriched in the EMT and MYC-TARGETS-V2 pathways, while the M2 subtype was enriched in the PI3K-AKT-mTOR-SIGNALING and TGF-BETA-SIGNALING pathways. Additionally, the SPP1 subtype was enriched in the MYC-TARGETS-V1, HYPOXIA, and DNA-REPAIR pathways. The population of dendritic cells was clustered into three subtypes (DC1, DC2, and DC3), although they were not clearly separated in ssGSEA Hallmark pathway. However, DC2 tended to be enriched in the DNA-REPAIR pathway, while DC showed a tendency to be enriched in the EMT pathway (Supplementary Fig. 1B). Additionally, fibroblasts were divided into three sub-clusters (myoCAF, iCAF, and SMC). The myoCAF subtype was enriched in immune-related pathways and the INFLAMMATORY-RESPONSE pathway, whereas iCAF and SMC subtypes were not clearly distinguished as shown in Supplemantary Fig. 1C. The venous subtype, a subset of endothelial cells, was enriched in immune-related pathways and the INFLAMMATORY-RESPONSE pathway, while the Tip-like sub-cluster was enriched in the MYC-TARGETS-V1 pathway. Additionally, the arterial subtype was enriched in the COMPLEMENT and ANGIOGENESIS pathways, whereas the Lymphatic sub-cluster was enriched in the NOTCH-SIGNALING pathway, as shown in Supplementary Fig. 1D.

Cell–cell interactions across malignant cells and TME cells

To investigate differences in cell–cell communication among various cell subpopulations, we analyzed interaction numbers and interaction strength using CellChat [16]. Our study included three conditions: No Progression (NP), Primary Before BCG, and Progression. Results in Fig. 4A illustrate variations in interaction patterns across these conditions. In the NP condition, interactions were relatively sparse, with notable communication occurring primarily between epithelial cells and other cell types. In the Primary condition (Before BCG), the interaction network became more complex, indicating increased communication among T cells, B cells, myeloid cells, and other subpopulations. The Progression condition showed the most extensive and robust interactions, suggesting a heightened level of cell–cell communication. We conducted a pathway-based ligand-receptor interaction analysis between TME cells. Results (Fig. 4A) demonstrated dynamic changes in cell–cell communication networks across different conditions, emphasizing the importance of epithelial cell interactions in NP, Progression Before BCG, and Progression. Notably, the interaction strength between epithelial cells and myeloid cells, as well as fibroblasts, increased from Progression Before BCG to Progression.

According to Browaeys et al. [17], a validation study can substantiate the proposed methodology, illustrating that the final prior model of ligand–target interactions can be broadly applied to various biological systems, thereby supporting the applicability of Nichenet to a wide range of biological contexts. Consequently, we employed the TGF-β signaling pathway identified in Fig. 2 to analyze Nichenet. We defined the gene set of interest as those genes within the receiver cell type that are likely to be influenced by the cell–cell communication event.

Results shown in Fig. 4A revealed that the interaction strength between epithelial cells and myeloid cells, as well as fibroblasts, increased from the Progression Before BCG stage to the Progression stage. Consequently, we analyzed ligand-receptor interactions during muscle regeneration in Progression samples using NicheNet. By applying stringent cutoffs, we identified differentially expressed ligands predicted to interact with receptors on malignant cell (top 20 by differential expression). We compiled a list of ligand-receptor pairs expressed in relevant cell types and predicted them to act upstream of TGF-b signaling specific genes. To narrow down the extensive list of candidates, we focused on ligands with high activation scores for TGF-b signaling genes and complementary targets in macrophages (Figs. 4B–4C) and fibroblasts (Figs. 4E–4F). In addition, we identified the top 30 ligand-receptor pairs in macrophages (Fig. 4D) and fibroblasts (Fig. 4G).

Establishment and validation of prognostic significance marker genes

Using TCGA data, we employed CIBERSORTx to estimate proportions of M2 macrophages and fibroblasts. As shown in Figs. 4D and 4G, top 30 ligand-receptor pairs were identified the. Based on these results, we examined the expression of the DSC2(L)-DSG2(R) pair in epithelial and myeloid cells and the expression of the ENG(L)-BMPR2(R) pair in epithelial and fibroblast cells from our snRNA-Seq data. We then analyzed their survival associations in TCGA BLCA data. Results are shown in Fig. 5. Patients with a higher average proportion of M2 macrophages and elevated expression of the DSC2(L)-DSG2(R) pair demonstrated a significant difference in survival with a p-value of 0.016 (Fig. 5A). Similarly, patients with a higher average proportion of fibroblasts and increased expression of the ENG(L)-BMPR2(R) pair also showed a significant survival difference, with a p-value of 0.017 (Fig. 5B). According to Nakauma-González et al. [32], DSC2 and DSG2 in bladder cancer are markers of intratumoral genomic and immunologic heterogeneity, particularly evident through squamous differentiation. This morphological heterogeneity can serve as a biomarker for intrinsic immunotherapy resistance in bladder cancer patients.

The black horse of Lloyds’ banks are a familiar sight on UK high streets, but the lender has quietly become one of the country’s biggest landlords too, amassing £2bn in residential property, according to new analysis.

The company has reached the landmark valuation on its property assets after a drive to buy more than 7,000 properties, according to the Financial Times.

Already the UK’s largest mortgage lender, it set out ambitious plans four years ago to acquire 50,000 rental homes by 2030 to become the country’s biggest landlord.

It launched its Lloyds Living division in July 2021, then called Citra Living, which provides homes for rent and shared ownership at 42 developments across the country. They are a mix of houses and low-rise apartment blocks, usually located in suburban areas, rather than city centres.

Lloyds Living now manages 7,500 homes, “from one bedroom apartments for young professionals to four bedroom houses for growing families”. This puts it among the UK’s biggest private sector landlords, which include the insurance and pension group Legal & General, the fund manager M&G and the property developer Grainger.

A spokesperson for the bank said: “We are pleased with the significant progress made to grow the Lloyds Living business since its launch in 2021, and how – in line with the strategic aims set out from the start – it is helping to increase access to good quality, affordable housing nationwide and is already contributing significantly to the group’s diversified income streams.”

Lloyds has said most of its acquisition activity has been funding its development partners to build more housing.

In June, the bank struck its second big property deal with Barratt Redrow, the UK’s largest housebuilder, as part of the companies’ build-to-rent partnership. It added 598 two-, three- and four-bedroom homes in 11 new and existing Lloyds Living sites including Berkshire, Oxfordshire, Buckinghamshire, Kent, Cheshire and Gloucestershire.

Last summer, Lloyds started turning former office buildings into social homes, at a site in Pudsey in West Yorkshire, with plans for 93 homes to be rented at half the usual rate, making it the first UK bank to enter the social housing market directly.

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The bank held a social housing conference in July, convening housing associations and then-housing secretary Angela Rayner, to discuss the desperate need for more affordable housing in the UK. Lloyds chief executive Charlie Nunn cautioned: “The challenge is considerable and complex,” but added that there are “countless brownfield sites that lie empty”.

Diversifying away from lending into private home rentals means Lloyds is less reliant on interest income, which was squeezed in recent years by record low UK interest rates.

Lloyds has reported “strong income growth” from Lloyds Living. However, its performance has been overshadowed by the ongoing car loans commission scandal. The lender is also the UK’s biggest car lender through its Black Horse division and is expected to foot the largest bill compensation bill for the scandal among its peers.

Introduction

Total joint arthroplasty (TJA) is widely recognized as one of the most effective surgical interventions for managing end-stage joint disease and offers significant pain relief and functional restoration.^1,2 However, the long-term success of TJA is often compromised by aseptic loosening caused by wear particle-induced periprosthetic osteolysis (PPO), which remains the leading cause of prosthesis failure and revision surgeries worldwide.^3,4 Among the various types of wear debris generated during implant use, ultra-high molecular weight polyethylene (UHMWPE) particles are the most prevalent in modern joint prostheses because of their widespread application as bearing materials.^5,6 However, prolonged mechanical wear releases a large number of UHMWPE particles into the peri-implant microenvironment, triggering chronic inflammation and progressive bone resorption.^3,7

Recent projections indicate that the demand for TJA will continue to increase significantly over the coming decades.⁸ For example, projected models based on Medicare data anticipate a 176% increase in total hip arthroplasty (THA) and a 139% increase in total knee arthroplasty (TKA) by 2040, with further growth expected by 2060, similar trends have been reported in other predictive models.^9–11 Concurrently, the incidence of osteoporotic fractures and other bone-related conditions is rising with demographic aging, emphasizing the urgent need for preventive and regenerative strategies to preserve bone integrity and long-term implant stability.^12–14

UHMWPE and titanium particles have been implicated in the pathogenesis of PPO, and despite differences in their physicochemical properties, they share similar macrophage-mediated inflammatory pathways.^15,16 Once released, wear particles are recognized and internalized by local immune cells, particularly macrophages, triggering a cascade of chronic inflammatory responses.¹⁷ These immune reactions disrupt the bone remodeling balance, primarily by promoting osteoclast differentiation and activity, ultimately leading to progressive bone loss around the prosthesis.^18,19 Mounting evidence suggests that the imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages plays a pivotal role in this pathological process.^20,21 M1 macrophages secrete tumor necrosis factor-alpha (TNF-α), interleukins (IL-1β, IL-6), and reactive oxygen species (ROS), which stimulate osteoclastogenesis.²² In contrast, M2 macrophages support inflammation resolution and tissue regeneration through IL-10 and TGF-β secretion.²³ Therefore, Macrophage polarization has emerged as a therapeutic target to prevent or reverse periprosthetic bone loss.²⁴

Various strategies have been explored to modulate this polarization, such as surface modification of implants, local delivery of anti-inflammatory agents, and use of immune-modulating biomaterials.²⁵ However, these approaches often lack long-term efficacy or are difficult to implement clinically.^26,27 Cell-based therapies, particularly those involving mesenchymal stem cells (MSCs), are gaining traction owing to their immunomodulatory potential and regenerative properties.

Bone marrow-derived mesenchymal stem cells (BMSCs) possess multipotent differentiation potential and exert profound immunomodulatory effects on both innate and adaptive immune systems. Through direct cell-cell contact and paracrine signaling, BMSCs can suppress M1 polarization and promote M2 macrophage differentiation.²⁸ By secreting a range of soluble factors, including IL-10, PGE2, TSG-6, and TGF-β, and modulating key inflammatory signaling pathways, such as the NF-κB, STAT3, and PI3K/Akt pathways.^29,30 Clinical and preclinical studies have demonstrated the efficacy of MSC-based therapy in promoting bone repair, reducing inflammation, and improving outcomes in degenerative and osteoporotic bone diseases.^31–33 In orthopedic applications, bone marrow-derived mesenchymal stem cells (BMSCs) have shown the ability to regulate the local immune microenvironment, inhibit osteoclastogenesis, and enhance osteogenesis, providing dual benefits in restoring bone balance and mitigating inflammation, which may be highly relevant in lesions involving both bone resorption and inflammation-driven bone loss.^34,35

However, the mechanistic understanding of how BMSCs regulate macrophage polarization and cytokine production in the presence of UHMWPE wear particle-induced osteolysis remains incomplete. Although BMSCs have demonstrated promising therapeutic effects in various inflammatory diseases, their specific roles in orthopedic settings, particularly in wear particle-induced periprosthetic osteolysis, remain to be fully elucidated. In particular, the influence of BMSCs on macrophage polarization and remodeling of the local immune microenvironment has not been systematically studied. The signaling networks and cellular interactions underlying these effects remain unclear and require further investigation.

Objective: Therefore, this study aims to conduct a comprehensive evaluation of the regulatory effects of BMSCs on macrophage polarization and inflammatory cytokine secretion in response to UHMWPE particles. By integrating an in vitro co-culture system with an in vivo murine calvarial osteolysis model, we assessed the immunomodulatory mechanisms of BMSCs, the impact of treatment frequency on therapeutic efficacy, and their influence on local bone microarchitecture and cytokine profiles.

By uncovering how BMSCs reprogram macrophage phenotypes and reshape the peri-implant immune milieu, this study seeks to provide a mechanistic foundation for the development of stem cell–based precision immunotherapies or advanced biofunctionalized materials for preventing UHMWPE wear particle-induced osteolysis.

Materials and Methods

Preparation of UHMWPE Wear Particles

The UHMWPE particles (Nanochemazone, Leduc, Alberta, Canada) had a mean particle diameter of 2.6 μm (range from <0.6 to 21 μm), measured by scanning electron microscopy according to the manufacturer’s specifications.^36,37 The particles were immersed in 70% ethanol for 24–48 hours, rinsed with sterile PBS, and UV-sterilized for 30 minutes. The particles tested negative for endotoxin using a Limulus Amebocyte Lysate Kit (Beyotime Biotechnology, Shanghai, China). Particles were suspended in DMEM (Gibco, NY, USA) and stored at 4 °C. To prevent aggregation, the suspension was ultrasonicated for 15–30 min before use.

Isolation and Culture of BMSCs

Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the femurs and tibias of 2-week-old C57BL/6 mice using the compact bone explant method, as previously described.^38,39 Cells were cultured in α-Minimum Essential Medium (α-MEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA). Cells at passages 3–5 were used for subsequent experiments. To verify their identity and multipotency, BMSCs were induced to differentiate into osteogenic, adipogenic, and chondrogenic lineages using a standard induction medium. Differentiation was confirmed by Alizarin Red S, Oil Red O, and Alcian Blue staining (Sigma-Aldrich, St. Louis, MO, USA).

Culture of RAW264.7 Macrophages

The RAW264.7 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were cultured in DMEM supplemented with 10% FBS (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA), and maintained at 37°C in a humidified incubator with 5% CO₂. The culture medium was refreshed every 2–3 days, and cells in the logarithmic growth phase were used for experiments.

Determination of UHMWPE Particle Concentration

To determine the appropriate concentration of UHMWPE particles for macrophage polarization assays, RAW264.7 cells were treated with varying concentrations (0.05, 0.1, 0.2, and 0.8 mg/mL) for 24 hours. The expression of the M1 marker CD80 and M2 marker CD206 was assessed using flow cytometry, and the CD80 ⁺/CD206 ⁺ cell ratio was calculated to evaluate the polarization shift.

Co-Culture of BMSCs and RAW264.7 Cells Using a Transwell System

A non-contact co-culture system was established using Transwell plates with 0.4 μm pore size membrane inserts (Corning, NY, USA). RAW264.7 macrophages were seeded into the lower chambers (1×10⁵ cells/well) in 1 mL DMEM with 10% FBS. For the particle-treated groups, the UHMWPE particles were added at a final concentration of 0.2 mg/mL. After 12 h, BMSCs (1×10⁵ cells/well) were seeded into the upper inserts in 1 mL α-MEM with 10% FBS and co-cultured for 24 h at 37°C with 5% CO₂. Five groups were included: A. RAW264.7 alone (M), B. BMSCs alone (BMSCs), C. co-culture without particles (M + BMSCs), D. RAW264.7 with particles (M + UPE), and E. co-culture with particles (M + UPE + BMSCs). After incubation, the supernatants were collected, centrifuged, and stored at −80°C for cytokine assays. RAW264.7 cells were harvested for flow cytometry and immunofluorescence analysis.

Flow Cytometry Analysis

Flow Cytometric Characterization of BMSCs

Flow cytometry (ACEA Biosciences Inc., Hangzhou, China) was performed using a panel of surface markers.BMSCs were harvested, washed, and stained with fluorochrome-conjugated antibodies (BioLegend, San Diego, CA, USA), including CD31-FITC, CD45-FITC, CD34-PE, Sca-1-PE, CD90-APC, CD105-APC, CD11b-PerCP-Cy5.5, and CD44-PerCP-Cy5.5. Isotype controls were used to assess nonspecific binding. At least 10,000 events per sample were acquired and analyzed using FSC/SSC gating to exclude debris and doublets.

Macrophage Polarization of RAW264.7 Cells

After co-culture, RAW264.7 cells were harvested, washed, and stained with F4/80-PerCP-Cy5.5, CD80-PE, and CD206-APC (BioLegend, San Diego, CA, USA). Isotype-matched controls were used to assess nonspecific binding. At least 10,000 events were recorded for each sample, and gating strategies were used to quantify the proportions of M1 (F4/80⁺CD80⁺) and M2 (F4/80⁺CD206⁺) macrophages.

Enzyme-Linked Immunosorbent Assay (ELISA)

The concentrations of the inflammatory cytokines TNF-α and IL-4 in the culture supernatants were quantified using ELISA kits (Cusabio, Wuhan, Hubei, China) according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA), and cytokine concentrations were calculated based on standard curves.

Immunofluorescence Staining

After co-culture, the cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China), permeabilized with 0.1% Triton X-100 (Solarbio, Beijing, China), and blocked in 1% bovne serum albumin (BSA, Solarbio, Beijing, China). The cells were then incubated with antibodies against F4/80 (red) and either CD80 or CD206 (green, BioLegend, San Diego, CA, USA), followed by nuclear counterstaining with DAPI (blue, Beyotime, Shanghai, China). Fluorescence images (Leica Microsystems, Wetzlar, Germany) were acquired, and merged channels were used to assess marker colocalization with F4/80-positive macrophages.

Mouse Calvarial Osteolysis Model Induced by UHMWPE Particles

C57BL/6 male mice (2–3 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The protocols implemented in this study adhered to internationally recognized standards and were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University. A murine calvarial osteolysis model was established using 4-week-old male C57BL/6 mice (n=4/group). After anesthesia with 4% chloral hydrate (Solarbio, Beijing, China), a midline incision was made to expose the parietal bone, and a UHMWPE particle suspension (0.2 mL, 10 mg/mL) or 0.2 mL PBS was applied to the calvarial surface. Mice were randomly assigned to four groups: A. Sham (PBS), B. PIO (UHMWPE), C. BMSCs-1 (UHMWPE + BMSCs on day 7), and D.BMSCs-2 (UHMWPE + BMSCs on days 7 and 14). BMSCs were subcutaneously administered at a dose of 1×10⁶ cells in 0.2 mL PBS. All mice were sacrificed on day 21 for analysis.

Micro-Computed Tomography (Micro-CT) Analysis

Calvarial samples were scanned using a VNC-102 micro-CT scanner (VINNO, Suzhou, China) at 90 kV and 0.09 mA with a voxel size of 10 μm. Soft tissue and residual particles were removed before scanning. Three-dimensional reconstructions were generated, and bone parameters were evaluated within a standardized region of interest near the sagittal suture.

Histological and Immunohistochemical (IHC) Analyses

After micro-CT scanning, calvarial samples were decalcified in 10% EDTA (Solarbio, Beijing, China) for 14 days at room temperature, dehydrated, embedded in paraffin, and sectioned at 5 μm. Hematoxylin and eosin (HE) staining was performed to evaluate histological changes.

For IHC, the sections were deparaffinized, antigen retrieval was performed in citrate buffer (pH 6.0, Beyotime, Shanghai, China), and nonspecific binding was blocked with 3% BSA. The Slides were incubated overnight at 4 °C with primary antibodies against CD80 (BioLegend, San Diego, CA, USA) and CD163 (Abcam, Cambridge, UK), followed by incubation with HRP-conjugated secondary antibodies (Abcam, Cambridge, UK). DAB was used for color development, and nuclei were counterstained with hematoxylin. IHC-staining was visualized by Leica DM microscope (Leica Microsystems, Wetzlar, Germany).

ELISA for Mouse Tissue Samples

Periosteal tissue samples were collected on day 21 post-surgery, homogenized in lysis buffer containing protease inhibitors, and centrifuged. Supernatants were used to quantify TNF-α and IL-4 levels using ELISA kits (Cusabio, Wuhan, Hubei, China). Subsequent steps followed the same procedure as the ELISA assay described earlier.

Flow Cytometry of Tissue-Derived Cells

Periosteal tissues were enzymatically digested with collagenase I, hyaluronidase, and Dnase (Solarbio, Beijing, China) at 37 °C, filtered through a 40 μm mesh, and washed with PBS. Single-cell suspensions (1×10⁶ cells/mL) were stained with F4/80-PerCP-Cy5.5, CD80-PE, and CD206-APC. After incubation and washing, ≥10,000 events per sample were collected and analyzed. Macrophage subsets were defined as M1 (F4/80⁺CD80⁺) and M2 (F4/80⁺CD206⁺).

Statistical Analyses

All experiments were performed in triplicate as independent biological replicates. Data are presented as mean ± standard deviation (SD). Data normality was assessed using the Shapiro–Wilk test in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Most data sets showed no significant deviation from a normal distribution (p > 0.05), while a few groups exhibited slight deviations (p < 0.05). Given that the overall data were approximately normally distributed, parametric tests were applied for statistical comparisons. Comparisons between two groups were made using an unpaired two-tailed Student’s t-test, and one-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons. Statistical significance was set at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001).

Results

Characterization of BMSCs Isolated from Mouse Bone Marrow

BMSCs were successfully isolated from mouse femurs and tibias using the compact bone adhesion method (Figure 1A). By day 3 of primary culture, fibroblast-like cells had migrated from the bone fragments and adhered to the flask surface. They proliferated rapidly and gradually formed whirlpool-like colonies, and by 10–14 days,  most cells exhibited a spindle-shaped morphology, characteristic of mesenchymal stem cells (Figure 1B).

Figure 1 Characterization of bone marrow-derived mesenchymal stem cells (BMSCs). (A) Surgical isolation of mouse femoral and tibial bone chips for BMSC extraction using the compact bone digestion–explant method. (B) Phase-contrast microscopy showing fibroblast-like morphology and spiral colony formation by 10–14 days. (C) Trilineage differentiation confirmed by Alizarin Red S staining for osteogenesis, Oil Red O staining for adipogenesis, and Alcian Blue staining for chondrogenesis. (D) Flow cytometry analysis showing high expression of CD44, CD90, CD105, and Sca-1, and negative expression of CD31, CD34, CD45, and CD11b.

To confirm their multipotency, BMSCs were subjected to trilineage differentiation. After 14 days, Alizarin Red S staining revealed extensive calcium deposition, confirming osteogenic differentiation. Oil Red O staining identified intracellular lipid droplets indicative of adipogenic differentiation, whereas Alcian Blue staining demonstrated glycosaminoglycan-rich extracellular matrix deposition, confirming chondrogenic potential (Figure 1C).

Flow cytometry further validated the immunophenotypic profiles of the isolated cells. BMSCs showed strong expression of CD44, CD90, CD105, and Sca‑1, whereas hematopoietic and endothelial markers (CD31, CD34, CD45, CD11b) were almost absent compared to the isotype controls, meeting the criteria for defining mesenchymal stem cells (Figure 1D).

BMSCs Alleviate UHMWPE Particle-Induced Calvarial Osteolysis in vivo

To evaluate the therapeutic effects of BMSCs on UHMWPE particle-induced osteolysis, micro-CT was used to assess the calvarial bone loss. As shown in Figure 2A–D), group A (sham) displayed intact cortical bone and dense trabeculae, whereas group B (PIO model) showed severe cortical erosion and trabecular destruction. The C group (single BMSC injection, BMSCs‑1) slightly reduced bone resorption but lacked clear structural restoration. In contrast, the D group (repeated BMSC injections, BMSCs‑2) demonstrated more evident preservation of trabecular architecture.

Figure 2 Micro-CT evaluation of UHMWPE particle-induced calvarial osteolysis and the effects of BMSC treatment. (A–D) Representative 3D micro-CT images of murine calvaria: Sham (A), PIO (B) single BMSC injection (BMSCs-1, C), and repeated injections (BMSCs-2, D). Bone resorption was observed in the PIO group and alleviated by BMSC therapy, especially in BMSCs-2. (E) Quantitative analysis of BMD, BV/TV, Tb.Th, and Tb.N showed significant improvement in bone quality after BMSC treatment compared with PIO. Data are mean ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs PIO. Abbreviation: ns, not significant.

The quantitative analysis (Figure 2E) confirmed these observations. UHMWPE particles significantly reduced BMD, BV/TV, and Tb. Th, and Tb. N compared to those in the sham (P<0.01–0.0001). A single BMSC injection did not produce significant improvements in any of these parameters. However, repeated BMSC delivery significantly increased BV/TV (P<0.05), Tb. Th (P<0.01), and Tb. N (P<0.05) compared to the B group, although BMD remained lower than that in the sham with only mild, non-significant recovery.

These findings suggest that repeated BMSC treatment alleviates UHMWPE-induced bone loss primarily by improving trabecular volume and architecture, whereas full mineral density recovery may require longer-term remodeling.

Histological and Immunohistochemical Evaluation of Calvarial Tissue

HE staining revealed marked periosteal thickening, dense inflammatory cell infiltration, and extensive bone surface erosion in the PIO group compared with the sham group (Figure 3A). In the BMSCs‑1 injection group, periosteal hyperplasia and inflammatory infiltration were noticeably reduced, and more intact cortical bone could be observed. BMSCs‑2 showed the greatest reduction in inflammatory infiltration and better preservation of bone architecture, with the periosteal morphology approaching that of the sham group.

Figure 3 Histological and immunohistochemical staining of calvarial sections. (A) HE staining showing periosteal inflammation and bone destruction in the PIO group, partially alleviated by BMSC treatment. (B) IHC staining of CD80 showing reduced pro-inflammatory macrophage infiltration following single and repeated BMSC administration. (C) IHC staining of CD163 showing increased anti-inflammatory macrophage recruitment after BMSC treatment. Scale bars = 100 μm and 50 μm.

IHC staining further demonstrated changes in macrophage polarization within periosteal tissue (Figure 3B). CD80 was strongly expressed in the PIO group, with numerous CD80⁺ cells distributed along the periosteal surface and within the inflammatory lesion. In the BMSCs‑1 group, CD80 expression was reduced compared with that in the PIO group, and the BMSCs‑2 group showed an even lower density of CD80⁺ cells.

Conversely, CD163 expression was markedly reduced in the PIO group compared with that in the sham group (Figure 3C). BMSC treatment restored CD163 expression, with more CD163⁺ cells visible in both single and repeated-injection groups. The BMSCs‑2 group showed the most abundant CD163⁺ macrophage distribution, particularly around the preserved cortical bone.

Taken together, the histological and IHC staining results suggest that BMSC treatment alleviates UHMWPE-induced inflammatory bone destruction, suppresses M1 macrophage infiltration, and promotes M2 macrophage recruitment, with repeated injection providing a more pronounced effect.

BMSCs Modulate Periosteal Inflammatory Cytokines and Macrophage Polarization in vivo

BMSCs Enhance M2 Macrophage Polarization in Periosteal Tissue

Macrophage polarization in the calvarial periosteum was analyzed using flow cytometry. For CD80 (M1 macrophage marker), the proportion of CD80⁺ macrophages was 25.02 ± 2.00% in the sham group and increased markedly to 36.25 ± 2.86% in the PIO group (P < 0.01). Both BMSC-treated groups showed reduced CD80 expression relative to PIO, with values of 23.48 ± 4.48% in the BMSCs-1 group and 24.98 ± 2.52% in the BMSCs-2 group (P < 0.01 vs PIO) (Figure 4A).

Figure 4 Flow cytometry and ELISA analyses of macrophage polarization and cytokine expression in periosteal tissue. (A–C) Quantitative analysis of CD80⁺ (M1) and CD206⁺ (M2) macrophages by flow cytometry showing increased CD80 and reduced CD206 in the PIO group, reversed by BMSC treatment, especially after repeated injections. (D and E) Representative gating plots illustrating M1/M2 distribution. (F and G) ELISA results showing elevated TNF-α and IL-10 in the PIO group and a reduction toward sham levels after BMSC therapy. Data are mean ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs PIO. Abbreviation: ns, not significant.

The proportion of CD206⁺ (M2) macrophages was significantly lower in the PIO group (14.71 ± 2.65%) than in the sham (24.94 ± 3.33%, P < 0.05). Both BMSC-treated groups showed increased CD206 expression compared with PIO, reaching 24.07 ± 4.98% in BMSCs-1 and 25.40 ± 2.35% in BMSCs-2 (P < 0.05 for both vs PIO) (Figure 4B).

In addition, the CD80⁺/CD206⁺ polarization ratio (Figure 4C) was markedly elevated in the PIO group (1.64 ± 0.03) compared with the sham (1.20 ± 0.19, P < 0.01), indicating enhanced M1 polarization. This ratio was significantly reduced after BMSC treatment. This ratio decreased to 1.15 ± 0.02 and 1.12 ± 0.05 in the BMSCs-1 and BMSCs-2 groups, respectively, restoring values close to those observed in the sham. Representative flow cytometry plots (Figure 4D and E) further illustrate these changes, showing a clear rightward expansion of CD80⁺ M1 cells under UHMWPE stimulation, which was substantially reversed following BMSC administration, alongside an increase in CD206⁺ M2 macrophages.

BMSCs Regulate Periosteal Inflammatory Cytokine Levels

The periosteal tissue was analyzed by ELISA on day 21. TNF-α levels were significantly higher in the PIO group than in the sham (P < 0.05). Single BMSC treatment (BMSCs‑1) slightly reduced TNF‑α but without statistical significance, whereas repeated treatment (BMSCs‑2) markedly suppressed TNF‑α to near-sham levels (P < 0.05 vs PIO) (Figure 4F). For IL‑10, the PIO group showed the highest levels (P<0.05 vs sham), indicating a compensatory anti-inflammatory response. BMSCs‑1 slightly lowered IL‑10 (ns), while BMSCs‑2 significantly reduced IL‑10 compared with PIO (P<0.05), reaching values comparable to those of sham (Figure 4G).

Overall, repeated BMSC administration more effectively modulated periosteal inflammation and suppressed excessive TNF‑α production, while normalizing dysregulated IL‑10.

UHMWPE Particles Induce Dose-Dependent Macrophage Polarization in vitro

RAW264.7 macrophages were exposed to UHMWPE particles at 0.05, 0.1, 0.2, and 0.8 mg/mL for 24 h, and M1 (CD80) and M2 (CD206) expression was analyzed by flow cytometry. Quantitative analysis (Figure 5A–C) showed that the proportion of F4/80⁺CD80⁺ (M1) macrophages significantly increased at 0.05, 0.1, and 0.2 mg/mL compared with control (****P < 0.0001, ***P < 0.001), while 0.8 mg/mL showed no significant difference. The proportion of F4/80⁺CD206⁺ (M2) macrophages remained unchanged at lower concentrations but slightly increased at 0.8 mg/mL (*P < 0.05). The CD80⁺/CD206⁺ polarization ratio was maximally elevated at 0.05 and 0.1 mg/mL (****P < 0.0001), moderately at 0.2 mg/mL (***P < 0.001), and minimally at 0.8 mg/mL (**P < 0.01), indicating a dose-dependent shift toward M1 polarization. Representative flow cytometry plots (Figure 5D and E) illustrate these trends, showing an expansion of the CD80⁺ population with increasing particle concentrations and partial restoration of CD206⁺ expression at the highest dose.

Figure 5 Dose-dependent effects of UHMWPE particles on macrophage polarization. (A–C) Quantification of CD80⁺, CD206⁺, and the M1/M2 ratio across particle concentrations, showing elevated M1 polarization and reduced M2 expression in a dose-dependent manner. (D and E) Representative flow cytometry plots of CD80 and CD206 expression. Data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 vs control. Abbreviation: ns, not significant.

Collectively, 0.2 mg/mL UHMWPE induced a stable and reproducible pro-inflammatory phenotype while maintaining macrophage viability, and was therefore selected for subsequent experiments.

BMSCs Suppress UHMWPE‑induced Macrophage Polarization and Inflammatory Cytokines in vitro

BMSCs Modulate Macrophage Polarization in Vitro

Macrophage polarization in RAW264.7 cells was analyzed using flow cytometry after UHMWPE particle stimulation (Figure 6A–E). The proportion of CD80⁺ (M1) macrophages increased from 44.17 ± 2.79% in the RAW264.7 control group to 89.63 ± 0.46% after UHMWPE stimulation (P < 0.0001). BMSCs co-culture with UHMWPE significantly reduced CD80⁺ expression to 46.94 ± 8.57% (P < 0.0001 vs UHMWPE), restoring values close to the RAW264.7 control group baseline. For CD206⁺ (M2) macrophages, UHMWPE exposure caused only a minor change (10.40 ± 0.11% vs 12.25 ± 0.29% in control, ns), whereas BMSCs alone markedly increased CD206⁺ expression to 35.27 ± 0.91% (P < 0.0001 vs RAW264.7), and BMSCs co-culture with UHMWPE-stimulated macrophages further elevated CD206⁺ expression to 30.69 ± 2.48% (P < 0.0001 vs UHMWPE).

Figure 6 Effects of BMSCs on UHMWPE‑induced macrophage polarization and cytokine secretion in vitro. (A–C) Quantitative analysis of F4/80⁺CD80⁺ (M1) and F4/80⁺CD206⁺ (M2) macrophages showing increased M1 and reduced M2 polarization after UHMWPE exposure, both reversed by BMSC co-culture. (D and E) Representative flow cytometry plots illustrating polarization shifts. (F and G) ELISA results showing UHMWPE-induced TNF-α elevation and mild IL-4 increase, both further modulated by BMSC co-culture. Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs control. Abbreviation: ns, not significant.

Consequently, the CD80⁺/CD206⁺ polarization ratio rose from 4.25 ± 0.31 in control to 7.32 ± 0.18 in the UHMWPE group (P < 0.0001), and was significantly reduced to 1.52 ± 0.17 by BMSC co-culture with UHMWPE (P < 0.0001 vs UHMWPE), indicating a clear restoration of the M1/M2 balance.

BMSCs Regulate Inflammatory Cytokine Secretion in vitro

ELISA analysis of culture supernatants (Figure 6F and G) showed that UHMWPE particles significantly elevated TNF‑α compared with the RAW264.7 group (P < 0.0001), whereas BMSC co‑culture markedly reduced TNF‑α to near‑baseline levels (P < 0.0001 vs UHMWPE). Conversely, IL‑4 secretion was only slightly increased by UHMWPE particles, but was further enhanced by BMSC co‑culture (P < 0.0001 vs UHMWPE).

Overall, these in vitro results demonstrate that BMSCs suppress UHMWPE-induced pro-inflammatory M1 polarization while promoting an anti-inflammatory M2 phenotype, accompanied by corresponding cytokine reprogramming.

BMSCs Modulate UHMWPE‑Induced Macrophage Polarization by Immunofluorescence

Immunofluorescence staining revealed macrophage polarization under UHMWPE stimulation and BMSC co‑culture (Figure 7). CD80 (green) indicates M1 macrophages, CD206 (green) indicates M2 macrophages, F4/80 (red) indicates all macrophage types, and DAPI (blue) indicates the nuclei. The groups included M (RAW264.7), M + UPE (RAW264.7 + UHMWPE), and M (UPE) + BMSCs (RAW264.7 + UHMWPE + BMSCs).

Figure 7 Immunofluorescence of macrophage polarization markers.(A) CD80 (green), F4/80 (red), and DAPI (blue) staining showed strong M1 marker expression in RAW264.7 + UHMWPE, reduced by BMSC co‑culture. (B) CD206 (green), F4/80 (red), and DAPI (blue) staining were suppressed by UHMWPE but enhanced after BMSC co‑culture. Scale bar: 50 μm.

For CD80, the M group showed weak staining, indicating a resting state. The UHMWPE particles markedly increased the CD80 fluorescence, confirming M1 polarization. BMSC co‑culture reduced CD80 staining compared to that in the M+UPE group, suggesting suppression of M1 activation. For CD206, baseline expression in the M group was moderate but further suppressed by UHMWPE particle, indicating reduced M2 polarization. Co‑culture with BMSCs restored CD206 fluorescence, promoting a shift toward the M2 phenotype.

These qualitative observations align with the flow cytometry results, supporting BMSCs’ role in reprogramming macrophages toward an anti‑inflammatory state.

Discussion

Periprosthetic osteolysis remains the leading cause of late implant failure and is primarily driven by macrophage-mediated inflammation in response to wear particles.^16,40 Among these, UHMWPE debris is clinically more relevant than titanium because of its higher generation rate in modern prostheses. This paper further indicates that UHMWPE particles elicit stronger sex-dependent macrophage responses than metallic debris, resulting in more persistent inflammation.⁴¹ In our study, UHMWPE particles induced robust M1 macrophage polarization, elevated TNF‑α, and significant bone loss, confirming previous findings that particulate debris skews macrophages toward a pro-inflammatory phenotype.^42,43

Consistent with prior studies on wear particle–induced osteolysis, our data demonstrated that UHMWPE particles strongly promote M1 macrophage polarization and pro-inflammatory cytokine secretion. Similar inflammatory profiles have been observed in titanium and nanoparticle exposure models, confirming macrophage activation as a key pathogenic mechanism in periprosthetic osteolysis.⁴⁴

However, our results extend these findings by showing that UHMWPE particles not only trigger stronger pro-inflammatory activation than metallic debris but also induce a dysregulated compensatory IL-10 response, suggesting sustained immune stress rather than transient inflammation. Importantly, repeated administration of BMSCs effectively alleviated bone destruction, reduced TNF‑α levels, and enhanced M2 macrophage polarization both in vivo and in vitro.

The RAW264.7 murine macrophage line, as used in our study, is a well-established in vitro model owing to its stable genotype, consistent phenotype, and reproducible responsiveness to immune stimuli, such as lipopolysaccharides (LPS).⁴⁵ These cells retain key macrophage functions, including phagocytosis, nitric oxide production, and cytokine secretion, which closely mirror the behavior of primary macrophages under inflammatory stress.⁴⁶ This supports their reliability in modeling wear particle–induced immune responses and osteolytic processes.

UHMWPE Particles Induce Persistent M1 Polarization and Dysregulated IL‑10

UHMWPE particles caused a pronounced increase in CD80⁺ macrophages and TNF‑α secretion, consistent with evidence that wear debris activates NF‑κB and MAPK signaling, amplifying osteoclastogenesis.^47,48 Indeed, UHMWPE debris specifically activates the Chemerin/ChemR23–NF‑κB axis, amplifying downstream AP‑1 signaling and sustaining TNF‑α release.^49,50 Interestingly, IL‑10, a classical anti-inflammatory cytokine, was also elevated in the particle-stimulated periosteum. Similar paradoxical IL‑10 upregulation has been reported in UHMWPE particle-induced osteolysis models as a transient compensatory response attempting to counterbalance TNF‑α-driven inflammation.⁵¹

Specifically, Loi demonstrated that UHMWPE-induced macrophage activation leads to simultaneous TNF‑α and IL‑10 secretion, reflecting a mixed but imbalanced immune state.⁵² Likewise, a murine UHMWPE model revealed that while TNF‑α and IL‑6 are strongly induced, IL‑10 also increases at sub-compensatory levels—a response further modulated by metformin treatment.⁵¹ However, these endogenous IL‑10 responses remain insufficient to restore immune balance, as the M1/M2 ratio stays skewed, sustaining osteoclast-driven bone resorption.²⁰

Interestingly, our study also revealed that IL-10 levels decreased following BMSC treatment, concurrent with the reduction of TNF-α. This seemingly paradoxical pattern likely reflects the self-limiting nature of the IL-10 response. During intense inflammatory activation, macrophages upregulate IL-10 as a negative feedback mechanism through STAT3-dependent induction of SOCS3, aiming to restrain excessive NF-κB signaling.^53,54 However, once the inflammatory drive subsides—such as after BMSC-mediated suppression of TNF-α and restoration of M2 polarization—the stimulus for IL-10 production is reduced, leading to normalization rather than persistent elevation.⁵⁴ In mathematical models of LPS-stimulated monocytes, IL-10 expression similarly peaks early and declines as pro-inflammatory signaling wanes.⁵⁵ Moreover, in wear particle–induced osteolysis, the NF-κB/let-7f-5p–IL-10 pathway has been shown to regulate macrophage polarization, where let-7f-5p suppresses IL-10 expression and reinforces M1 activation.⁵⁶ Moreover, macrophage models have demonstrated that IL-10 transcription can be initiated within minutes but is transient and subject to remodeling.⁵⁷ Collectively, these findings indicate that IL-10 expression is dynamically tuned by feedback circuits and does not remain persistently elevated once inflammatory homeostasis is restored.⁵⁸

Our flow cytometry and ELISA data confirmed a significant rise in CD80⁺ macrophages, supporting a model-dependent inflammatory shift. These findings reinforce the idea that wear particles generate a “mixed but predominantly pro-inflammatory” immune microenvironment, where IL-10 is present but insufficient to counteract the dominant M1-mediated inflammatory cascade.

BMSCs Reprogram Macrophage Phenotypes and Modulate Cytokines

BMSC co-culture significantly suppressed M1 polarization while promoting M2 differentiation, as shown by decreased CD80 and increased CD206 expression. This aligns with previous studies demonstrating that BMSCs exert immunosuppressive effects through paracrine secretion of prostaglandin E2 (PGE2), TSG‑6, IL‑10, and TGF‑β, which inhibit NF‑κB activation and promote STAT3 signaling.^29,59 Notably, repeated BMSC administration in vivo achieved more pronounced effects than a single injection, likely due to enhanced local retention and sustained release of immunomodulatory factors.⁶⁰ Similar trends were observed in rheumatoid arthritis and bone defect models, where multiple BMSC doses improved therapeutic efficacy compared with single dosing.^34,61

Immunofluorescence further confirmed these effects: UHMWPE-stimulated macrophages showed intense CD80 fluorescence colocalizing with F4/80, whereas BMSC co-culture markedly reduced this signal. Conversely, CD206 fluorescence, which was suppressed by UHMWPE, was restored by BMSCs treatment. These findings support the idea that BMSC-derived soluble factors and extracellular vesicles directly drive macrophage phenotype switching.^23,28

Mechanistic Insights and Emerging Pathways

Based on our flow cytometry and ELISA findings showing a marked TNF‑α reduction but normalized IL‑10 levels after BMSC treatment, we speculate that BMSCs restore immune homeostasis through the NF‑κB/STAT3 feedback network rather than merely amplifying M2 polarization. Besides classical NF‑κB inhibition, recent work suggests IL‑17 signaling and ferroptosis contribute to particle-induced osteolysis.⁶² Interestingly, some studies link IL‑17 to LCN2-mediated chronic inflammation and bone loss, raising the possibility that IL‑17/LCN2 signaling might underlie the “mixed but imbalanced” immune state we observed.^63,64 In our model, UHMWPE stimulation caused robust TNF‑α secretion accompanied by a compensatory but insufficient IL‑10 elevation, suggesting a prolonged low-level inflammatory stress rather than acute necrotic damage, which is consistent with ferroptosis-associated chronic macrophage injury reported in wear debris models.⁶⁵ Although we did not directly detect ferroptosis markers, the partial restoration of M2 macrophages after BMSC treatment implies their action may converge on rebalancing NF‑κB/STAT3 rather than completely blocking IL‑17 signaling. Furthermore, BMSC-derived exosomal miR‑146a has been shown to suppress TRAF6/NF‑κB, facilitating macrophage repolarization toward M2.^60,66

Compared to titanium debris, UHMWPE particles induced both stronger TNF‑α production and compensatory IL‑10 elevation. This aligns with reports that UHMWPE debris preferentially activates monocyte/macrophage-mediated inflammation with limited lymphocyte involvement, whereas metallic debris triggers a broader innate and adaptive immune response due to ion release and oxidative stress.⁶⁷ Therefore, the type of wear particle may influence the required immunomodulatory strategy.

Translational Relevance

Our study adds two clinically meaningful insights: (1) UHMWPE-induced PPO requires prolonged immune regulation because particle release is chronic; (2) repeated BMSC delivery yields superior bone preservation compared with single dosing. Compared with single-target approaches such as surface-modified titanium implants or small-molecule anti-inflammatory drugs, BMSCs exhibit a dual advantage by simultaneously regulating immune polarization and promoting osteogenic microenvironments, which may explain the superior bone preservation observed with repeated dosing. This supports the concept of optimized BMSC treatment schedules or cell-free therapies using preconditioned exosomes as a future direction. The latest USC-derived exosome and MSC-EV therapies showed immunomodulatory efficacy equivalent to that of live MSCs, suggesting engineered EVs could reduce the need for repeated cell transplantation.^68,69 Exosome-based therapies offer several practical advantages over direct stem cell transplantation, including their “off-the-shelf” availability, absence of tumorigenic or differentiation risks, greater stability during storage and transport, and easier standardization for clinical manufacturing.^70,71 These features make exosome therapy an attractive and scalable alternative for translational applications in orthopedic and inflammatory bone diseases.

By combining in vivo periosteal macrophage flow cytometry, cytokine assays, and in vitro transwell co-culture, we provided multi-layer evidence of BMSC-driven macrophage reprogramming. Few PPO studies integrate these complementary approaches, making this work more comprehensive in evaluating immune–bone crosstalk.

Limitations and Future Directions

This study has some limitations. First, the 21-day observation period may not capture long-term remodeling, as bone mineral density recovery lagged behind trabecular structure restoration. Second, we did not directly trace BMSC survival or exosome release at the calvarial site. Advanced tools, such as single-cell RNA sequencing and live-cell tracking, can clarify the temporal dynamics of macrophage reprogramming. Third, although previous studies have implicated NF κB, TLR4/MyD88, ferroptosis-related NF κB-SLC7A11, and chemerin/ChemR23-AP 1 signaling in wear particle-induced immune dysregulation, this study did not directly investigate the underlying signaling pathways, which is a notable limitation; nevertheless, subsequent research is planned to elucidate these mechanisms in detail.

Additionally, exploring BMSC-derived exosomes as a cell-free alternative may overcome logistical barriers to repeated cell transplantation.⁷² Compared with live cell delivery, exosome-based approaches offer improved delivery consistency, lower immunogenicity, and reduced safety risks associated with uncontrolled differentiation or vascular occlusion.⁷³ Future studies could leverage engineered BMSCs or bone-targeting exosomes (eg, collagen-binding EVs) to achieve “low-dose, high-efficiency” or sustained release, thereby enhancing therapeutic precision and reproducibility while maintaining safety and controllability. Finally, validating these findings in larger, load-bearing models is essential for clinical translation.

Conclusion

This study demonstrates that local delivery of bone marrow mesenchymal stem cells (BMSCs), particularly through repeated administration, effectively attenuates UHMWPE wear particle–induced osteolysis by reprogramming macrophage polarization from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype. We confirmed that BMSCs exert these effects mainly via paracrine-mediated immune modulation, leading to reduced cytokine secretion and improved bone microarchitecture. These findings provide a mechanistic foundation for developing MSC-based immunomodulatory therapies against periprosthetic osteolysis and related inflammatory bone disorders.

Despite these promising results, further studies are required to address current challenges, including long-term efficacy, detailed signaling mechanisms, and optimization of delivery strategies. Future work focusing on BMSC-derived exosomes and bone-targeted cell-free systems may offer a more practical, safe, and scalable therapeutic approach for clinical translation.

Abbreviations

PPO, periprosthetic osteolysis; UHMWPE, ultra-high molecular weight polyethylene; BMSCs, bone marrow mesenchymal stem cells; TJA, total joint arthroplasty; HE staining, hematoxylin and eosin staining; IHC, immunohistochemical; ELISA, enzyme-linked immunosorbent assay; IF, immunofluorescence; BMD, bone mineral density; BV/TV, bone volume fraction/total volume; TNF-α, tumor necrosis factor alpha; Tb.Th, trabecular thickness; Tb.N, trabecular number; IL-10, Interleukin-10; IL-4, Interleukin-4.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics State

All animal procedures were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University. The study strictly followed the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

The authors report no funding associated with the study described in this article.

Disclosure

The author(s) report no conflicts of interest in this work.

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Industrikraft in Sweden AB was formed in June 2024 to support the expansion of the Swedish electricity supply. Industrikraft is now entering the next phase of nuclear power investment with the following companies – ABB, Alfa Laval, Boliden, Hitachi Energy, Höganäs AB, SSAB, Saab, Stora Enso, and the Volvo Group.

Vattenfall and Industrikraft have now signed an agreement that allows the parties to progress in enabling new nuclear power on the Värö Peninsula at Ringhals. The agreement covers various areas such as responsibilities, influence, and financing.

The next step in the collaboration is for Industrikraft to become a 20 percent shareholder in Videberg Kraft AB. The government has previously announced that the state also intends to become a shareholder in the company.

“Industrikraft and Vattenfall have reached consensus on the most important issues”, says Tom Erixon, chairman of Industrikraft and CEO of Alfa Laval. In a first step, Industrikraft will invest SEK 400 million in the project. In addition to co-financing the project company, the industry contributes with resources, expertise in project management and selection of technologies. Swedish technology has a place in a competitive environment to drive the development of a strong supplier cluster in Europe.

“It is very positivenews that Vattenfall and Industrikraft are now taking the next step to move the project forward together. The industry is an important partner in enabling new nuclear power in Sweden. The commitment and willingness of these companies to co-own the project on the Värö Peninsula is a sign that there will be a demand for the new fossil-free electricity production capacity. In the next step, Videberg Kraft AB is working to prepare an application for state risk-sharing”, says Anna Borg, CEO of Vattenfall.