Macrophage-associated biocompatibility of titanium revealed by analyzing foreign body reaction at bone-implant interface

Abstract

PURPOSE

Titanium implants are widely used to replace pathological joints, bones, and teeth, with successful engraftment requiring osteoblast attachment to the metal surface for bone regeneration. However, the immune response at the bone–implant interface remains unclear, and few studies have examined why titanium elicits a reduced foreign-body reaction (FBR) compared to other metals. This study aimed to elucidate the mechanism underlying titanium biocompatibility by characterizing the immune response at the bone–implant interface in a rat model.

MATERIALS AND METHODS

Copper, machined titanium, and sandblasted/acid-etched titanium rods were fabricated for implantation into rat tibiae. Topographical and chemical features of each rod surface were evaluated. Rods were inserted into rat tibiae, and immune cell subtypes were analyzed by flow cytometry, histomorphometry, and immunohistochemistry. Statistical analyses were performed at a significance level of 0.05.

RESULTS

Flow cytometry of bone marrow cells collected on Days 1, 7, and 35 post-implantation revealed recruitment of macrophages and neutrophils at all implant sites. Histological analysis confirmed immune cell infiltration at the metal–bone interface, with a pronounced FBR surrounding copper rods. Immunohistochemistry demonstrated an abundance of osteoclast-like M1 macrophages at the copper–implant interface. In contrast, M1 macrophages were absent near titanium implants, where reparative M2 macrophages were present.

CONCLUSION

The recruitment of M1 macrophages at the copper implantation site, but not at the titanium implant, indicates the FBR to copper and underlies the biocompatibility of titanium. Titanium may affect the differentiation of intrabony macrophages to increase its biocompatibility.

INTRODUCTION

Osseointegration refers to the direct connection between living bone and a metal implant surface.¹,² Brånemark first proposed this concept in the 1950s after observing that rabbit bones fully incorporated Ti, which could only be removed by fracturing the bone.¹ Since then, advances in metal surface treatment technologies have improved the safety and efficacy of metal implants in humans.³,⁴,⁵,⁶,⁷,⁸,⁹ Osseointegration is conventionally defined as the process by which bone marrow (BM) cells differentiate into osteoblasts to initiate new bone formation at the metal-bone interface. Metal implants are defined in terms of biocompatibility and inertness.

Histological evaluation of the bone-implant interface, which can reveal information about the cellular reaction at the metal surface, suggests that osseointegration is not related to an advantageous biological response but rather to the lack of an adverse immune reaction.²,¹⁰,¹¹ Osseointegration does not occur if a metal implant induces a strong foreign body reaction (FBR). FBR is characterized by the accumulation of inflammatory cells around the implant and formation of granulation tissue.¹²,¹³,¹⁴,¹⁵ Hence, osseointegration is not a biological property of metal implants, but acts as an innate immune host defense mechanism against foreign bodies. This explains why some metal implants are more inert and biocompatible than others.

Although our understanding of osseointegration has improved in recent years, the exact immune responses that occur when Ti is recognized as a foreign body within the bone remain unclear. Similarly, the cellular components involved in this process are yet to be identified. Furthermore, the mechanisms underlying the suppression of FBR and promotion of bone formation following Ti implantation remain largely understudied. In this study, we evaluated the cellular immune response at the interface between the bone and different types of metal rods implanted in rat tibiae. By identifying the major immune cells that affect the biocompatibility of Ti versus other metal surfaces, we aimed to elucidate the role of immune cells in the suppression of FBR in Ti dental implants.

MATERIALS AND METHODS

Female Sprague-Dawley rats (130 – 140 g) were purchased from Orient Bio (Gapyeong, Korea) and bred in the animal laboratory of the Dental Research Institute, Seoul National University. The study was approved by the Institutional Animal Care and Use Committee (SNU-210823-3-2). Animal experiments were performed on 75 rats (8 – 16 weeks old).

The rods were cylindrical, 1.5 mm in diameter, and 2.0 mm in length. They were composed of grade 4 commercially pure Ti or Cu. All rods were generated using computer numerical control machining. Half of the Ti rods were sandblasted and acid-etched (SLA), whereas the other half were not modified further and remained as machined surfaces. Field-emission scanning electron microscopy (FE-SEM; Hitachi S-4700; Hitachi, Tokyo, Japan) was used to image the metal surface. Next, metal surface roughness was measured through confocal laser scanning microscopy (LSM 800; Carl Zeiss AG, Oberkochen, Germany). The surface roughness parameter, Sa (arithmetic mean height), was measured for each rod. The chemical compositions of the rods were analyzed using energy dispersive spectroscopy (Sigma Probe, Thermo VG, East Grinstead, UK).

The rats were anesthetized with 87.5 mg/kg ketamine (Yuhan, Seoul, Korea) and 12.5 mg/kg xylazine (Rompun; Bayer Korea Ltd., Seoul, Korea) before implantation. The hind legs of the rats were shaved and disinfected. Next, 2% lidocaine hydrochloride and 1 : 100,000 epinephrine (Yuhan) were applied to the surgical site as local anesthetics. The upper tibial epiphytic area was exposed using full-thickness incisions from the skin to the periosteum. The surgical sites on the tibiae were prepared with rotating implant drills (1.5 mm diameter at 1,000 rpm) while irrigating with sterile saline solution (Fig. 1A). An appropriate metal implant (or no implant) was grafted into the drilled hole according to a randomized experimental group design (Fig. 1B). The muscle and fascia were closed with resorbable 4-0 Vicryl sutures (Coated Vicryl; Ethicon, Raritan, NJ, USA), and the skin was closed with nylon (Blue nylon; Ailee, Busan, Korea) after surgery. The rats were euthanized on postoperative days 1, 10, and 35 to extract the tibiae for flow cytometry or histological analyses (Fig. 1A). A 1-cm tibial segment was extracted from the site of the drilled hole. All animal experiments were conducted in accordance with the ARRIVE guidelines.¹⁶

Fig. 1. Schematic drawing of experimental procedures and the randomized experimental group design. (A) Female rats (n= 75; 8 – 16 weeks old) underwent metal rod implantation surgery. The appropriate metal implant according to the group (or no implant) was grafted into a drilled hole at the tibia upper epiphysis. The animals were then euthanized and subjected to flow cytometry or histological analysis on Days 1, 7, and 35 post-implantations. (B) The appropriate metal implant (or no implant) was grafted according to the randomized experimental group design. Each experiment was done with five rats and repeated two to three times for each post-implantation time point. In total, 45 and 30 rats were used for flow cytometry and histological analysis, respectively.

Bone fragments (1 cm) were incubated with 20% EDTA for two weeks and paraffinized for decalcified tibial bone analysis. Paraffin-embedded bones were sectioned (4 µm) for histological analysis. The tissue sections were deparaffinized using NeoClear (Merck Millipore, Burlington, MA, USA) and rehydrated in an ethanol series (100%, 95%, and 70%). The tissue slides were then stained with hematoxylin and eosin (H&E) or with anti-CD3 (clone SP7; Abcam, Cambridge, UK), anti-CD68 (clone EPR23917-164; Abcam), anti-HIS48 (clone HIS48; Abcam), anti-RANKL (clone C1; Abcam), anti-CD86 (clone SJ20-00; Thermo Fisher Scientific, Waltham, MA, USA), anti-CD163 (clone EPR19518; Abcam), and anti-CD206 (clone MR5D3; BioRad) for immunohistochemistry. The bones and connective tissues surrounding the implant were surgically harvested en bloc for non-decalcified tibial bone analysis. The blocks were fixed in 10% neutral-buffered formalin for two weeks, dehydrated with ethanol, and embedded in light-curing resin (Technovit 7200 VLC; Kulzer, Wehrheim, Germany). The EXAKT system (EXAKT Apparatebau, Norderstedt, Germany) was used to cut and grind the embedded blocks into sections of approximately 50 µm. The sections were stained with H&E and modified Goldner’s Masson’s trichrome staining solution and examined under a light microscope. This staining technique effectively differentiates between newly formed (red) and existing mature (blue) bone. The slides were scanned using a slide scanner (Aperio CS2; Leica Biosystems, Nussloch, Germany), and the scanned images were captured using an Aperio Imagescope (Leica Biosystems).

The cancellous tissue was removed from the 1-cm tibial sections and washed three to five times with 1 × phosphate-buffered saline. Next, the BM was flushed out using RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum. The eluted cells were subsequently collected in 15-mL flow cytometry tubes. Control leukocytes were collected from the cervical lymph nodes and venous blood as previously described.¹⁷,¹⁸

The isolated immune cells were incubated with fluorochrome-conjugated anti-rat antibodies for 30 min at 4℃ and washed twice with flow cytometry wash buffer. Antibodies targeting the following markers were used for cell surface staining: CD8α (clone OX-8), interleukin (IL)-10 (clone A5-4), all from BD biosciences (Franklin Lakes, NJ); CD161 (clone 3.2.3), CD11b/c (clone OX-42), CD45 (clone OX-1), CD3 (clone 1F4), T cell receptor (TCR)α/β (clone R73), TCRγ/δ (clone V65), CD4 (clone W3/25), Foxp3 (clone 150D), IL-4 (clone OX-81), all from Biolegend (San Diego, CA); granulocyte marker (clone HIS48), interferon (IFN)γ (clone DB-1), tumor necrosis factor (TNF)α (clone N3-19.1), IL-17A (clone Bio17B), B220 (clone HIS24), CD3 (clone UCHT1), CD25 (clone OX-39), all from Thermo Fisher Scientific. Intracellular staining was performed using the Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher Scientific) according to the manufacturer’s instructions. Before intracellular cytokine and transcription factor staining of IFNγ, IL-4, IL-17A, and Foxp3, the immune cells were stimulated in vitro with phorbol-12-myristate-13-acetate and ionomycin for 4 h at 37℃. The live immune cells were gated using forward scatter exclusion of dead cells stained with a Ghost Dye™ (Tonbo Biosciences, San Diego, CA, USA). Data were acquired on a Cytoflex LX (Beckman Coulter, Brea, CA, USA) or an LSR Fortessa X-20 (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometer and analyzed using FlowJo software (v10.8.1; BD Biosciences).

GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. Differences between multiple groups were evaluated using the Kruskal-Wallis test. A two-tailed Mann-Whitney U test was used for comparisons between two groups. P < .05 was considered statistically significant (^*P < .05; ^**P < .01; ^***P < .001; NS, not significant). All data are presented as the mean ± standard error of the mean (sem).

RESULTS

FE-SEM images were used to confirm that the rods had typical machined (Ti) or SLA surfaces (Fig. 2A). The mean Sa value of the Ti-SLA surface was higher than those of the machined Ti and Cu surfaces, which had similar Sa (Fig. 2B). The major surface elements on the Ti rods were Ti and oxygen, whereas the machined Cu rods predominantly contained Cu (Fig. 2C). To investigate the effect of Ti and Cu on immune response at the bone-metal interface, three types of metal rods (Cu, Ti, and Ti-SLA) were manufactured and implanted into the rat tibia to generate the Cu, Ti, and Ti-SLA groups, respectively. Tibial cross-sections were collected and histologically evaluated on Days 1, 10, and 35 post-implantations. Rats in the control group did not undergo surgery, whereas those in the sham group had a drill hole but without rod implantation.

Fig. 2. Surface topographical features and surface chemistry. (A) Field-emission scanning electron microscopy (n = 3). The machined grooves on the Cu (top) and Ti (middle) surfaces are shown. The Ti-SLA (bottom) surface showed a representative irregular pattern in the microtopography. (B) Graph showing the Sa values for each group. Data are expressed as the mean ± standard deviation (SD) (n = 3, ^**P < .01). (C) Graph showing the energy dispersive spectroscopy analysis results. Data are shown as the mean ± SD (n = 3).

Undecalcified histological observations of sham rats showed partial bone regeneration on Day 10 and almost complete bone healing on Day 35 (Fig. 3A). On post-implantation Day 10, the tibiae of rats with Cu rods presented no evidence of new bone formation but a clear inflammatory immune response resembling an FBR inside and outside the drilled hole (Fig. 3A). This inflammatory response was not observed in the Ti and Ti-SLA groups on Day 10. Instead, new bone formation and near-complete bone healing were observed on Days 10 and 35, respectively (Fig. 3A). Higher magnification of the bone-metal interface confirmed that the Cu and Ti rods elicited distinct immune responses (Fig. 3B). The Cu group had extensive immune cell infiltration at the site of implantation and no tissue interposition between the tibia and Cu rods on Days 10 and 35 (Fig. 3B), suggesting a pro-inflammatory response against the metal implant. Conversely, no prominent immune cell infiltration was observed at the bone-metal interface of the Ti and Ti-SLA groups. New bone formation and maturation were evident on Days 10 and 35 in these groups (Fig. 3B). These results indicate that Cu and Ti metal rods produce markedly different immune responses within the bone, with only Cu inducing FBR at the site of implantation.

Fig. 3. Implantation of metal rods results in various degrees of intrabony FBR. (A) The tibiae and connective tissues that surrounded the implant on Days 10 (10D) and 35 (35D) post-implantations were surgically harvested en bloc for non-decalcified bone analysis. After fixation, 1-cm thick crosscut tissues were embedded in light-curing resin and cut into 50-µm sections. The sections were stained with modified Goldner’s Masson trichrome (MT) staining (the upper two rows) and H&E (bottom row) with low magnification. The red arrows show inflammatory cell accumulation, whereas the black arrows show new bone formation and near-complete bone healing. The black scale bar presents 1 mm. (B) A higher magnification of the bone-metal interface is shown, as observed through MT staining (the upper two rows) and H&E (bottom row). The Cu and Ti rods elicited distinct immune responses, with immune cell accumulation observed (red arrows). Conversely, no prominent immune cell infiltration was observed at the bone-metal interface of the Ti and Ti-SLA groups. Rather, new bone formation and maturation were evident on Days 10 and 35 in these groups (black arrows). The white scale bar presents 200 µm.

Immune cells were isolated from the tibial BM at 1, 10, and 35 days post-implantation to identify those that responded to the implanted metal. Isolated immune cell subsets were characterized through flow cytometry (Fig. 4). Analysis of the control rats confirmed that the frequency of BM-derived CD45+ immune cells did not change significantly across the three time points. Additionally, surgery or the type of metal implant did not significantly alter immune cell frequencies at any given time point. However, the frequencies of HIS48+TCRγδ-TCRαβ- neutrophils were significantly higher in the Cu group than in other groups on Day 1. This suggests that neutrophils play a major role in the early inflammatory immune response to Cu implants. The frequencies of other immune cell subtypes, including αβ T cells, macrophages, and natural killer cells, were not significantly different between the groups (Fig. 5).

Fig. 4. Characterization of the immune cells in early intrabony foreign body reaction. (A) Immune cells were isolated from the tibial bone marrow (BM) at 1, 10, and 35 days post-implantation and characterized through flow cytometry to identify the immune cells that respond to the implanted metal. The frequency of BM-derived CD45+ immune cells did not change significantly across the three timepoints (n = 6, 5, 8, P = .265). (B) The frequency of CD45+ immune cells on Day 10 post surgery showed that the type of metal implant did not significantly alter immune cell frequencies (P = .059). The numbers represent frequencies of CD45+ cells among total harvested cells. Data are expressed as the mean ± standard error of the mean (sem) from the tibiae samples (n = 5, 5, 5, 4, 6), as obtained in three independent experiments. (C) BM neutrophils were identified as γδTCR-HIS48+ cells among FSC/SSC gated CD45+ immune cells (bottom row, red box). The data shown are those of cells harvested from the BM block on Day 1 post surgery. The numbers in boxes indicate percentages of gated cells. Data are representative of three independent experiments and a total of 5, 5, 5, 4, and 6 tibiae samples for each group. (D, E) Frequencies of TCRαβ+ T cells on Day 1 post surgery were comparable among the types of metal implant. However, the frequency of HIS48+ neutrophils showed statistically significant differences among the groups (P = .028). Data are expressed as the mean ± sem from the tibiae samples (n = 5, 5, 5, 4, 6), as obtained in three independent experiments for each group. Differences between multiple groups were evaluated using the Kruskal-Wallis test. P < .05 was considered statistically significant (^*P < .05; ^**P < .01; ^***P < .001; NS, not significant).

Fig. 5. Histological analysis of the tibia on Days 1 and 35 post-implantations. Cross-sections of the tibiae from the control (no surgery), Sham, Cu, Ti, and Ti-SLA groups on Days 1 (A) and 35 (B) post-implantation were stained with H&E and shown at lower and higher magnification. The area designated by the red box (first row, × 40) was magnified (second row, × 200). Serial specimens were immunohistochemically stained with anti-rat CD68 for total macrophages, anti-rat HIS48 for neutrophils, anti-rat CD3 for T cells, and anti-rat RANKL for osteoclast-like cells (third to sixth rows, × 200).

We further sought to determine whether Cu-reactive neutrophils persisted long-term or mediated FBR by recruiting or promoting the differentiation of other immune cells. To this end, we evaluated the frequencies of immune cell subsets on Days 10 and 35 post-implantation using flow cytometry (Fig. 6A). Although the frequencies of BM TCRαβ-CD11b+ macrophages were comparable among the groups on Day 1, they were significantly higher in the Cu group on Day 10 (Fig. 6B). However, the number of macrophages in the Cu group had decreased by Day 35, indicating that there were no significant differences between the groups at this time point (Fig. 6C). These results indicated that the number of HIS48+ neutrophils in the BM increased immediately (Day 1) after Cu rod implantation. However, the frequency of neutrophils decreased on Day 10, whereas the relative frequency of CD11b+ macrophages increased. This contrast indicates that these cells play a major role in the FBR against Cu implants.

Fig. 6. Macrophages are strongly implicated in the FBR on Day 10 post-implantation. (A) BM macrophages were identified as αβTCR-CD11b+ cells among FSC/SSC gated CD45+ immune cells (red box). Data show dot plots obtained through flow cytometry on Days 1, 10, and 35 post surgery. Numbers in boxes indicate percentages of gated cells. Data are representative of three independent experiments and a total of 5, 5, 5, 4, and 6 tibiae samples for each group. (B, C) Frequencies of αβTCR-CD11b+ macrophages on Days 1 and 10 post-implantations, as determined using flow cytometry. Although the frequencies of macrophages were comparable among the groups on Day 1, they were significantly higher in the Cu group on Day 10 (P = .006). However, the frequency of macrophages in the Cu group had decreased by Day 35 (P = .037). These results demonstrate that the relative frequency of CD11b+ macrophages increased on Day 10, indicating that these cells play a major role in the FBR against the Cu implant. Data are expressed as the mean ± sem from the tibiae samples (n = 5, 5, 5, 4, 6) and are representative of three independent experiments for each group. Differences among multiple groups were evaluated using the Kruskal-Wallis test. P < .05 was considered statistically significant (^*P < .05; ^**P < .01; ^***P < .001; NS, not significant).

We performed immunohistochemical staining to characterize the macrophages at the bone-metal interface on Day 10. We used antibodies against CD68 (a pan-macrophage marker) and those directed against M1 and M2 macrophages. Briefly, a 1-cm section of the rat tibia was harvested from around the implant site and decalcified. H&E staining showed evidence of inflammatory cell infiltration and bone remodeling around the drilled hole (Fig. 7). In particular, the tibial sections of the sham and Cu groups were enriched in CD68+ macrophages but devoid of CD3+ T cells and HIS48+ neutrophils (Fig. 7). The number of RANKL+ giant multinucleated cells was elevated in the sham and Cu groups, but not in the control, Ti, and Ti-SLA groups (Fig. 7). Serial histological sections under high magnification in the Cu group revealed that these macrophages expressed CD86 but not CD206, which is characteristic of the M1 phenotype. Moreover, the macrophages did not express the monocyte and neutrophil marker HIS48 but exhibited RANKL+ expression (Fig. 7). This suggests that M1 macrophages present an osteoclast marker and can potentially absorb newly formed bone at the implant site. No osteoclast-like M1 macrophages were observed in the Ti and Ti-SLA groups, suggesting that the type of metal used in the implants modulates host immune cells (especially neutrophils and macrophages) to elicit or prevent FBR. In addition, FBR-promoting M1 macrophages disappeared by Day 35 after implantation (Fig. 8), indicating that they may influence bone regeneration after immune response induction. These results elucidate the mechanism underlying the immune response to Cu implants in the bone and confirm the strong immunosuppressive properties of Ti in vivo.

Fig. 7. Histological analysis of the tibia on Day 10 post-implantation. (A) The 1-cm bone fragments were cut from the rat tibiae, decalcified, and paraffinized (upper left). A 4-µm thick cross-sectional slide was stained with H&E from the control (no surgery), Sham, Cu, Ti, and Ti-SLA groups on Day 10 and shown in lower and higher magnification (upper two rows). The area designated by the red box (the first row, × 40) was magnified (the second row, × 200). The serial specimens were immunohistochemically stained with anti-rat CD68 for total macrophages, anti-rat HIS48 for neutrophils, anti-rat CD3 for T cells, and anti-rat RANKL for osteoclast-like cells (third to sixth rows, × 200). (B) To identify the phenotype of multinucleated giant cells in the Cu group, immunohistochemical staining was performed with anti-rat CD86 for M1 macrophages, anti-rat CD206 for M2 macrophages, anti-rat HIS48 for neutrophils, and anti-rat RANKL for osteoclast-like cells and shown in higher magnification (× 400). Red and black arrows indicate CD86+CD206- M1 macrophages with RANKL+ multinucleated giant osteoclast-like cells observed at the tibia in the Cu implantation group.

Fig. 8. Immunohistochemical analysis of the tibia for identification of macrophage subtypes on Day 35 post-implantation. Cross-sections of the tibiae from the control (no surgery), Sham, Cu, Ti, and Ti-SLA groups on Day 35 post-implantation were immunohistochemically stained with anti-rat CD86 for M1 macrophages, as well as anti-rat CD163 and CD206 for M2 macrophages (× 200).

DISCUSSION

Host reactions to implanted biomaterials include injury, blood-material interactions, FBR, and fibrosis/fibrous capsule development.¹⁰,¹³,¹⁹,²⁰,²¹,²²,²³ In the present study, a rat model demonstrated that different metal implants induce diverse intrabony immune reactions at the bone-metal interface. Furthermore, we elucidated the dynamics of the immune response at the bone-implant interface to demonstrate why certain metal implants induced osseointegration whereas others did not. For instance, neutrophils promoted the formation of fibrous granulation tissue at the bone-metal interface instead of orchestrating healthy bone regeneration in the present study. This inflammatory FBR was not observed in the Ti and Ti-SLA implantation groups, suggesting that Ti has immunosuppressive properties when implanted into the bone.

The key questions of why and how Ti suppresses neutrophil recruitment and M1 macrophage differentiation following implantation into the bone are currently under our investigation. In this study, we propose several possible mechanisms. Injury to the vascularized connective tissue initiates inflammatory responses and causes thrombus formation in the very early stages of implantation. This process activates extrinsic and intrinsic coagulation systems, the complement system, the fibrinolytic system, the kinin-generating system, and platelets.²⁰,²⁴ Our histological and flow cytometric analyses showed an accumulation of red blood cells near the hole drilled in the tibia on Day 1 post-implantation. The high frequency of HIS48+ monocytes or neutrophils in tibial BM indicates that neutrophils can be initially recruited to the implant surface from blood vessels after surgical injury. From a wound-healing perspective, blood protein deposition on a biomaterial surface constitutes provisional matrix formation.²⁴,²⁵ The provisional matrix facilitates wound healing and FBR by supplying structural, biochemical, and cellular components. Different metals may induce varying immune reactions through the formation of different provisional matrices. Thus, the Cu implant surface may induce a protein matrix that preferentially promotes the differentiation of M1 macrophages. However, the potential mechanism underlying Ti biocompatibility in bone warrants further study.

Acute and chronic inflammation occurred in the later post-implantation stages. The acute inflammatory response is characterized by extensive neutrophil infiltration. Mast cell degranulation, which results in histamine release and fibrinogen adsorption, also mediates acute inflammatory responses following implantation of biomaterials.²⁶,²⁷,²⁸,²⁹ IL-4 and IL-13 released from mast and T helper (Th)2 cells also modulate the degree of subsequent FBR.¹³,²¹,²⁴ Thus, the neutrophils recruited to the Cu implant and proinflammatory Th2 cytokines may collectively participate in the recruitment of macrophages. Histamine release and fibrinogen absorption may further affect the inflammatory response at the implant-bone interface by mediating phagocyte recruitment and adhesion to the implant surface.¹⁴,²⁰,²⁷,³⁰ Therefore, the addition of H1 and H2 histamine receptor antagonists may reduce the recruitment of neutrophils and macrophages to Cu implants in the tibia. Moreover, we plan to investigate whether immune cell adhesion to the implant surface immediately after transplantation determines the biocompatibility of Ti implants.

Implanted biomaterials are coated with host proteins that facilitate implant-cell interactions after the initial inflammatory stage.³¹,³² It is likely that the surface properties of the biomaterial dictate the type and concentration of the bound protein. Neutrophils, macrophages, and foreign-body giant cells (FBGCs) interact with proteins adsorbed onto implant surfaces via adhesion receptors.³⁰,³³,³⁴ Implant-bound proteins include albumin, fibrinogen, the complement system, fibronectin, vitronectin, and γ-globulin. One or more of these proteins may bind to Cu implants and participate in FBR, whereas their absence from the Ti implant surface promotes bone repair. Moreover, these adsorbed proteins may desorb rapidly (e.g., via the Vroman effect), which may explain the rapid decrease in neutrophil and M1 macrophage counts at the bone-Cu implant interface on Day 35 post-implantation.¹³,³⁵,³⁶

To our knowledge, this study is the first to detect multinucleated giant cells at the interface between the bone and the Cu implant on Day 10 post-implantation. These cells were characterized as FBGCs based on their CD68+CD86+RANKL+CD206-HIS48- expression profile. FBGCs have an immune cell phenotype similar to that of monocytes and macrophages, suggesting that they are formed by the fusion of monocyte-derived macrophages.¹²,³⁷,³⁸,³⁹,⁴⁰,⁴¹ Moreover, FBGCs in tissues removed from human implant surgeries express macrophage-associated immune cell surface markers such as CD45, HLA-DR, CD11(a, b, and c), CD18 (part of the leukocyte function-associated antigen family), CD54 (ICAM-1), and CD44, and further exhibit strong cytoplasmic CD68 expression.¹⁹,³⁴,³⁷,³⁸,⁴² FBGCs isolated from tissues surrounding total joint arthroplasties also express the osteoclast markers tartrate-resistant acid phosphatase and vitronectin receptor.³⁹,⁴³,⁴⁴ These characteristics were also observed in macrophages of the Cu implant group on Day 10, and they were identified as the M1 subtype in the present study.

The observed biological differences between Ti and Cu may be primarily attributed to variations in surface chemistry.⁴⁵ Based on the energy dispersive spectroscopic findings of this study, the Cu surface was nearly pure Cu, whereas the Ti and Ti-SLA surfaces consisted of Ti and oxygen due to the spontaneous formation of an oxide layer on Ti. A previous study reported a high rate of cell death surrounding Cu after two days, a phenomenon absent in grade 4 commercially pure Ti.⁴⁵ These distinct chemical compositions likely account for the differences in biocompatibility between Ti and Cu. Another possible explanation involves the variation in surface microtopography.⁴⁵ Earlier research has indicated different bone responses when comparing smooth and roughened Ti surfaces.⁴⁶ However, in this study, the microtopographic effects might be negligible since the Ti and Cu specimens exhibited similar microtopographic characteristics, as shown by the FE-SEM and surface roughness results. Nonetheless, further investigation is necessary, as similar Sa values and FE-SEM images do not ensure an identical three-dimensional topography.⁴⁵

CONCLUSION

Our findings suggest that the presence of FBGC-like M1 macrophages at the Cu implantation site, but not at the Ti implant, drives the FBR to Cu and underlies the biocompatibility of Ti implants. However, future studies are required to elucidate the roles of the provisional matrix, Th2 inflammatory cytokines, and implant surface-adsorbed proteins at various post-implantation stages, as well as identify protein targets to increase the biocompatibility of other implant materials.