Degradation of WE43 Magnesium Alloy in Vivo and Its Degradation Products on Macrophages

Abstract

Due to their biocompatibility, biodegradability, and suitable mechanical properties, magnesium-based biodegradable implants are emerging as a promising alternative to traditional metal implants. The Mg-4Y-3RE (WE43) biodegradable alloy is among the most extensively studied and widely utilized magnesium alloys in clinical applications. As an absorbable and degradable metallic material, magnesium alloys undergo gradual degradation, wear, and fracture within the body. These alloys reduce the long-term risks associated with permanent implants but generate insoluble byproducts that accumulate in surrounding tissues. Following the implantation of magnesium alloys, granulation tissue and fibrous encapsulation typically form around the material. However, limited research has addressed the interaction between insoluble byproducts of magnesium alloys and macrophages. This study focused on the biological effects of macrophages during the second stage of the host inflammatory response in the degradation process of magnesium alloy. Using subcutaneous implantation of WE43 magnesium alloy sheets, observations were made regarding the degradation components, morphological changes in surrounding tissues, and the biological effects of macrophages upon phagocytosis of insoluble byproducts. The primary degradation products of WE43 in vivo were identified as Ca₃ (PO₄)₂, Mg₃(PO₄)₂, Na₃PO₄, NaCa (PO₄), MgSO₄, MgCO₃, NaCl, Mg24Y5, and Mg12YNd. Postimplantation, levels of IL-1β and IL-18 in adjacent tissues significantly increased (p < 0.05). By 8 weeks, compared to nitinol alloy, significant thickening of the fibrous capsule (p < 0.05) was observed, accompanied by substantial inflammatory cell infiltration, vascularization, and the presence of macrophages and multinucleated giant cells. Macrophages were observed extending pseudopodia to enclose and phagocytose particles, forming phagosomes and creating a relatively isolated microenvironment around the engulfed substances, where further particle degradation occurred. Following the phagocytosis of degradation products, macrophages exhibited increased lysosome numbers, mitochondrial swelling and damage, phagolysosome formation, and autophagosome development. Furthermore, the degradation products were observed to induce elevated reactive oxygen species (ROS) production in macrophages, activation of P2X7 receptors, enhanced IL-6 secretion, endoplasmic reticulum stress, autophagy, and activation of the NLRP3 inflammasome pathway. This study provides novel insights and contributes a theoretical foundation for a more comprehensive understanding of magnesium alloy degradation in vivo.

Introduction

Due to the biocompatibility, biodegradability, and favorable mechanical properties of magnesium-based temporary implants, they have emerged as a promising alternative to traditional metal implants. Magnesium implants offer the advantage of degrading over time and being safely absorbed by the body, thereby eliminating the need for secondary resection surgery and minimizing long-term complications. Currently, these implants are being explored for various applications, including bone fixation and cardiovascular stents.

In the field of cardiovascular stents, percutaneous coronary intervention (PCI) remains the primary treatment for coronary atherosclerotic disease (CAD), aimed at opening occluded or stenotic coronary arteries.¹ Traditionally, drug-coated stainless steel (SS) stents are used for this purpose. While drug-coated stents reduce abnormal neointimal hyperplasia caused by foreign body implantation,² they can also lead to late and very late stent thrombosis due to factors such as eosinophil-mediated anaphylaxis, lack of endothelialization, and persistent or acquired stent malapposition. To address these complications, researchers have sought to develop devices that not only dilate coronary artery obstructions but also provide temporary vascular support, prevent elastic recoil and remodeling, inhibit neointimal hyperplasia, and eventually degrade and disappear.²⁻⁴ Among the materials investigated, the Mg-4Y-3RE (WE43) biodegradable alloyis the most extensively studied and clinically applied magnesium alloy.⁴⁻⁶ A similar alloy, Magmaris, has been approved for clinical trials and human use.⁷

In orthopedic clinical applications, the development of degradable bone implants, particularly those made from metal materials, represents a new frontier. Degradable bone screws, plates, and prostheses, such as knee implants, are gaining attention. The key advantage of degradable implants is their elimination of the need for removal after fulfilling their function. Magnesium’s Young’s modulus closely matches that of natural bone, and magnesium ions have been shown to promote bone growth, making magnesium highly attractive for medical applications involving bone contact.⁸⁻¹⁰ Several magnesium alloy implants, including screws, nails, anchors, and pins, have been approved by regulatory bodies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA).¹¹

Despite magnesium’s potential in temporary implant applications, several challenges remain. During the biodegradation of magnesium and its alloys, the primary byproducts include hydrogen, a localized alkaline environment, Mg²⁺ ions, and insoluble degradation products. While there has been extensive research on hydrogen, local alkalinity, and Mg²⁺ ions, the insoluble degradation products have been less explored. Mg²⁺ promotes the attachment, proliferation, and differentiation of osteoblasts.¹²⁻¹⁴ However, if the local pH rises above 7.8, alkalosis may occur, resulting in local toxicity. Moreover, hydrogen is released as gas bubbles during magnesium biodegradation, potentially causing tissue necrosis¹⁵ and posing a risk of gas embolism. Although the degradation process of magnesium alloys is relatively slow, insoluble byproducts form and accumulate around blood vessels or bone tissue. Current research on these local insoluble degradation products remains limited.

The host tissue response following the implantation of biodegradable scaffolds typically occurs in three distinct stages.¹⁶ The first stage involves acute and chronic inflammation initiated by the host to degrade the biomaterial, a process that tends to be similar regardless of the degradation rate of the biomaterial. In the second stage, inflammatory cells such as granulocytes and macrophages infiltrate the implantation site, forming a fibrous capsule around the implant. The third stage is marked by the development of a fibrous capsule enriched with blood vessels.^16,17 Throughout this process, various multifunctional cells are recruited to the site and interact with one another. Macrophages play a pivotal role in the foreign body response (FBR) to biomaterials,¹⁸ including both resident tissue macrophages and monocyte-derived macrophages.¹⁹ These cells share a functional phenotype, characterized by their ability to phagocytose foreign bodies, participate in tissue repair, and release enzymes and factors that contribute to sustained inflammation.^20,21 In addition, they are involved in both innate and adaptive immune responses.²² During the degradation of magnesium alloys, degradation products or microfragments are released and persist in the tissues surrounding the implant for extended periods. However, research on the interaction between insoluble magnesium alloy products and macrophages remains limited. This study focused primarily on macrophages during the second stage of the host inflammatory response, aiming to explore their biological effects during the degradation of magnesium alloys.

As an absorbable and degradable metal, magnesium alloy gradually degrades, wears down, and fragments within the body, reducing the long-term risks associated with permanent implants. However, this process generates insoluble byproducts that accumulate in surrounding tissues. Previous studies by Hanzi et al.^23,24 have shown that granulation tissue and fibrous encapsulation often form around certain organs following magnesium alloy implantation. Despite this, there is still a lack of detailed research on the interaction between macrophages and magnesium alloy’s insoluble products for the first time. On the one hand, macrophages can phagocytose degradation products and form phagosomes, which are further degraded into smaller particles. On the other hand, after phagocytizing degradation products, macrophages produce a series of biological effects, including increased production of intracellular reactive oxygen species (ROS), activation of P2X7 receptor, increased secretion of IL-6, endoplasmic reticulum stress, autophagy, and activation of NLRP3 pathway. The findings provide novel insights and a theoretical foundation for a deeper understanding of magnesium alloy degradation in vivo.

2. Materials and Methods

2.1. Implant Materials

The nickel–titanium alloy and magnesium alloy WE43 (4% Y-3.3% RE (Nd, Gd) −0.5% Zr) were supplied as cast specimens by the Department of Materials Science and Engineering at Harbin Institute of Technology, China. Cylindrical samples (10 mm in diameter, 1 mm thick) were sectioned from the as-cast WE43 magnesium alloy and nickel–titanium alloy rods.

2.2. Animal Models

All SD rats, 30 males weighing 230–250g, were sourced from the Animal Center of the Second Affiliated Hospital of Harbin Medical University. The rats were housed in the animal facility of the Experimental Center at the First Affiliated Hospital of Harbin Medical University, under well-ventilated conditions with a controlled temperature of 20–22 °C and relative humidity of 28%–50%. The lighting followed a 12-h light-dark cycle. Each rat was housed in an individual cage with unrestricted access to food and water. All experimental procedures adhered strictly to the Guidelines for the Management of Muscle Use in Experimental Animals and followed medical ethical standards. Approval for this study was granted by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University, ensuring all procedures met ethical compliance.

The 30 male SD rats were randomly assigned to three groups: control, nickel–titanium alloy (NiTi group), and magnesium alloy WE43 (WE43 group), with 10 rats in each group. Prior to the experiment, the rats were fasted for 24 h and then anesthetized via intraperitoneal injection of barbital. They were positioned prone on a foam board, their dorsal fur shaved, and the skin disinfected with iodophor. A longitudinal incision was made along the midline of the back to expose and separate the right subcutaneous cavity.

At 0, 4, and 8 weeks postimplantation, the rats were placed supine on the operating board with limbs secured. The dorsal skin was incised to fully expose the subcutaneous tissue. Fibrous capsules encapsulating the metal implants were identified, and the capsules, along with surrounding subcutaneous tissue, were excised. The excised capsules were divided into four parts: one-quarter was fixed in paraformaldehyde for histopathological analysis, another quarter was stored at −80 °C for Western blot analysis, a third quarter was fixed in glutaraldehyde for electron microscopy observation, and the final quarter was embedded in Cryo-Gel (BL557A, Bisosharp, China) for fluorescence microscopy examination.

Each WE43 sample was weighed (W0) before implantation, and the final weight (W1) was weighed at 4 and 8 weeks after implantation. The samples were washed with deionized water and ethanol in order to eliminate degradation products before weighing, dried naturally for about 2 h. The corrosion rate was calculated according to formula 1.

2.3. Scanning Electron Microscopy (SEM) and X-ray Diffraction (XRD) Analysis

After the metal sheets were implanted, they were retrieved subcutaneously at 0, 4, and 8 weeks for analysis via scanning electron microscopy (SU8000; Hitachi, Japan), equipped with an electron backscatter diffraction (EBSD) probe. Phase composition was assessed using X-ray diffraction (X’pert PRO, PANalytical Inc., Netherlands), under an experimental voltage of 40 kV with a Cu target. The scanning range was set to 5–90° with a scanning speed of 2°/min. Additionally, scanning electron microscopy (SU8000; Hitachi, Japan) was utilized to further examine the metal samples.

2.4. ROS Analysis

Frozen sections of rat subcutaneous tissues were stained with a DCFH-DA probe (Beyotime, China) to measure ROS levels. Tissue samples from the control, NiTi, and WE43 groups were incubated in 20 μM/L DCFH-DA (diluted 1:500 in PBS) at 37 °C for 20 min following the manufacturer’s protocol. The sections were then washed three times with PBS and visualized using a fluorescence microscope (Leica, Germany). Composite color images were generated using Image Pro-Plus software (Media Cybernetics, Silver Spring).

2.5. Transmission Electron Microscope (TEM)

At 8 weeks, the subcutaneous tissue from the WE43 group was sectioned into 3 mm pieces, immediately immersed in 3% glutaraldehyde, and fixed for over 72 h at 4 °C. Sections were then prepared and stained for examination under transmission electron microscopy (HT7800; Hitachi, Japan).

2.6. Pathological Analysis

Pathological analysis was conducted on the subcutaneous tissues surrounding the metal plates in the control, NiTi, and WE43 groups. All tissue samples were fixed in 10% paraformaldehyde for 48 h, followed by paraffin embedding, sectioning, and Hematoxylin-Eosin (HE) staining.

The pathological evaluation of fibrous connective tissue around the subcutaneous metal sheets was based on the following grading system: grade 1: Immature granulation tissue with diffuse infiltration of acute and chronic inflammatory cells (score 5); grade 2: Granulated fibrous connective tissue with mild chronic inflammatory cell infiltration (score 4); grade 3: Granulation fiber connective tissue with fibrosis (score 3); grade 4: Fibrous connective tissue with low cellular composition (score 2); grade 5: Formation of foreign body granulation tissue with macrophage phagocytosis of foreign particles and infiltration of foreign body giant cells (score 1).

2.7. Immunohistochemical (IHC) and Immunofluorescence (IF) of Subcutaneous Tissue

Slices from the NiTi and WE43 groups, at 0 and 60 days, were prepared and incubated overnight at 4 °C with commercially available antibodies, including CD68 (Abcam, USA), P2X7 (Abclone, China), NLRP3 (Wanlei, China), IL-18 (Abclone, China), and IL-1β (Abclone, China), following the manufacturers’ protocols. The two-step universal kit (PV-8000, ZSGB-BIO, China) and the biotin–streptavidin-enhanced DAB detection kit (ZLI-9018, ZSGB-BIO, China) were used according to the provided instructions. For immunofluorescence analysis, TRITC-labeled rabbit antigoat IgG (ZSGB-BIO, China) was applied. The results were observed, and the intensity of positive staining was quantified using Image Pro-Plus 6.0 (IPP6.0, Media Cybernetics, Inc., USA) to calculate the integrated optical density (IOD) values.

2.8. Western Blot (WB) Analysis

Protein extracts from the NiTi and WE43 groups, taken at 0 and 8 weeks, were analyzed using the enhanced BCA Protein Assay Kit (Beyotime, China). Cell lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes (Pall Corporation, NY), and blocked with a rapid blocking buffer (Seven, Beijing, China) for 10 min. Membranes were then incubated overnight at 4 °C with primary antibodies against IL-6, IL-1β, IL-18, NLRP3, ASC, Caspase-1, Beclin-1, p62, LC3α/β, LAMP-1, Atg-7, Atg-5, Atg-16L1, CHOP, GRP94, GRP78, TXNIP, CD86, CD163, and P2X7 (diluted 1:500–1:1000). Afterward, membranes were treated with secondary antibodies (1:10,000 dilution) for 2 h at room temperature and visualized using a Bio-Rad Universal Hood II gel imaging system. Protein levels were quantified using Image Lab software (NIH) to determine relative gray intensities.

2.9. Enzyme-Linked Immunosorbent Assay (ELISA)

The subcutaneous tissues and serum from the NiTi and WE43 groups, at 0 and 8 weeks, were analyzed using IL-18 Mouse ELISA Kits and IL-1β Mouse ELISA Kits (both from Invitrogen). Samples were incubated in precoated 96-well plates for 30 min and analyzed with an ELISA instrument, as directed by the manufacturer’s protocols.

2.10. Statistical Analysis

Data were presented as the mean ± SD, based on at least three independent experiments unless stated otherwise. Statistical analysis was performed using one-way ANOVA followed by the Newman–Keuls multiple comparison test. GraphPad Prism 9.5 (GraphPad Software) was used for statistical evaluation and graphical representation. Statistical significance between groups was indicated by *p < 0.05, **p < 0.01, & p < 0.05, and #p > 0.05.

3. Results

3.1. Observation of X-ray Imaging

The X-ray imaging was conducted at 0, 4, and 8 weeks following metal plate implantation. At 0 weeks, minimal differences were observed between the WE43 and NiTi groups, except for the brighter appearance of the nickel–titanium alloy due to its inherent properties. By 4 weeks, the NiTi group exhibited negligible changes, while in the WE43 group, fibrous encapsulation with distinct boundaries around the metal was visible, along with fragmented structures and opaque regions surrounding the implant. At 8 weeks, the NiTi group retained a fully intact, clear, and transparent metal structure with no significant changes. In contrast, the WE43 group displayed cystic encapsulation with extensive opaque areas, indicating metal degradation, with numerous fragmented remnants clearly delineated from the surrounding tissue (Figure 1a).

Figure 1.

Characteristics of the metal foils in the body of rats with WE43 and NiTi. (a) X-ray examination of rats of the WE43 group and NiTi group at 4 and 8 weeks. (b) Observation of the metal foils in the body of rats with WE43 and NiTi at 4 and 8 weeks. The red arrow indicates the WE43 metal and NiTi.

3.2. Observation of Metal Sheets in the Body

At 8 weeks postimplantation, no tissue proliferation was observed around the NiTi metal sheet, and the implant remained clearly visible. However, in the WE43 group, a cystic structure approximately 1.3 cm in diameter formed, exhibiting a dark color and a distinct boundary from adjacent tissues. Compared to the 8-week time point, the NiTi group showed minimal changes in fibrous proliferation, with the metal sheet still clearly discernible. In the WE43 group, a larger cystic formation of about 2.0 cm in diameter was observed, with a darker hue and well-defined boundaries (Figure 1b).

3.3. Observation of Fibrous Sacs and the Metal Sheets

Upon dissecting the fibrous sacs around the implants, it was observed that after 8 weeks in the WE43 group, gas was present within the cyst, and the capsule wall was approximately 0.2 mm thick but irregular in texture. Corrosion was evident on the WE43 metal surface, although the overall structure remained visible. After further incision at 8 weeks, excess gas was detected, with the cyst wall thickness increasing to 0.3 mm. The wall remained uneven, and although the metal structure was still discernible, a significant amount of white fragmented material was present. In contrast, the NiTi group formed thin-walled fibrous sacs at both 4 and 8 weeks, with no visible changes to the metal sheet (Figure 2a). Upon dissection, no alterations or corrosion were observed on the NiTi metal surface. At 4 weeks, corrosion and white substance deposition were detected on the WE43 metal surface, although the structure was largely intact. By 8 weeks, significant damage to the metal structure was observed, with the surface heavily coated in white material (Figure 2b). The corrosion rate curve trend of WE43 shows that the corrosion was rapid within 8 weeks, and the experimental time of this study was short, so the degradation rule of WE43 magnesium alloy in vivo cannot be completely displayed in this experiment (Figure 2c).

Figure 2.

Characteristics of the metal foils of WE43 and NiTi. (a) Fibrous capsules surrounding the metal foils of WE43 and NiTi at 4 and 8 weeks. (b) General observation of the metal foils of WE43 and NiTi at 0, 4, and 8 weeks. (c) Corrosion rate of WE43 at 4 and 8 weeks.

3.4. IL-1β and IL-18 of Subcutaneous Tissue and Serum of Rats

The IL-1β and IL-18 levels in subcutaneous tissue and serum were measured at 8 weeks in both the WE43 and NiTi groups using ELISA. The results indicated a slight increase in serum IL-1β and IL-18 in both groups; however, the differences were not statistically significant (Figure 3a). In contrast, IL-1β and IL-18 levels in the subcutaneous tissues of both the WE43 and NiTi groups were significantly elevated compared to the control group (p < 0.05) (Figure 3b), with the WE43 group showing a notably higher increase than the NiTi group(p < 0.05) (Figure 3b).

Figure 3.

IL-1β and IL-18 were estimated by the ELISA assay of rat serum and subcutaneous tissue. All other groups were compared to the control group (n = 3, *p < 0.05 vs control group; &p < 0.05 vs NiTi group).

3.5. Surface Analysis of the Alloy Plates by SEM and XRD

First, we analyzed the microstructure of WE43 material. The microstructure of grain of WE43 is shown in Figure 4c. The minimum grain size was 5.35 μm, and the maximum grain size was 102.27 μm. The average grain size was 41.86 μm (Figure 4a,b). Basal recalculated pole figures of the WE43 alloy was shown in Figure 4c. Scanning electron microscope (SEM) analysis of the nickel–titanium alloy and WE43 magnesium alloy implanted in vivo revealed distinct differences. The NiTi alloy’s structure remained stable after 4 and 8 weeks, though surface irregularities and roughness were observed at 8 weeks. In the WE43 magnesium alloy, surface corrosion was detected as early as 4 weeks, with rod-shaped structures of relatively regular morphology appearing. By 8 weeks, these regular structures had disappeared, and a large amount of amorphous material was present, indicating severe corrosion. Energy spectrum analysis showed that at 4 weeks, the primary elements detected were C, O, P, S, Cl, Ca, Y, Nd, and Gd. By 8 weeks, the elements identified included C, N, O, P, S, Na, K, Mg, Cl, Ca, Y, Nd, and Gd, with notable increases in K and N (Figure 5a). XRD analysis of the material at 8 weeks identified the primary components as Ca₃(PO₄)₂, Mg₃(PO₄)₂, Na₃PO₄, NaCa(PO₄), MgSO₄, MgCO₃, NaCl, Mg24Y5, and Mg12YNd (Figure 5b). Due to the relatively low concentrations of K and N, no specific compounds containing these elements were detected via XRD. These findings align with previous studies, which suggest that HPO₄^2– and HCO^3– contribute to the formation of a protective layer on the surface, delaying the progression of corrosion.²⁵

Figure 4.

SEM images and the IPF maps of microstructures of the WE43 alloy. (a) Distribution of crystallographic direction parallel to CD. (b) Distribution map of different rolled grain sizes.(c) SEM images. (d) Basal recalculated pole figures of the WE43 alloy.

Figure 5.

Observation of metal foils of WE43 and NiTi. (a) Morphology and EDS of the WE43 and NiTi of WE43 and NiTi at 0, 4, and 8 weeks. (b) XRD patterns of the WE43 alloy at 8 weeks.

3.6. Histopathological Findings and Histomorphometry

Following the implantation of nickel–titanium alloy and WE43 magnesium alloy, subcutaneous tissue exhibited mild inflammatory and foreign body reactions, consistent with a typical wound healing process. At later stages, both metal sheets were encapsulated by fibrous sacs predominantly composed of surrounding fibrous connective tissue. A grading system was developed based on standard histological features, with corresponding scores assigned (Table 1). Histological results from the hematoxylin and eosin (HE) staining of both groups were analyzed at 4 and 8 weeks, with the specific scoring criteria detailed in Section 2.6. Pathological scores for the NiTi and WE43 groups showed a significant decline over time. At 4 and 8 weeks, the NiTi group had significantly lower pathological scores than the WE43 group Histological results from the hematoxylin and eosin (HE) staining of both groups were analyzed at 4 and 8 weeks, with the specific scoring criteria detailed in Section 2.6 (Figure 6). Changes in granulation tissue thickness and the fibrous connective tissue capsule surrounding the metal are illustrated in Figure 6, with both groups showing significant increases over time. There was no significant difference in fibrous capsule thickness between the NiTi group at 4 and 8 weeks, while the WE43 group displayed significantly thicker fibrous capsules at 8 weeks compared to 4 weeks. At both 4 and 8 weeks, a statistically significant difference in fibrous capsule thickness was observed between the NiTi and WE43 groups.

Table 1. Histopathological Scoring Criteria^a.

Histopathological grading	Cell infiltration	Vascular numbers	Collagen fibers	Score
Grade 1	++	+	–	4
Grade 2	+	++	–	3
Grade 3	–	+	+	2
Grade 4	–	–	++	1

^aThe capsule around the implants was scored histopathologically according to the characteristic features of wound healing.

Figure 6.

Histopathological scores and capsule thickness of the capsule tissues around the WE43 alloy and NiTi alloy at 4 and 8 weeks. (a) Histopathological scores of both groups at 4 and 8 weeks. (b) Capsule thickness of both groups at 4 and 8 weeks. *p < 05 compared with the 4 and 8 weeks of different groups, &p < 05 compared with WE43 alloy and NiTi alloy of different times. Data represent the mean ± SD (n = 3).

In normal rats, subcutaneous tissue consists primarily of the dermis, with visible collagen fibers, a few fibroblasts, and a small number of blood vessels. At 8 weeks, the NiTi group displayed intact fibrous sacs surrounding the metal, characterized by smooth walls, inflammatory cell infiltration, abundant fibroblasts, prominent vascular proliferation, and occasional macrophages. In the WE43 group, fibrous capsule formation was observed at 8 weeks (Figure 7b), with unsmooth cyst walls and substantial infiltration of inflammatory cells. Additionally, vascular formation, as well as the presence of macrophages and multinucleated giant cells, was noted. Blue foreign body particles, likely degradation products from the WE43 magnesium alloy, were visible within the interstitial spaces between fibers and were also detected within macrophages and multinucleated giant cells (Figure 7a,b).

Figure 7.

Representative images of the respective rat tissue models. Representative images of the control, NiTi group, and WE43 group of rats at 4 and 8 weeks. Black arrow, macrophages cells; blue arrow, multinucleated giant cells; and red arrow, foreign body particles.

3.7. Expression of ROS and P2X7 and Immunofluorescence Results

At 8 weeks, ROS were detected in the tissues surrounding both the subcutaneous nitinol alloy and magnesium alloy implants. The nitinol group showed a slight increase in ROS levels compared to the control group, though the increase was not statistically significant. This may be due to the predominance of fibrous tissue hyperplasia and the relative absence of inflammatory cells in the nitinol group at 8 weeks, leading to lower ROS production. In contrast, the WE43 group exhibited a significant increase in ROS compared to both the control and nitinol groups (Figure 8a). This rise in ROS is likely a result of continuous degradation of the WE43 alloy, which induces macrophages to phagocytose the degradation products, consequently producing elevated ROS levels.

Figure 8.

Main biological effects of the macrophages of the subcutaneous tissue of inserting WE43 for 8 weeks. (A) ROS of the macrophages of the subcutaneous tissue was assessed with fluorescence microscopy. (B) P2X7 receptor was estimated by immunofluorescence. (C) Representative proteins of macrophages and P2X7 were measured with WB. Representative images from three experiments were shown.

Additionally, P2X7 receptor expression was evaluated. No notable increase was observed in the nitinol group compared to the control. However, the WE43 group showed significantly higher P2X7 staining intensity than both the control and nitinol groups (Figure 8b). It is speculated that this upregulation of P2X7 receptors is triggered by macrophages engulfing insoluble degradation products.

3.8. WE43 Metal Implantation Causes an Increase in Macrophages and an Increase in P2X7 Receptors

Histological analysis through HE staining and immunofluorescence revealed an increase in macrophages in the WE43 group. In this study, WB was used to detect macrophage-specific proteins CD163, CD86, and P2X7 receptors (Figure 8c). Results indicated a significant increase in these markers in the WE43 group compared to the control, and levels were also higher than in the NiTi group. The results of immunohistochemical showed that the expression of macrophage specific protein CD163 was positive in WE43 group, but negative in control group and NiTi group (Figure 11). This suggests that macrophages and P2X7 receptors played a key role in the metabolism of WE43 alloy degradation products in the WE43 group.

Figure 11.

Immunohistochemistry of CD163, IL-1β, IL-18, Caspase-1, NLRP3, CHOP, and Beclin-1 of macrophages in the subcutaneous tissue of the WE43 group for 8 weeks. Black arrow, Staining positive cells.

3.9. Observation of Macrophages in Subcutaneous Tissue with a Transmission Electron Microscope (TEM)

To further investigate the impact of insoluble particles generated from WE43 degradation on macrophages, scanning electron microscopy was employed to observe ultrastructural changes. Macrophages extended pseudopodia to envelop and phagocytose particles, forming phagosomes that created a relatively isolated environment around the engulfed material (Figure 9b,c,g,e,i). Large gaps and reduced particle density were observed, indicating particle degradation (Figure 8c,e,i). After particle engulfment, macrophages exhibited an increase in lysosomes, swollen mitochondria, and damage or fusion of some mitochondrial cristae (Figure 9c,f,i). The endoplasmic reticulum also expanded and increased in density (Figure 8d,e,f,i). Some particles were enveloped by lysosomes, forming phagolysosomes (Figure 9b,c,e), which participated in the degradation process. Transmission electron microscopy (TEM) revealed the presence of autophagosomes with a characteristic double-layer membrane structure (Figure 9a,b,c,e,i).

Figure 9.

TEM images of macrophages in the subcutaneous tissue of the WE43 group at 8 weeks. After phagocytosis of degradation products of WE43, macrophages form different sizes of phagocytic/lysosomes (a–i). A, autophagosome; B, lysosome; E, endoplasmic reticulum; ED, endosome; M, mitochondria; N, nucleus; P, phagosome/phagolysosome; black arrow, UCPs; five-pointed star, pseudopodia; triangle, phagosome black space.

3.10. WE43 Metal Implantation Induces Endoplasmic Reticulum Stress (ERS) in Macrophages

Based on the electron microscopy analysis, macrophages in the WE43 group exhibited a widening and increased endoplasmic reticulum. To investigate endoplasmic reticulum stress, WB was used to detect the levels of stress-related proteins CHOP, GRP94, GRP78, and TXNIP. The results showed that all these proteins were significantly elevated in the WE43 group compared to the control and NiTi groups (Figure 10a). The results of immunohistochemical showed that the expression of endoplasmic reticulum associated protein CHOP was strongly positive in WE43 group, but negative in control group and NiTi group (Figure 11). These findings, combined with transmission electron microscopy results, suggest that macrophage phagocytosis of WE43 degradation products induces endoplasmic reticulum stress.

Figure 10.

Results of Western blot were as shown. (a) Endoplasmic reticulum stress-related proteins of macrophages were measured. (b) The proteins of macrophages associated with the NLRP3 inflammatory pathway were measured. (c) Autophagy-related proteins of macrophages were assessed. Representative images from three experiments were shown.

3.11. WE43 Metal Implantation Induces Macrophage Autophagy and Apoptosis

Electron microscopy also revealed that autophagy was triggered after macrophages engulfed WE43 metal degradation products. Autophagy-related proteins, including LC3α/β, LAMP-1, Atg-7, Atg-5, Atg-16L1, Beclin-1, and P62, were analyzed. Compared to the control and NiTi groups, the WE43 group showed a significant increase in LC3α/β, LAMP-1, Atg-7, Atg-5, Atg-16L1, and Beclin-1, while P62 levels significantly decreased (Figure 10b). The results of immunohistochemical showed that the expression of autophagy associated protein Beclin-1 was strongly positive in WE43 group, but negative in control group and NiTi group (Figure 11). These results further confirm that autophagy is induced following the engulfment of WE43 degradation products by macrophages. Compared to the control and NiTi groups, the WE43 group showed a significant increase in Bax, a apoptosis marker (Figure 11).

3.12. WE43 Metal Implantation Induces Activation of the ROS-NLRP3 Pathway in Macrophages

In addition, WB analysis was used to assess proteins related to the ROS-NLRP3 pathway, including IL-1β, NLRP3, ASC, IL-18, and Caspase-1. The results demonstrated a significant increase in these proteins in the WE43 group compared to the control group, and the difference was also statistically significant when compared to the NiTi group (Figure 10c). Furthermore, inflammation-related factor IL-6 was markedly elevated in the WE43 group (Figure 10c). Immunohistochemical results showed that the expression of NLRP3 pathway related proteins IL-1 β, NLRP3, IL-18, caspase-1 was strongly positive in the WE43 group, but negative in the control group and NiTi group (Figure 11). These results supporting the involvement of inflammatory responses in this process.

Discussion

The WE43 alloy treated by different processing technologies has the same phase composition but different microstructure. In this study, the sample treated by forging technology has a strong segregation tendency and a large mismatch energy because it contains rare earth elements, which were larger than magnesium atoms. The uneven segregation of rare earth elements during forging reduces the pinning effect of solute atoms, and the grains show abnormal grain growth, so the average grain size is larger, but it is basically consistent with the grain size in other articles.^26,27

Host tissue responses following the implantation of biodegradable scaffolds can generally be categorized into three stages. This study focuses on the secondary and tertiary responses, particularly examining the role of macrophages in these phases. In the presence of biocompatible materials, early resolution of acute and chronic inflammatory responses is typical, with chronic inflammation driven by monocytes usually subsiding within 2 weeks.^28,29 Inflammatory responses persisting beyond 3 weeks often indicate infection. After 4 weeks of subcutaneous implantation of WE43 magnesium alloy, the absence of significant neutrophil and lymphocyte infiltration indicates the alloy’s favorable biocompatibility as a biodegradable material.

As acute and chronic inflammation resolves, macrophage and fibroblast infiltration, along with neovascularization, leads to the formation of granulation tissue,³⁰ which serves as a precursor to fibrous capsule development. This granulation tissue acts as a barrier between the implant and biomaterials, mediated by a foreign body reaction involving monocytes, macrophages, and foreign body giant cells.³¹ In this study, at 4 and 8 weeks following nitinol implantation, neutrophils and monocytes dominated the granulation tissue, while macrophages and foreign body giant cells were less prevalent. However, following WE43 magnesium alloy implantation in rats, significant metal corrosion was observed at 4 weeks, and extensive degradation was evident by 8 weeks, accompanied by a substantial accumulation of corrosion particles. Surrounding these particles were numerous multinucleated giant cells, macrophages, neutrophils, and fibroblasts, along with extensive angiogenesis, consistent with prior studies. As magnesium alloy degrades, it disintegrates into smaller particles.³² Therefore, investigating the biological effects of these degradation particles in vivo is of crucial importance.

The particles of different sizes and shapes have different biological effects.³³ When the particle size exceeds the phagocytic ability of a single macrophage, such as when the particle diameter is 10–100 μm, macrophages will form multinucleated macrophages, multinucleated giant cells (MnGC), or foreign body giant cells (FBGC), and then undergo phagocytic degradation.³⁴ In vitro studies, osteoblasts MC3T3 were incubated with pure magnesium particles with a diameter of 100 nm-50 μm. Almost all cells were killed at 1500 μg/mL, and the cell survival rate was greater than 70% after 24 h at 500 μg/mL. The survival rate of osteosarcoma SAOS2 cells incubated with pure magnesium particles with a diameter of 20–30 μm for 48 h at 500 μ g/mL is approximately 90%.³⁵

Previous studies have identified several biological environmental factors that influence the corrosion rate of magnesium (Mg) materials, including local pH fluctuations, ion species and their concentrations, oxygen presence,³⁶ protein concentration,³⁷ and the adhesion of cells and tissue components to the magnesium surface. Additionally, the formation of fibrous capsules on the surface of magnesium alloys plays a critical role in modulating corrosion behavior.³⁸ The combined effect of these factors governs the corrosion dynamics of magnesium and its alloys within biological systems, which in turn impacts their performance and potential as biodegradable materials. Electrolyte flow also significantly contributes to corrosion acceleration.^39,40 In vivo, tissue fluid and plasma surrounding the magnesium alloy function as electrolytes, with the circulation of these fluids dependent on the density of blood vessels and the level of blood flow. It has also been observed that the corrosion rate varies according to the site of implantation.^23,41 In this study, WE43 magnesium alloy was implanted into subcutaneous tissue, and although the corrosion rate may differ from that in vascular or bone tissues, the basic composition of the corrosion products remained relatively unchanged. Analysis revealed that the insoluble corrosion products consisted of Ca₃(PO₄)₂, Mg₃(PO₄)₂, Na₃PO₄, NaCa(PO₄), MgSO₄, MgCO₃, NaCl, Mg₂₄Y₅, and Mg₁₂YNd.

Studies have shown that Ca₃(PO₄)₂ can activate the inflammatory response of macrophages and stimulate the pro-inflammatory phenotype. In addition, it can induce the repolarization of M2 macrophage phenotype to M1 phenotype gene by increasing the expression of TNF-α and IL-1β.⁴² The transformation of macrophages involves changes in energy metabolism,⁴³ M1 macrophages exhibit metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation (OXPHOS) to generate energy (ATP).^44,45 Extracellular PO₄^3– is transported into macrophages via slc20a1 transporter and then enters the cell. High levels of intracellular PO₄^3– drive the TCA cycle and oxidative phosphorylation, resulting in rapid and efficient energy production. Provide biochemical and biochemical basis for M2 macrophage polarization.⁴⁶ In terms of specific mechanism, PO₄^3– involves AMPK mTOR axis in macrophage polarization, which may be achieved by increasing energy metabolism. The AMPK mTOR axis acts as a central metabolic switch in cellular metabolism and as a signaling molecule in macrophages. When cellular energy increases, the activation of AMPK decreases, leading to the opening of the mTOR signaling cascade.⁴⁶ In addition, intracellular ATP can be secreted into the extracellular space and metabolized to adenosine, which subsequently promotes macrophage polarization toward the M2 phenotype through autocrine and/or paracrine signaling via A2B adenosine receptors.⁴⁶ The MgSO₄ particles may play an anti-inflammatory and tissue repair role by activating M2 macrophages and secreting a large number of cytokines.⁴⁷ The Mg24Y5, Mg12YNd, and MgCO₃ mixed particles can cause M1 polarization of macrophages and play a pro-inflammatory role.⁴⁸

Histopathological examination demonstrated that both magnesium alloy and nickel–titanium exhibited good biocompatibility. Over time, vascularized fibrous capsules formed around the implants and matured progressively. Notably, the volume loss of the implant correlated with the thickness of the fibrous capsule at different anatomical locations.⁴⁹ Across all implantation sites and time points, tissues surrounding both magnesium and nickel–titanium alloys displayed a typical wound healing response, with minimal inflammation and foreign body reactions. The surrounding tissues developed a cystic structure consisting of fibrous connective tissue encapsulating particles, a finding consistent with this study.

Histopathological analysis of each group revealed that the scores for the WE43 group significantly decreased over time compared to the 4-week mark (p < 0.05) (Figures 5 and 6). Similarly, the histopathological scores for the NiTi group also decreased significantly over time (p < 0.05). At 8 weeks, vascularized fibrous capsules were observed around both magnesium and nickel–titanium alloys at all implantation sites. The formation of encapsulation around implants is generally part of the wound-healing process and a reaction to foreign bodies.⁵⁰ Initially, these fibrous capsules consist of immature granulation tissue, rich in cells and blood vessels. Over time, the number of infiltrating cells and blood vessels decreases, leading to the development of mature fibrous capsules.⁵¹ Magnesium alloy may degrade more rapidly in immature granulation tissue, where blood flow is abundant, but more slowly in mature fibrous tissue, which contains more extracellular matrix and exhibits reduced blood circulation.

In line with these observations, a thicker fibrous capsule at a similar level of maturity is expected to contain more blood vessels, facilitating the diffusion of Mg²⁺ and OH^– ions, which can result in greater volume loss. Previous studies have also reported that the width of the fibrous capsule surrounding magnesium alloy or nitinol implants increases over time,^50,52 consistent with the findings of this study. The presence of blood vessels within the fibrous capsule contributes to greater volume loss, and even at later observation points, an air cavity can be seen between the capsule and the magnesium alloy. This cavity, which forms due to gas absorption and tissue deformation, may collapse over time as the capsule contacts the magnesium alloy, further influencing volume loss. These results suggest that variations in wound healing, depending on the implantation site, play a crucial role in determining the corrosion rate of magnesium alloy implants. However, by 4 weeks postimplantation, the envelope surrounding the magnesium alloy becomes thinner, and over time, the impact of envelope thickness may diminish. Nitinol alloys, in contrast, tend to form thinner and more mature fibrous capsules compared to other biomaterials.⁵³ In this study, inflammatory cell infiltration was observed around the magnesium alloy, and the histological characteristics of the fibrous capsule differed from those formed around NiTi implants.

Furthermore, the formation of a gas cavity beneath the skin was noted, with its size dependent on the balance between the gas generated by the corrosion reaction and the amount that is circulated or diffused by the surrounding tissue fluid. It has been documented that the volume of these cavities typically decreases over time.⁵⁴ In the present study, prominent gas cavities were observed at the 8-week mark. Some research suggests that air cavity size decreases after 4 weeks of implantation, a finding that differs from the results of this study. The primary reason for this discrepancy may be the use of WE43 magnesium alloy in this research, whereas other studies employed Mg-1.0Al magnesium alloy, which likely led to different corrosion rates due to the variation in alloy composition.

Magnesium corrosion tends to proceed most rapidly at the edges of scaffolds. Subsequently, the formation of an insoluble salt layer around the magnesium alloy is thought to shield the alloy from blood circulation, thus slowing the corrosion process. During magnesium corrosion, insoluble salts form on the surface of the alloy, such as Mg(OH)₂, MgCO₃, magnesium phosphate, calcium phosphate, and hydroxyapatite.⁵⁵ which are consistent with the findings of this study. Notably, calcium phosphate has low solubility in body fluids, which allows it to inhibit further corrosion of magnesium alloys by physically coating their surfaces.^54,55 After magnesium alloy vascular stents are implanted, insoluble salts fill the voids once occupied by the magnesium alloy struts.^53,56 Additionally, corrosion can be delayed through surface modifications such as anodizing or applying coatings, with corrosion inhibition lasting between 2 and 12 months depending on the alloy and surface treatment.^57,58 While bioresorbable stents mitigate long-term risks associated with permanent implants, they introduce new challenges related to metabolite buildup and their accumulation in the arterial wall. This accumulation can lead to macrophage recruitment to the arterial wall.²¹ In this study, numerous macrophages and multinucleated giant cells were observed surrounding the fibrous capsule, as macrophages engulfed degradation products and contributed to the formation of insoluble salts.

Histological examination using HE staining revealed blue foreign body particles inside macrophages and multinucleated giant cells, confirming that they had engulfed degradation products. Transmission electron microscopy further showed macrophages extending pseudopodia to encapsulate particles, forming phagosomes that provided a relatively isolated environment for particle degradation. Large gaps were observed around the particles, and their density decreased, indicating progressive degradation. After particle exposure, macrophages exhibited increased lysosome numbers, swollen mitochondria with damaged or fused cristae, and expanded endoplasmic reticulum. Lysosomes encapsulated some particles, forming phagolysosomes that played a role in particle degradation. Autophagosomes with a characteristic bilayer membrane structure were also observed, indicating active autophagy. These findings suggest that macrophages participate in the degradation of magnesium alloy insoluble products in vivo, producing specific biological effects.

Following the phagocytosis of insoluble degradation products, macrophages triggered an increase in IL-1β, IL-18, and IL-6, which induced a localized pro-inflammatory response without causing a systemic inflammatory reaction. Some studies have suggested that the metal levels released by implanted scaffolds are relatively low and insufficient to cause systemic responses.^59,60 IL-1 β, IL-6 and IL-18 are mainly pro-inflammatory factors produced by M1 macrophage activation, which may cause damage to surrounding cells or tissues and enhance the phagocytic capacity of macrophages.⁶¹ However, the biological effect of IL-6 controls the differentiation of monocytes into macrophages by regulating the expression of macrophage colony-stimulating factor,⁶² which is also a cytokine produced by M2b macrophages, and realizes immune regulation by driving anti-inflammatory Th2 immune response.⁶³ The role of IL-18 is complex,which can stimulate innate immunity and Th1 and Th2 mediated responses, and can also directly inhibit angiogenesis.⁶¹ IL-1β induces endothelial cells to express adhesion molecule ligands to promote further circulation of monocytes in the blood and recruitment of tissue resident macrophages.⁶⁴ However, the local accumulation of degradation products in tissues and cells of the vessel wall could potentially result in adverse effects and has been associated with preclinical and clinical ISR.

Results from a recent large-scale clinical study on magnesium alloy stents (SCT incidence at 1-year follow-up: 0.5% in the BIOSOLVE-IV group) demonstrate that while sirolimus-eluting coatings effectively reduced abnormal smooth muscle cell proliferation poststent implantation, the Magmaris scaffold remains susceptible to SCT.⁶⁵ This risk is linked to uneven scaffold degradation, which compromises vessel wall stability and support.⁶⁶ Furthermore, chronic inflammation associated with neoatherosclerotic changes, well-known thrombogenic factors, exacerbates this risk.⁴ The study’s findings offer theoretical insights into the mechanisms behind SCT development in magnesium alloy scaffolds.

Organelle changes, particularly after macrophages phagocytose magnesium alloy degradation products, were observed through transmission electron microscopy. Further analysis revealed that phagocytosis of insoluble products induces an increase in intracellular ROS. Mitochondria, known to generate ROS during respiration, become damaged during this process, further escalating ROS levels. Studies have confirmed that mitochondria, as a central signaling platform, can promote macrophage polarization to M1 phenotype and play a pro-inflammatory role through the production of ROS.⁶⁷ Additionally, macrophage phagocytosis of particles not only triggers the formation of phagolysosomes for degradation but also leads to lysosomal swelling, instability, and rupture. The release of lysosomal contents, including cathepsin B, into the cytoplasm contributes to increased ROS production. Moreover, this study confirms the activation of P2X7 purinergic receptors following macrophage phagocytosis of magnesium alloy degradation products. These purinergic receptors, a family of transmembrane proteins, recognize extracellular nucleotides and nucleosides, mediating cellular responses to extracellular ATP. The P2X7 receptor forms a pore that permits the influx of small cations, such as Na⁺, K⁺, and Ca²⁺,⁶⁸ further driving ROS production.^69,70

ROS play a key role in the initiation of autophagy. On the one hand, pathogens or other stimuli can activate ROS and induce autophagy,⁷¹ on the other hand, oxidatively damaged organelles are also transported to lysosomes for degradation and recycling by autophagy.⁷² ROS can initiate autophagy, and after autophagy activation, it can exert antioxidant function and clear excessive ROS in the body.⁷³ There are complex interactions between ROS and autophagy. The autophagy induced by ROS can reduce oxidative stress and lead to cell survival, while the lack of autophagy will lead to increased oxidative stress and ROS accumulation. Macrophages can polarize to present different phenotypes and exhibit different functions in different microenvironments. Lipopolysaccharide (LPS), ROS and other factors promote the differentiation of macrophages into M1 macrophages, which play the function of clearing pathogens and releasing pro-inflammatory factors in the early stage of inflammation. Anti-inflammatory cytokines such as IL-4 and IL-10 induce macrophages to differentiate into M2 macrophages, which can inhibit immune response, promote tissue repair and reconstruction, and regulate angiogenesis in the late stage of inflammation.⁷⁴ When autophagy perturbs the polarization of macrophages, autophagy in macrophages is dysfunctional and promotes polarization toward the M1 phenotype. However, the activation of autophagy promotes M2 macrophage polarization, thereby attenuating the inflammatory response.⁷⁵ In this study, autophagy is activated. Macrophages have both M1 and M2 types. We speculate that macrophages clear the insoluble products of phagocytosis through autophagy, and can promote angiogenesis and tissue repair.

The endoplasmic reticulum (ER) is a vital multifunctional organelle that plays a crucial role in maintaining cellular homeostasis. In this study, transmission electron microscopy and WB identified ER stress-related proteins, including CHOP, GRP94, GRP78, and TXNIP. Phagocytosis of insoluble degradation products by macrophages was found to cause morphological and functional changes in the ER, leading to the accumulation of misfolded proteins and disruption of homeostasis. These alterations triggered ER stress and activated the unfolded protein response (UPR) pathway. As protein folding and refolding within the ER lumen are highly energy-dependent processes, ATP depletion resulting from protein misfolding stimulates mitochondrial oxidative phosphorylation, which increases both ATP and ROS production. Additionally, ER stress promotes mitochondrial ROS production due to two key factors: the proximity of mitochondria and the ER, which facilitates direct physical interaction between the two organelles,⁷⁶ and disruptions in Ca²⁺ regulation,^77,78 affecting mitochondrial membrane potential, ATP consumption, and ROS formation.⁷⁹ Previous studies have demonstrated that macrophages experience elevated intracellular Ca²⁺ levels following phagocytosis of insoluble products. Ultimately, ROS production exceeds the cellular threshold, further amplifying ROS levels.

After macrophages engulf magnesium alloy degradation products, a complex crosstalk occurs between mitochondrial damage, lysosomal content leakage, cathepsin B release, and ER stress. These processes converge to activate inflammasomes through multiple pathways, including ROS generation, lysosomal rupture, mitochondrial dysfunction, and activation of P2X7 receptor channels (leading to intracellular K⁺ efflux). One of the most well-characterized inflammasomes is the nucleotide-binding oligomerization domain (NOD)-like receptor inflammasome containing pyridine domain 3 (NLRP3) inflammasome, which plays a critical role in the body’s immune defense. Inflammasomes are multiprotein complexes that protect the host from foreign pathogens and tissue damage by releasing cytokines. They typically consist of three components: sensors, adaptors, and effectors. The sensors, also known as pattern recognition receptors (PRRs), serve as sensors that typically detect exogenous or endogenous signals and subsequently recruit apoptosis-associated speck-like protein containing a CARD (ASC) components, initiating the immune response. Through interactions between the caspase activation and recruitment domain (CARD), ASC filaments recruit pro-caspase-1, which becomes autoproteolyzed to form active caspase-1.⁸⁰⁻⁸² Active caspase-1 enzymatically cleaves pro-inflammatory cytokine precursors, such as pro-IL-1β and pro-IL-18, to generate the active cytokines IL-1β and IL-18.

Following the engulfment of magnesium alloy degradation products by macrophages, autophagosomes with a double-layer membrane structure were observed, and key autophagy-related proteins, including Beclin-1, P62, LC3α/β, LAMP-1, Atg-7, Atg-5, and Atg-16L1, exhibited statistically significant differences when compared to the NiTi group. Autophagy is a fundamental process in which cytoplasmic materials are degraded in lysosomes.⁸³ Under physiological conditions, autophagy is induced to generate energy and essential building blocks by breaking down pre-existing intracellular components and waste.^84,85 Under stress conditions, such as ER stress and oxidative stress, autophagy eliminates abnormal protein aggregates and damaged organelles.⁸⁶ After macrophages engulf magnesium alloy products, lysosomes contribute to the degradation of these materials, while autophagosomes fuse with lysosomes to form autolysosomes, initiating autophagy.^87,88 This process is crucial for maintaining cellular turnover and homeostasis.⁸⁴ In addition to autophagy, magnesium alloy degradation products can also cause macrophage apoptosis, consistent with previous studies.⁴⁸

Additionally, after macrophages engulfed insoluble degradation products, intracellular ROS levels increased, activating the ROS-NLRP3 (Nod-like receptor protein 3) pathway. This activation led to elevated secretion of downstream IL-18 and IL-1β, resulting in a pro-inflammatory response. Research suggests that ROS upregulation is linked to mitochondrial dysfunction and ER stress.^86,87 In this study, macrophage engulfment of UCPs caused morphological changes, such as mitochondrial swelling, expansion of the inner membrane, fusion, and dysfunction, all of which contributed to elevated ROS levels. The subsequent activation of the NLRP3 inflammasome involved interactions with ASC, leading to recruitment, activation, and proteolysis of pro-caspase-1. The resulting active caspase-1 triggered the activation and release of IL-1β and IL-18.⁸⁸ Additionally, increased ER size, expansion of the ER lumen, and the presence of ER stress further contributed to ROS-NLRP3 pathway activation, amplifying the biological effects observed.

Research has established that magnesium alloy degradation products can modulate inflammation and exhibit immunomodulatory effects by promoting M2 macrophage polarization.^89,90 These effects are primarily mediated by magnesium alloy extracts, whose main component is Mg²⁺.⁹⁰ However, evidence indicates that excessive magnesium concentrations impair cell viability,^91,92 with over 50% of magnesium alloy extract causing macrophage cytotoxicity, partially accounting for its anti-inflammatory effects. Magnesium has also been shown to suppress immune responses by downregulating the MAPK pathway.⁹³ and reducing NF-κB pathway activity in various cell types^94,95 When macrophages were directly cultured with magnesium alloy metal, phenotypic changes were observed, leading to M2 polarization with inflammation-inhibitory properties.⁹⁶ However, these findings are predominantly based on in vitro studies. This study, for the first time, investigates the effects of magnesium alloy degradation products on macrophages in vivo. The results indicate a pro-inflammatory response, largely attributable to the phagocytosis of nondegradable degradation products by macrophages. This finding diverges from prior studies, which primarily highlighted anti-inflammatory effects under in vitro conditions.

It has been suggested that a carefully controlled degradation rate and composition of magnesium alloys can effectively reduce inflammatory responses and enhance the material’s biocompatibility. One approach involves the addition of silver (Ag) as a grain-refining agent and an anti-inflammatory component, while another focuses on grain refinement to improve the alloy’s mechanical strength and deformation capabilities.^89,97 These strategies present innovative directions for optimizing the clinical application of magnesium alloys.

Conclusions

As a bioabsorbable metal, magnesium alloy undergoes gradual degradation, wear, and fragmentation within the body. The investigation uniquely explored the interaction between macrophages and WE43 degradation products. Following the phagocytosis of these products, particles were enclosed in phagosomes, leading to increased lysosomal activity, which facilitated further degradation. This process triggered endoplasmic reticulum stress, mitochondrial dysfunction, P2X7 receptor activation, autophagy, and a rise in intracellular ROS levels. The subsequent activation of the NLRP3 inflammasome pathway led to the release of pro-inflammatory cytokines IL-1β, IL-18, IL-6.

In this study, WE43 magnesium alloy was implanted subcutaneously to observe the foreign body reaction of the body. We will consider the specific application sites of magnesium alloy in different parts, such as intravascular and bone, to further explore the biological effects of local macrophages in the future research. In addition, more in-depth analysis, especially other cytokines such as macrophage typing, will be carried out to further support these findings.

1

A is the surface of the sample implanted subcutaneously (cm²) and T is the corrosion time (days)

The authors declare no competing financial interest.