CD4⁺ and CD8⁺ T Cells Reduce Inflammation and Promote Bone Healing in Response to Titanium Implants

Abstract

T cells are adaptive immune cells essential in pathogenic response, cancer, and autoimmune disorders. During the integration of biomaterials with host tissue, T cells modify the local inflammatory environment by releasing cytokines that promote inflammatory resolution following implantation. T cells are vital for the modulation of innate immune cells, recruitment and proliferation of mesenchymal stem cells (MSCs), and formation of functional tissue around the biomaterial implant. We have demonstrated that deficiency of αβ T cells promotes macrophage polarization towards a pro-inflammatory phenotype and attenuates MSC recruitment and proliferation in vitro and in vivo. The goal of this study was to understand how CD4⁺ and CD8⁺ T cells, subsets of the αβ T cell family, impact the inflammatory response to titanium (Ti) biomaterials. Deficiency of either CD4⁺ or CD8⁺ T cells increased the proportion of pro-inflammatory macrophages, lowered anti-inflammatory macrophages, and diminished MSC recruitment in vitro and in vivo. In addition, new bone formation at the implantation site was significantly reduced in T cell-deficient mice compared to T cell-competent mice. Deficiency of CD4⁺ T cells exacerbated these effects compared to CD8⁺ T cell deficiency. Our results show the importance of CD4⁺ and CD8⁺ T cells in modulating the inflammatory response and promoting new bone formation in response to modified Ti implants.

Graphical Abstract

1. Introduction

Titanium (Ti) and its alloys are the material of choice for bone-dwelling implants due to their mechanical properties and high biocompatibility [1–4]. Osseointegration, the generation of functional new bone around the implant, is a complex process that requires a timely cascade of events orchestrated by immune cells [4–6]. Damage-associated molecular patterns (DAMPs) resulting from the surgical procedure initiate the chemotaxis of innate immune cells such as neutrophils and macrophages. We have demonstrated that neutrophils and macrophages are highly responsive to changes in biomaterial physicochemical properties such as surface roughness, chemistry, and hydrophilicity [1,7–14]. The initial interaction between innate immune cells and biomaterials results in a modification of the inflammatory microenvironment rich in cytokines and chemokines that recruit other immune cells, including T cells [8,11,15]. Studies from our group have shown that ablation of macrophages either by clodronate liposomes or by administration of AP20187 in the Macrophage Fas-Induced Apoptosis (MaFIA) transgenic mouse model severely reduces the recruitment of T cells and MSCs to the site of injury [11]. While the role of innate cells in biomaterial integration is clear, the role of adaptive immune cells is less clear.

T cells are classically activated by antigen-presenting cells (APCs), such as dendritic cells and macrophages, and activation occurs through the T cell receptor (TCR) and CD3 coreceptor. T cells can also be activated via innate immune cell production of pro-inflammatory cytokines, autonomous of antigen presentation [15–17]. After activation, T cells polarize into various subsets that release cytokines into the inflammatory microenvironment, altering the inflammatory milieu [18]. The two major subsets of T cells are helper T cells (CD4⁺) and cytotoxic T cells (CD8⁺), differentiated through the expression of the CD4 or CD8 coreceptor. CD4⁺ and CD8⁺ T cells, subsets of αβ T cells, are commonly studied in the context of infection, cancer, autoimmune disorders, skin healing, and bone disorders [18–22]. Additionally, T cells play a role in wound healing and fibrotic tissue formation and appear to have tissue-specific functions [23–26]

CD4⁺ T cells are the most significant immune cells in orchestrating adaptive immune responses. In pathogenic infection and cancer, CD4⁺ T cells are essential for antibody production by B cells for antigen recognition on foreign bodies for neutralization or elimination by the immune system. Naïve CD4⁺ T cells can be polarized into Th1, Th2, Th9, Th17, Th22, and CD4⁺ regulatory T cell (Treg) subsets. CD4⁺ T cells have previously demonstrated significance in tissue injury, with subsets such as Th1 and Th17 promoting a pro-inflammatory microenvironment through secretion of IFN-γ, IL-17, and TNF-α, inducing pro-inflammatory macrophage polarization and osteoclastogenesis [27–29]. On the other hand, CD4⁺ T cell subsets Th2 and Treg reduce and modulate inflammation through the production of IL-4, IL-10, and TGF-β. Th2 cells also promote anti-inflammatory macrophage polarization, and Tregs suppress inflammatory cells in the foreign body response. CD4⁺ T cells also promote activation of CD8⁺ T cells via secretion of cytokines IL-2 and IFN-γ [20,30]. Like CD4⁺ T cells, CD8⁺ T cells can be differentiated into Tc1, Tc2, Tc9, Tc17, and CD8⁺ Treg subsets. These cells produce the same inflammatory cytokines as their CD4⁺ T cell counterpart. CD8⁺ T cells are considerably more abundant than CD4⁺ T cells, but their role in the inflammatory response to biomaterial implantation is unknown. T cells have been explored briefly in fracture healing and bone regeneration, as elevations in proportions of CD8⁺ T cells relative to CD4⁺ T cells have been associated with delayed or poor healing in these applications [16]. Despite these findings, the significance of CD4⁺ T cells and CD8⁺ T cells in the inflammatory response to biomaterial implantation remains understudied.

We have previously demonstrated the role of αβ T cells in the inflammatory response to biomaterial implantation. CD4⁺ and CD8⁺ T cell polarization is impacted by Ti physicochemical cues such as roughness and wettability in vivo [8,15]. Additionally, we have demonstrated that deficiency of αβ T cells results in higher pro-inflammatory macrophage phenotype, lower anti-inflammatory macrophages, and significantly reduced recruitment and proliferation of MSCs in response to modified Ti [15]. Furthermore, we have demonstrated that the deficiency of αβ T cells significantly reduces new bone formation post-implantation [15]. Despite these findings, the individual role of the αβ T cell subsets, CD4⁺ and CD8⁺ T cells, remains understudied in response to biomaterial implantation.

The aim of this study was to understand how macrophage polarization, MSC recruitment and proliferation, and new bone formation are impacted by CD4⁺ or CD8⁺ T cell deficiency in response to modified Ti implants. Elucidating the role of these individual subsets in the foreign body response will demonstrate the significance of CD4⁺ and CD8⁺ T cells in the healing response and bone regeneration post-implantation.

2. Materials and Methods

2.1. Ti Disks and Implants

Smooth, rough, and rough hydrophilic Ti disks and implants were provided by Institut Straumann AG (Basel, Switzerland), as previously described [14,15]. In brief, 15 mm disks were created from 1 mm thick unalloyed titanium grade 2. Disks were degreased in an ultrasound acetone bath and then immersed in a 2% ammonium fluoride/ 2% hydrofluoric acid/ 10% nitric acid bath to elicit smooth Ti surfaces. Smooth Ti disks were sand-blasted with 0.25–0.50 mm coarse-grit corundum, then acid etched in a solution of HCl and H2SO4 to generate rough Ti disks. After processing, smooth and rough Ti disks were washed in deionized water and dried. To create rough hydrophilic Ti disks, the same surface modification was applied as was used for rough Ti disks, but following modification, disks were rinsed under a nitrogen atmosphere and sealed in an isotonic NaCl solution to prevent air exposure. 1 mm diameter Ti rods were used to generate 4 mm implants and modified using the same protocols as disks. γ-irradiation was used to sterilize all Ti disks and implants before use.

2.2. Titanium Disk and Implant Characterization

Scanning electron microscopy was used to qualitatively assess surface roughness (SEM, Zeiss Auriga, Carl Zeiss, Germany) and quantitatively using confocal microscopy (LSM 980 Laser Scanning Microscope, Germany) with a 20x objective and a total measurement area of 790 μm × 790 μm, as described previously [13,15]. A moving average Gaussian filter with a cut-off wavelength of 30 μm was used to calculate the arithmetic mean height of the scale-limited surface (Sa). Six areas of three samples for each surface type were measured to calculate mean surface roughness. Sessile drop contact angle was utilized to assess surface hydrophilicity using a Ramé-Hart goniometer (Model 100–25a, Rame-Hart Instrument Co., Succasunna, NJ). Measurements using 5 μl drops of deionized water were performed at six locations per sample (n=3). X-ray photoelectron spectroscopy (XPS) using a PhI5000 VersaProbe spectrometer was used to determine the composition of the oxide layer (ULVAC-PHI, Inc., Chanhassen, MN). Spectra were acquired at a base pressure of 1×10−7 Pa using a focused scanning monochromatic Al-Ka source (1,486.6 eV) with a spot size of 200 μm. The instrument was run in the FAT analyzer mode. The pass energy used for survey scans was 187.85 eV and 46.95 eV for detail spectra. Data were analyzed using the program CasaXPS. The signals were integrated following Shirley background subtraction. Measurements were performed in three separate areas on each sample.

2.3. CD4 and CD8 Knockout Mouse

To demonstrate the significance of CD4⁺ T cells and CD8⁺ T cells in the response to modified Ti, transgenic mice null for the CD4 coreceptor (Cd4−/−, The Jackson Laboratory, Stock # 002663) were crossed with C57BL/6J (WT, The Jackson Laboratory, Stock #000664) mice for ten generations to obtain same genetic background. Similarly, transgenic mice null for the CD8 coreceptor (Cd8−/−, The Jackson Laboratory, Stock # 002665) were crossed with WT mice for ten generations to obtain the same genetic background.

2.4. CD4⁺ and CD8⁺ T Cell Response to Femoral Implants In Vivo

Twelve-week-old male Cd4−/−, Cd8−/−, and WT mice were anesthetized using isoflurane delivered in O₂ gas and monitored for unconsciousness by pedal reflex as described previously [14,15]. The femoral condyles were revealed by opening the skin overlaying the knee and using a scalpel to bisect the patellar tendon longitudinally. A surgical dental bur was used to access the medullary canal, and a cylindrical smooth, rough, or rough hydrophilic Ti implant (Ø=1mm) was then press fit (n=6 mice per implant condition). The implant placement was confirmed by x-ray. To had relieve post-operative pain, mice were administered 1mg/kg buprenorphine ER-LAB (ZooPharm, Laramie, WY) before anesthesia recovery for 72 hours. Animals were monitored until initial ambulation and every 24 hours afterward. All animals access to food and water ad libitum for the duration of the study. No signs of infection were seen in this study. Animal procedures were performed in accordance with a protocol approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee (Protocol: AD10001108).

On postoperative days 3, 7, or 14, mice were euthanized by CO₂ asphyxiation, and femur bones and blood were harvested. Peri-implant tissue was isolated by cutting the femur mid-shaft, and the implant and surrounding marrow were flushed using Accutase (Innovative Cell Technologies, San Diego, CA). Isolated bone marrow was incubated in ACK Lysing Buffer (Quality Biological Inc., Gaithersburg, MD) to remove erythrocytes from the marrow isolates. Cells isolated from bone marrow were washed with staining buffer (BioLegend, San Diego, CA) before staining for flow cytometry analysis.

2.5. Flow Cytometry

Single-cell suspensions from bone marrow were used for flow cytometry analysis. Before fluorescent staining, red blood cells were lysed with ACK lysis buffer (ThermoFisher), and Fc receptors were blocked with TruStain FcX (anti-CD16/32, BioLegend). Cell suspensions were then incubated with fluorescent antibodies (Supplemental Table 1) to identify populations of neutrophils (CD45+/CD11b+/Ly6G+/Ly6C+), macrophages (CD45+/CD11b+/MHCII+/CD11c⁻/F4/80+), pro-inflammatory macrophages (CD45+/CD11b+/MHCII+/F4/80+/CD80+/CD206⁻), anti-inflammatory macrophages (CD45+/CD11b+/MHCII+/F4/80+/CD206+/CD80⁻), CD4⁺ T cells (CD45+/CD11b-/CD3+/CD4⁺), CD8⁺ T cells (CD45+/CD11b-/CD3+/CD8⁺), and MSCs (CD45⁻/Sca-1+/CD90+/CD105+) (BioLegend). Viability dye (BioLegend), single color, and fluorescence minus one control were used to establish the gating strategy, as we recently showed [15]. Samples were analyzed using a BD LSRFortessa-X20 Flow Cytometer (BD Biosciences, San Jose, CA), and a minimum of 250,000 events were collected per sample. Results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR).

2.6. Impact of CD4⁺ and CD8⁺ T Cells on Inflammatory Cytokine Production

To understand the role of CD4⁺ and CD8⁺ T cells in cytokine production in response to modified Ti implantation, analysis of the cytokine microenvironment in the peri-implant tissue was performed by flushing the implant and surrounding marrow with 500 μL chilled PBS without Ca⁺⁺ or Mg⁺⁺. Samples were vortexed for 2 minutes and centrifuged at 500g for 10 minutes. Supernatants were transferred into 1.5 mL tubes and stored at −80°C. Supernatants from peri-implant tissues were thawed, and levels of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ, and TNF-α were measured using Mouse T Helper Cytokine Panel LEGENDplex (BioLegend) following manufacturer’s instructions.

2.7. Macrophage Isolation

Primary murine macrophages were differentiated from bone marrow harvested from 12-week-old male WT mice as described previously [14,15]. Mice were euthanized by CO₂ asphyxiation, and femur bones were removed. PBS and treated with ACK buffer (ThermoFisher Scientific) were used to flush bone marrow. Cells were cultured in RPMI 1640 (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (ThermoFisher Scientific), 50 U/mL penicillin-50 μg/mL streptomycin (ThermoFisher Scientific), and 30 ng/mL macrophage colony stimulating factor (M-CSF, BioLegend) for four days at 37°C, 5% CO₂, and 100% humidity. On day 4, macrophages in culture were supplemented with fresh media. Accutase (Innovative Cell) was used to detach macrophages on day 7 for use in subsequent experiments.

2.8. T Cell Isolation and Activation

Splenocytes harvested from 12-week-old Cd4^−/−, Cd8^−/−, and WT male mice were used for primary murine T cell differentiation as described previously [15]. Mice were euthanized by CO₂ asphyxiation, and spleens were removed. The spleens were homogenized, flushed with PBS, and treated with ACK buffer. Naïve T cells were derived by seeding splenocytes at a density of 1,000,000 cells/well in 6-well plates and cultured in RPMI 1640 Medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (ThermoFisher Scientific), 50 U/mL penicillin-50 μg/mL streptomycin (ThermoFisher Scientific), 0.05 mM β-mercaptoethanol (ThermoFisher Scientific), 25 μL of prewashed and resuspended Dynabeads Mouse T-Activator CD3/CD28 (ThermoFisher Scientific) and 30 U/mL recombinant mouse IL-2 (BioLegend) at 37°C, 5% CO₂, and 100% humidity. Activated T cells were harvested after three days for use in subsequent experiments.

2.9. Impact of CD4⁺ and CD8⁺ T Cell Polarization on Macrophage Phenotype In Vitro

To understand the role of CD4⁺ and CD8⁺ T cells on the inflammatory phenotype of macrophages. We performed coculture experiments with cells from Cd4^−/−, Cd8^−/−, and WT mice. Naïve macrophages were seeded in 24-well plates on either TCPS, smooth, rough, or rough hydrophilic Ti disks at a density of 200,000 cells/well. After 24 hours, T cells from Cd4^−/− or Cd8^−/− mice were added to the macrophage cultures at a cell seeding density of 300,000 cells/well. All cells were cultured in RPMI 1640 Medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (ThermoFisher Scientific), 50 U/mL penicillin-50 μg/mL streptomycin (ThermoFisher Scientific), and 0.05 mM β-mercaptoethanol (ThermoFisher Scientific). After 48 hours of cell interaction, media was removed, and macrophages were detached with Accutase (Innovative Cell Technologies) for flow cytometry analysis. Single-cell suspensions were incubated with TruStain FcX (anti-CD16/32, BioLegend). Cell suspensions were then incubated with fluorescent antibodies to identify pro-inflammatory and anti-inflammatory macrophages as described above. A minimum of 50,000 events were collected per sample. Results were analyzed using FlowJo software.

2.10. Macrophage-T Cell-MSC Co-culture Model

To understand CD4⁺ and CD8⁺ T cell deficiency on the crosstalk between macrophage and T cells for MSC recruitment and proliferation, we conducted co-culture experiments between macrophages and activated T cells isolated from Cd4^−/−, Cd8^−/−, or WT mice as described above. After 36 hours of co-culture, conditioned media was removed, and starvation media was added (RPMI 1640, 1% FBS). At 12 hours of incubation, conditioned media was collected and transferred onto a new 24-well plate. Adipose-derived MSCs (m17.ASC, Sigma-Aldrich) were fluorescently tagged (CellTracker^™ Green CMFDA Dye, ThermoFisher Scientific) and seeded at 50,000 cells/cm² on an 8 μm pore transwell inserts in RPMI 1640 medium supplemented with 1% FBS. Transwells were removed after 12 hours, and MSC migration was quantified using the Synergy HTX Multimode Reader (Biotek Instruments) to measure cell fluorescence. Cell fluorescence was quantified at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

For MSC proliferation, adipose-derived MSCs (Sigma-Aldrich) were seeded at 50,000 cells/cm² in RPMI 1640 medium supplemented with 1% FBS. After 12 hours, MSCs were incubated with Click-iT^™ EdU (ThermoFisher Scientific). Conditioned media from macrophage-T cell co-cultures on Ti surfaces was added after 1 hour. After 12 hours, cells were fixed, permeabilized, and treated with Click-iT^™ EdU buffer and reaction cocktail. Cells were analyzed by flow cytometry for the formation of new DNA. A minimum of 20,000 events were collected per sample.

We also analyzed secreted proteins involved in MSC recruitment and proliferation within the conditioned media before culture with MSCs. Conditioned media was transferred into 1.5 mL tubes and stored at −20°C. Conditioned media from macrophage-T cell cocultures were then analyzed, and levels of IGF-1, WNT3A, TGF-β1 (R&D Systems), IL-10, CXCL12 (BioLegend), and WNT10B (Aviva Systems Biology, San Diego, CA) were measured by ELISA following manufacturer’s instructions.

2.11. Effect of CD4⁺ or CD8⁺ T Cell Deficiency in de novo Bone Formation on Titanium Implants.

To elucidate the role of CD4⁺ or CD8⁺ T cells in de novo bone formation, 12-week-old male WT, Cd4^−/−, and Cd8^−/− mice (n=6) were anesthetized using isoflurane/O₂ gas and instrumented to access the medullary canal and smooth, rough, or rough-hydro Ti rods were implanted as described before. After 21 days, femurs were removed from mice following euthanasia and fixed in 10% formalin (ThermoFisher) for subsequent microCT analysis.

2.12. MicroCT Analysis

The distal femoral metaphysis was scanned at a resolution of 4032 × 2688 pixels (voxel size of 4.00μm) over 180° using a high-resolution aluminum-copper filter, scanning energies of 80kV and 60 μA, 3940 ms exposure time. An image was acquired every 0.5° and frame averaging was conducted for every five frames using SkyScan 1276 (Bruker, Billerica, MA). Serial two-dimensional cross-sections were assembled into 3-D reconstructions and analyzed using SkyScan software and CTAn (Bruker). For bone-to-implant contact analysis, a tubular region of interest (ROI) with a length of 3.75mm, an outer diameter of 1.12mm, and an inner diameter of 1.00mm, creating a volumetric sleeve aligned with the centroid of the implant was generated. For bone formation, ROIs of lengths of 3.75mm, with an inner diameter of 1.12 mm and an outer diameter of 1.35mm, were generated, creating a volumetric washer. All images were individually evaluated, and the ROI mask was applied to each image. A threshold of 1000.00–2500.00 Hounsfield units in the ROI was used to identify bone. Morphometric indices of bone volume selected for representation from the binarized volume of interest (VOI) included the bone volume/tissue volume (BV/TV) ratio, which quantifies the bone amount relative to the VOI size. 3D representations of bone formation were modeled using Dragonfly (Comet Technologies Canada Inc., Montréal, QC).

2.13. Statistical Analysis

Data are presented as mean ± SD. Statistical analysis was performed using Prism9 (GraphPad Software, San Diego, CA). Data were first subjected to the Shapiro-Wilk normality test. Results from this test indicate that the data were normally distributed. A one-factor, equal analysis of variance (ANOVA) was used to test the null hypothesis that group means were equal at a significance level of α=0.05, with post-hoc Tukey’s HSD test for multiple comparisons.

3. Results

3.1. Titanium Disks and Implant Characterization

Increased surface roughness and topography on rough and rough hydrophilic Ti in comparison to smooth Ti was seen by SEM (Figure 1A). Higher roughness for rough (Sa = 3.28 μm) and rough hydrophilic Ti (Sa = 3.30 μm) was shown by confocal microscopy when compared to smooth Ti (Sa = 0.60 μm). Rough and rough hydrophilic Ti surfaces did not demonstrate quantitative or qualitative differences in surface roughness. Water contact angle measurements (Figure 1B) showed that rough Ti (θ = 91°) was the most hydrophobic and rough-hydrophilic Ti (θ = 0°) was the most hydrophilic. Smooth Ti was normal (θ = 63°) compared to rough and rough hydrophilic Ti. The carbon content of the oxide layer was correlated with the wettability of our Ti surfaces. Rough hydrophilic Ti had the lowest carbon content in the oxide layer, while rough Ti had the highest carbon content in the oxide layer, demonstrated by surface chemical composition (Figure 1C).

Figure 1:

Characterization of smooth, rough, and rough hydrophilic Ti samples. (A) Surface topography was analyzed qualitatively via scanning electron microscopy. B) Measurement of water contact angle. (C) Chemical composition of the material oxide layer for each sample (n=6).

3.2. Deficiency of CD4⁺ or CD8⁺ T Cells Skews MSC Recruitment and Innate Immune Cell Response to Modified Ti Implants In Vivo

On day 3 post-implantation, neutrophils were the highest in WT mice (Figure 2). Neutrophils were lower in response to rough and rough hydrophilic Ti. From Cd4^−/− mice, neutrophils were the lowest in response to smooth and rough Ti implants. Pro-inflammatory macrophages were the highest in Cd4^−/− mice compared to WT and Cd8^−/− mice and did not demonstrate differences between modified Ti implants. Anti-inflammatory macrophages were higher in WT mice, producing the highest populations in response to rough hydrophilic Ti. Anti-inflammatory macrophages were the lowest in Cd4^−/− mice but were higher in response to rough hydrophilic Ti. MSC recruitment was the highest in WT mice, with no significant differences between modified Ti implants. MSC recruitment in Cd4^−/− and Cd8^−/− mice was lower than in WT mice but did not demonstrate significant differences between groups.

Figure 2:

Mice deficient in CD4⁺ or CD8⁺ T cells have altered populations of neutrophils, pro-inflammatory macrophages, anti-inflammatory macrophages, and MSCs in response to modified Ti implants. Flow cytometry analysis of peri-implant bone marrow at three days post-implantation (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. smooth Ti, $ vs. rough Ti.

On postoperative day 7, neutrophils and pro-inflammatory macrophages were the highest in Cd4^−/− mice in response to modified Ti implants (Figure 3). Neutrophils did not demonstrate significant differences between WT and Cd8^−/− mice in response to smooth and rough Ti implants. Pro-inflammatory macrophages were the lowest in response to rough hydrophilic Ti for all mice. Pro-inflammatory macrophages were the highest in response to smooth and rough Ti from Cd4^−/− mice. Anti-inflammatory macrophages were the highest in response to smooth and rough Ti in WT mice. Anti-inflammatory macrophages were the lowest in Cd4^−/− mice and did not show differences between modified Ti. Anti-inflammatory macrophages did not show differences between modified Ti in Cd8^−/− mice either. MSC recruitment was the highest in WT mice when compared against Cd4^−/− and Cd8^−/− mice. MSC recruitment was higher in response to rough hydrophilic Ti implants for Cd4^−/−, Cd8^−/−, and WT mice than smooth and rough Ti implants. Recruitment of MSCs was the lowest in Cd4^−/− mice, specifically in response to smooth and rough Ti implants.

Figure 3:

Deficiency of CD4⁺ or CD8⁺ T cells significantly changes the presence of neutrophils, polarization of macrophages, and recruitment of MSCs at seven days post-implantation. Flow cytometry analysis from peri-implant bone marrow at seven days post-implantation (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. smooth Ti, $ vs. rough Ti.

On day 14, neutrophils and pro-inflammatory macrophage populations were the highest in Cd4^−/− mice in response to all modified Ti implants (Figure 4). Neutrophils were lower in response to rough hydrophilic Ti implants for all mice. The lowest population of neutrophils was observed in WT mice. Pro-inflammatory macrophages were lower in response to rough hydrophilic Ti implants in all mice. Anti-inflammatory macrophages were the highest in response to smooth Ti implants in WT mice and the lowest in response to rough hydrophilic Ti. Cd4^−/− and Cd8^−/− mice receiving rough hydrophilic Ti implants demonstrated higher anti-inflammatory macrophages when compared to WT mice. Cd4^−/− and Cd8^−/− mice also had higher anti-inflammatory macrophages in response to rough hydrophilic Ti when compared to smooth and rough Ti. MSC recruitment was the highest in response to rough hydrophilic Ti implants, specifically in WT mice. Smooth Ti implants did not show significant differences between groups. MSC recruitment was higher in Cd8^−/− mice in response to rough Ti implants when compared against WT and Cd4^−/− mice. Cd4^−/− mice demonstrated lower MSC recruitment in rough hydrophilic Ti implants than WT and Cd8^−/− mice.

Figure 4:

Mice deficient in CD4⁺ or CD8⁺ T cells demonstrate higher pro-inflammatory macrophages and neutrophils and lower anti-inflammatory macrophages. Flow cytometry analysis from peri-implant bone marrow at 14 days post-implantation (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. smooth Ti, $ vs. rough Ti.

3.3. Macrophage Polarization is Impacted by the Deficiency of CD4⁺ or CD8⁺ T Cells on Modified Ti In Vitro

Macrophages polarized more towards a pro-inflammatory phenotype when cultured with Cd4^−/− T cells in response to all modified Ti substrates (Figure 5). Pro-inflammatory macrophages were the highest in response to smooth Ti when cultured with T cells from Cd4^−/− mice. Pro-inflammatory macrophages were also higher when cultured with T cells from Cd8^−/− mice when compared to WT mice. Pro-inflammatory macrophage polarization was the lowest in response to rough hydrophilic Ti when cultured with T cells from WT mice.

Figure 5:

Pro-inflammatory and anti-inflammatory macrophages are significantly impacted by CD4⁺ or CD8⁺ T cell deficiency. (A) Schematic demonstrating a co-culture model of macrophages with T cells. (B) Flow cytometry analysis of macrophages cultured in direct contact with T cells from either WT, Cd4^−/−, or Cd8^−/− mice. Cells were co-cultured on either TCPS, smooth Ti, rough Ti, or rough hydrophilic Ti for 48 hours (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. TCPS, $ vs. smooth Ti, % vs. rough Ti.

Macrophages polarized less towards an anti-inflammatory phenotype when cultured with T cells from Cd4^−/− mice in response to all modified Ti surfaces. Anti-inflammatory macrophages from Cd4^−/− mice were the lowest in response to TCPS and smooth Ti. Anti-inflammatory macrophage polarization was higher in response to rough hydrophilic Ti when cultured with T cells from WT mice. Macrophage polarization towards an anti-inflammatory phenotype was higher when cultured with T cells from Cd8^−/− mice compared to Cd4^−/− mice but lower when compared to WT mice.

3.4. CD4⁺ or CD8⁺ T Cell Deficiency Significantly Attenuates MSC Recruitment and Proliferation

MSC recruitment was the highest from the conditioned media of cells from WT mice, specifically on rough hydrophilic Ti surfaces (Figure 6). Recruitment of MSCs was lower for conditioned media of cells from Cd4^−/− mice when compared against Cd8^−/− and WT mice. MSC recruitment was higher from cells from Cd8^−/− mice when compared to Cd4^−/− mice but lower than WT mice. MSC recruitment did not demonstrate significant differences for cells cultured on smooth Ti.

Figure 6:

MSC recruitment and proliferation are attenuated by the CD4⁺ or CD8⁺ T cell deficiency. (A) Analysis of cell fluorescence for MSCs cultured in indirect contact with conditioned media from cells from coculture experiments. (B) Analysis of new DNA formation via flow cytometry from MSCs cultured with the conditioned media from WT, Cd4^−/−, Cd8^−/− mice (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. TCPS, $ vs. smooth Ti, % vs. rough Ti.

Similarly, MSC proliferation was also the highest from the conditioned media of cells from WT mice. Proliferation of MSCs was lower when cultured with the conditioned media from Cd4^−/− mice. MSC proliferation did not demonstrate differences between TCPS and smooth Ti. MSC proliferation was the lowest when cultured with the conditioned media from Cd4^−/− mice in response to TCPS and smooth Ti surfaces.

3.5. Deficiency of CD4⁺ or CD8⁺ T Cells Alters Peri-Implant Cytokine Profile in Response to Modified Ti In Vivo

We identified production of pro-inflammatory (IL-2, IL-6, IL-9, IL-17A, IL-17F, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-5, IL-10, IL-13, IL-22) cytokines in peri-implant tissue. At postoperative day 3, levels of pro-inflammatory IL-9 and IL-17A and anti-inflammatory IL-4, IL-5, and IL-13 were significantly lower in Cd4^−/− mice (Figure 7). IL-2 and TNF-α production was higher in Cd4^−/− mice when compared to WT mice, specifically in response to smooth and rough Ti implants. IL-22 production was the highest in WT mice but did not show significant differences between Cd4^−/− and Cd8^−/− mice. Production of IL-6, IL-10, IL-17F, and IFN-γ did not demonstrate significant differences between Cd4^−/−, Cd8^−/−, and WT mice.

Figure 7:

Inflammatory cytokine production specific to T cells is attenuated in mice deficient in CD4⁺ or CD8⁺ T cells in response to modified Ti implants. Cytokine analysis of peri-implant tissue from either WT, Cd4^−/−, Cd8^−/− mice at 3-, 7-, and 14-days post-implantation (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. smooth Ti, $ vs. rough Ti.

At 7 days post-implantation, production of pro-inflammatory IL-2, IL-9, and IL-17A, as well as anti-inflammatory IL-4, IL-5, and IL-13, were significantly lower in Cd4^−/− mice when compared against WT mice. Levels of IFN-γ, IL-6, IL-10, and IL-22 did not show significant differences between Cd4^−/−, Cd8^−/−, and WT mice. TNF-α production was the highest in response to smooth Ti implants, specifically in Cd4^−/− mice.

On day 14 post-implantation, circulating levels of pro-inflammatory IL-6 and IFN-γ, as well as anti-inflammatory IL-4 and IL-10, were higher in Cd4^−/− and Cd8^−/− mice compared to WT mice. Production of IL-4 was the lowest in response to rough hydrophilic Ti implants in both WT and Cd8^−/− mice, although IL-4 levels were the highest in response to these substrates in Cd4^−/− mice. IL-5 levels were significantly lower in Cd4^−/− mice. Production of other inflammatory cytokines did not show significant differences between Cd4^−/−, Cd8^−/−, and WT mice.

3.6. CD4⁺ or CD8⁺ T Cells Enhance Bone Formation and Bone Implant Contact in Response to Modified Ti Implants

We analyzed bone-to-implant contact and identified that WT mice produced significantly more bone around all modified Ti implants than Cd4^−/− and Cd8^−/− mice (Figures 8A and 8B). Cd4^−/− mice showed the lowest peri-implant bone in response to smooth Ti implants, while the WT mice receiving rough-hydrophilic Ti implants demonstrated the highest bone-implant contact. We also quantified new bone formation by analyzing bone volume as a proportion of total volume, and similarly, WT mice showed more new bone formation in response to all modified Ti implants when compared to Cd4^−/− and Cd8^−/− mice (Supplemental Figure 1). Cd4^−/− and Cd8^−/− mice receiving rough or rough hydrophilic Ti implants produced more new bone than those receiving smooth Ti implants. Cd4^−/− mice receiving rough hydrophilic implants showed lower bone formation than Cd8^−/− mice and WT mice.

Figure 8:

Bone implant contact is significantly lower in mice deficient in CD4⁺ or CD8⁺ T cells. Analysis of bone-implant contact around modified Ti implants from WT, Cd4^−/−, or Cd8^−/−mice at 21 days post-implantation (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. smooth Ti, $ vs. rough Ti.

3.7. CD4⁺ and CD8⁺ T Cells Deficiency Attenuates Secreted Factors Involved in MSC Recruitment, Proliferation, and Differentiation In Vitro

We identified secreted proteins within the conditioned media from macrophage-T cell coculture experiments that were utilized in experiments for MSC recruitment and proliferation. Secretion of IL-10, CXCL12, TGF-β1, IGF-1, and WNT10B was the highest in cells from WT mice, specifically in response to rough hydrophilic Ti (Figure 9). IL-10, TGF-β1, and IGF-1 levels were the lowest in cells from Cd4^−/− mice compared to WT mice. Additionally, the production of these proteins was not significantly different between modified Ti surfaces. CXCL12 and WNT10B levels were also lower in cells from Cd4^−/− mice, specifically on rough and rough hydrophilic Ti implants. CXCL12 secretion was the highest in response to rough hydrophilic Ti implants from cells from all groups of mice. WNT3A production was the lowest in cells from Cd8^−/− mice in response to all modified Ti surfaces.

Figure 9:

Secreted cytokines, proteins, and growth factors within the conditioned media from macrophage-T cell coculture experiments are altered by the deficiency of CD4⁺ or CD8⁺ T cells in response to modified Ti. Protein analysis of the conditioned media from macrophages cultured with T cells from either WT, Cd4^−/−, or Cd8^−/− mice (n=6). p<0.05: A vs. WT, B vs. Cd4^−/−, # vs. smooth Ti, $ vs. rough Ti.

4. Discussion

We have recently shown that both innate and adaptive immune cells are altered by the physicochemical cues on biomaterial implants [8,15]. We have previously investigated the inflammatory response to modified Ti implants, including the modifications in this study [1,8–11,15]. Rough and rough-hydrophilic Ti implants are utilized clinically due to their ability to attenuate the pro-inflammatory microenvironment and improve the formation of new bone in vivo when compared to smooth implants, promoting healing and implant success [1,3]. Varying the roughness of Ti implants provides two distinct inflammatory phenotypes of the immune response when examining the response to smooth and rough implants. Rough Ti implants upregulate the polarization of anti-inflammatory macrophages, production of anti-inflammatory cytokines, and differentiation of MSCs to osteoblasts, further enhanced by increasing wettability [1,31–33]. Our group has demonstrated that surface roughness and wettability on Ti implants significantly affect T cell polarization. T cells significantly contribute to the inflammatory response and new bone formation post-implantation. Previously, we have demonstrated that a deficiency of αβ T cells results in a higher presence of neutrophils and pro-inflammatory macrophages at the implantation site. This study aimed to deconvolute the individual significance of CD4⁺ and CD8⁺ T cells, subsets of the αβ T cell family, in the inflammatory response to modified Ti implants. Our study identified that CD4⁺ and CD8⁺ T cells play different roles in the inflammatory response, and deficiency of either subset significantly impacts the behavior of innate immune cells, recruitment and proliferation of MSCs, and new bone formation around Ti implants.

Neutrophils are the first and most abundant immune cells recruited to the injury site by DAMPs after implantation [13,34,35]. Previously, we have demonstrated that neutrophil recruitment at the site of implantation is the highest at early time points (days 1 and 3), and proportions are reduced at later time points (days 7 and 14) [13,15,36]. However, mice with deficiency in αβ T cells have shown significantly elevated neutrophil populations at all time points analyzed, suggesting that αβ T cells are essential for the suppression and clearance of neutrophils in the inflammatory microenvironment [15]. Neutrophil survival is mediated by death receptors expressed on the cell membrane, and apoptosis of neutrophils can be induced by T cell secretion of TNF-α [37]. Our study showed a lower proportion of neutrophils in mice lacking CD4⁺ or CD8⁺ T cells at early time points and an increased proportion at later time points compared to T cell-competent mice. These results suggest that CD4⁺ and CD8⁺ T cells play a significant role in neutrophil behavior by recruiting additional neutrophils early and resolving the inflammatory response later.

Neutrophils have previously been shown to be modulated by CD4⁺ and CD8⁺ T cells via secretion of inflammatory cytokines [37–40]. IL-8 and IL-17A production by human CD4⁺ T cells has been shown to promote neutrophil recruitment in an in vitro co-culture model [39]. IL-17A has been shown to play a vital role in regulating neutrophil behavior in several tissues, including lung, kidney, brain, spleen, gut, and liver, as mice deficient in interleukin 17 receptor (IL-17R) have shown significantly reduced recruitment of neutrophils in various immune responses [37,39,41]. Our data shows lower levels of IL-17A in Cd4^−/− mice compared to T cell-competent mice, demonstrating that CD4⁺ T cells, specifically Th17, are the primary source of IL-17A after biomaterial implantation [42–44]. The role of IL17A may be tissue-dependent, as a prior study involving murine skin wound healing showed that IL-17A deficiency also resulted in decreased neutrophil recruitment but accelerated wound healing in this model [45]. Additionally, secretion of IFN-γ and TNF-α by CD8⁺ T cells has been shown to promote higher activation and viability of neutrophils compared to CD4⁺ T cells in a neutrophil-T cell co-culture model [40,46]. Here, we observed increased levels of TNF-α in CD4^−/− mice in all time points measured, coinciding with persistently elevated populations of neutrophils even at 14 days post-implantation, suggesting that CD4⁺ T cells are essential for immunomodulating the local environment. CD4⁺ Tregs have shown a suppressive effect on neutrophils in a prior in vitro study analyzing neutrophil response to lipopolysaccharide (LPS)-activated Tregs. Tregs promote neutrophil apoptosis via direct cell-cell contact and production of IL-10 and TGF-β, as well as lower neutrophil secretion of reactive oxygen species and inflammatory cytokines [47,48]. Populations of neutrophils were most significantly impacted by the deficiency of CD4⁺ T cells. This suggests that CD4⁺ T cells play an important role in neutrophil recruitment and survival in the inflammatory response.

Macrophages play a significant role in immune cell crosstalk with T cells, as our group has previously shown that the presence of T cells can significantly increase the inflammatory polarization of macrophages in vitro [15,36]. T cells do not directly interact with the implant surface; therefore, T cell response to implantation is mediated by T cell interaction with the adherent cells on the biomaterial substrate [17,49,50]. This study demonstrates the impact of CD4⁺ or CD8⁺ T cell deficiency on the macrophage response to Ti implantation. Here, we show that CD4⁺ T cells are critical in controlling macrophage polarization, as mice deficient in CD4⁺ T cells have significantly higher pro-inflammatory and lower anti-inflammatory macrophages both in vitro and in vivo. While our results show pro- and anti-inflammatory macrophages as a proportion of total macrophages, given the deficiency of either CD4⁺ or CD8⁺ T cells, we also quantified pro- and anti-inflammatory macrophages as a proportion of all immune cells (CD45+), which demonstrated fewer differences between Cd4^−/− and Cd8^−/− mice (Supplemental Figure 2). CD4⁺ T cells produce several factors that promote pro-inflammatory macrophage activation, such as MIP-1α, MIP-1β, and ATAC [51]. IFN-γ production by T cells has been shown to increase the proliferation of macrophages and nitric oxide production. Additionally, Th2 cells and CD4⁺ Tregs appear to play a vital role in suppressing pro-inflammatory macrophages and activating anti-inflammatory macrophages via secretion of IL-4 and IL-10 [8,52–56]. Here, we show decreased production of IL-4 in CD4⁺ T cell-deficient mice, along with lower populations of anti-inflammatory macrophages, demonstrating that CD4⁺ T cells are a significant source of this anti-inflammatory cytokine in the inflammatory response that contributes to macrophage activity. We also found diminished secretion of IL-9, a cytokine produced primarily by Th9 cells, in CD4⁺ T cell-deficient mice. Macrophage polarization has been shown to be impacted by IL-9 in a human in vitro model, showing that recombinant IL-9 treatment reduced pro-inflammatory marker expression and promoted TGF-β secretion [57]. This upregulation of TGF-β has been shown to elicit an anti-inflammatory effect by reducing the oxidative burst ability of macrophages [57]. Additionally, a model of antigen-induced arthritis examined IL-9 deficiency and found that IL-9 is an important cytokine for the activation of Tregs, enhancing the suppressive capacity of these cells and promoting the resolution of the inflammatory response [58]. In our study, we believe that reduced production of IL-9 in CD4⁺ deficient mice impairs the resolution of the inflammatory response.

While mice deficient in CD8⁺ T cells did not demonstrate drastic differences in macrophage populations compared to CD4⁺ deficient mice, these cells still impact the inflammatory polarization of macrophages. Our study shows that CD8⁺ deficient mice demonstrate higher proportions of pro-inflammatory macrophages and lower anti-inflammatory macrophages in vitro and in vivo. CD8⁺ T cell interactions with macrophages are poorly characterized compared to CD4⁺ T cells. A previous study involving cardiotoxin-induced muscle injury found that adoptive transfer of CD8⁺ T cells increased macrophage recruitment into the injury site via increased expression of MCP-1 [59]. Additionally, in studies using a high-fat diet-induced obesity mouse model, CD8⁺ T cells have been shown to significantly contribute to tissue inflammation associated with obesity, recruiting and activating macrophages within adipose tissue [60]. CD8⁺ T cells have also been shown to produce other factors, such as MCP-3 and RANTES, which can impact the migration and differentiation of macrophages [60]. Furthermore, CD8⁺ T cells have demonstrated a suppressive function on osteoclast differentiation from macrophages, previously shown in a co-culture model where CD8⁺ T cells prevented the formation of multi-nucleated osteoclasts via secretion of soluble molecules such as osteoprotegerin [61]. On the other hand, CD4⁺ T cells showed the ability to activate and differentiate osteoclasts. Future studies should examine the role of CD8⁺ T cells in isolation, as we demonstrate that deficiency of these cells impacts the polarization of macrophages in the peri-implant microenvironment.

Recruitment of MSCs after the initial inflammatory response post-implantation is imperative to facilitate new bone formation and osseointegration. Previously, we have demonstrated that the deficiency of αβ T cells attenuates MSC recruitment, proliferation, and peri-implant bone contact at 14 days post-implantation [15]. Interestingly, our data shows that the lack of CD4⁺ or CD8⁺ T cells significantly reduces new bone formation and bone-implant contact at 21 days post-implantation, with bone-implant contact significantly lowered in Cd4^−/− mice. We found reduced levels of insulin-like growth factor 1 (IGF-1) and WNT10B in mice deficient in either CD4⁺ or CD8⁺ T cells, two secreted products that have previously shown essential roles in new bone formation [62,63]. IGF-1 is a hormone primarily produced in the liver, but both IGF-1 and WNT10B have been previously reported to be secreted locally by T cells and have demonstrated important functions that modulate osteoblastogenesis [64–66]. Here, we observed the lowest production of IGF-1 and WNT10B in cells, specifically from the results of the CD4⁺ deficient group. Other studies have reported that CD4⁺ and CD8⁺ T cells partially or entirely inhibit bone regeneration by blocking the osteogenic differentiation of MSCs. Yet, these reports utilize nude mice deficient in γδ T cells, a CD3⁺ T cell subset shown to significantly affect bone regeneration in vitro and in vivo [67–72]. In our study, our mice have competent γδ T cells, and we show that recruitment of MSCs is attenuated both in vitro and in vivo in mice deficient in either CD4⁺ or CD8⁺ T cells. These differences are only apparent at day 7 post-implantation, where CD4^−/− mice demonstrate significantly reduced recruitment of MSCs, whereas at day 14 post-implantation, differences become less pronounced. CD4⁺ and CD8⁺ T cells have previously shown a role in MSC recruitment via secretion of soluble factors involved in MSC chemotaxis to the injury site [51,73–76]. Differences in bone formation may result from adherent progenitor cells differentiating on the implant surface that would be modulated by either CD4⁺ and CD8⁺ T cells or their effector subsets. We have shown that MSCs cultured on rough or rough-hydrophilic Ti surfaces undergo osteogenic differentiation via integrin signaling. This creates an osteogenic microenvironment that increases osteogenic differentiation on cells not in contact with the biomaterial surface [77–80].

Tregs and Th17 cells, CD4⁺ T cell subsets, have demonstrated a role in MSC recruitment, proliferation, and differentiation. In a prior co-culture model between CD4⁺ T cells and MSCs, the presence of lymphocytes led to higher alkaline phosphatase production by MSCs, indicating differentiation towards osteogenic lineage [43,81]. Tregs have been shown to accelerate the healing of bone via increased MSC osteogenic differentiation and mineral deposition in a critical-sized cranial defect model [81]. TGF-β1, a signature Treg cytokine, has demonstrated a function to reduce bone resorption and promote new bone formation, as TGF-β1 stimulates the proliferation and differentiation of MSCs to osteoblasts and reduces apoptosis of these cells [82,83]. Several in vivo studies performed in dogs and minipigs have demonstrated that recombinant TGF-β1 treatment on implants composed of Ti and Ti alloys significantly increases osseointegration and new bone formation [82,84]. In our study, levels of TGF-β1 within macrophage-T cell conditioned media were significantly lower in mice deficient in CD4⁺ T cells. We also observed reduced MSC recruitment and proliferation in Cd4^−/− mice. Our results suggest that CD4⁺ T cells may contribute to the recruitment and proliferation of osteoprogenitor cells through TGF-β1. Furthermore, in a model of chronic ischemia, transplantation of MSCs with Tregs into the myocardium showed that Tregs promote the viability and proliferation of MSC post-transplantation [85].

Th17 cells have traditionally demonstrated a pro-inflammatory phenotype, with previous findings showing that IL-17A stimulates osteoclastogenesis, leading to bone destruction [27,28,43,86–89]. However, a murine model utilizing IL-17A-deficient mice found that lack of this cytokine resulted in delayed bone healing [69]. IL-17A secretion has been shown to contribute to MSC differentiation to osteoblasts and CXCL12 production, a powerful chemoattractant for recruiting immune cells and MSCs to the injury site [90,91]. An in vitro model for rheumatoid arthritis found that coculture of fibroblast-like synoviocytes with CD4⁺ T cells or treatment with recombinant IL-17A significantly enhanced the production of CXCL12 [91]. CD4⁺ T cells alone are not a primary source of CXCL12. However, CD4⁺ T cells may synergize with other cell types, like macrophages, to upregulate CXCL12 secretion and promote MSC recruitment and proliferation. In our study, we demonstrated significantly attenuated secretion of IL-17A and CXCL12 from mice deficient in CD4⁺ T cells, suggesting that CD4⁺ T cells play a fundamental role either in the secretion of these molecules or influencing other cells, such as macrophages, in producing these molecules. CXCL12 is a chemoattractant for other immune cells and has been associated with the progression of the inflammatory response. It has also been recognized as a critical regulator of cell migration during tissue healing and regeneration.

Additionally, previous studies have shown that CD8⁺ T cells inhibit MSC bone formation; therefore, mice deficient in CD4⁺ T cells may have an exacerbated reduction of MSC recruitment and new bone formation due to both the lack of CD4⁺ T cells and heightened levels of CD8⁺ T cells [16,92,93]. We note this compensation of either CD4⁺ or CD8⁺ T cells for the deficiency of the other subset in our transgenic knockout models as a limitation of this study (Supplemental Figure 3).

Our study has an application to other clinical states outside of implantation. Absence or diminishment of CD4⁺ T cells is the diagnosis of patients with human immunodeficiency virus (HIV). Studies have found that patients with HIV have significantly delayed or nonunion healing of bone fractures [94]. These patients also have higher levels of serum TNF-α, which we see in Cd4^−/− mice in our study [94]. Prior literature has speculated that an upregulation of TNF-α can promote desensitization to the inflammatory response, inhibiting the healing process [71,95]. Alterations in helper and cytotoxic T cells are characteristics of many conditions that can impact healing and regeneration in response to injury. In the context of bone healing and implantation, the changes in these T cell populations should be considered in patients who fall under this category to promote the long-term success of implants or fracture healing.

5. Conclusion

Our results show the importance of both CD4⁺ and CD8⁺ T cells in the inflammatory response to Ti implantation. Deficiency of either of these αβ T cell subsets results in a skewed innate immune cell profile in the inflammatory microenvironment and reduced new bone formation. These results demonstrate that CD4⁺ T cells play a more significant immunomodulatory role in neutrophil clearance, macrophage polarization, recruitment and proliferation of MSCs, and promoting bone-implant contact at the peri-implant site. CD8⁺ T cells demonstrate similar results to CD4⁺ T cells but do not appear to impact innate immune cells and MSCs to the same degree. Both αβ T cell subsets play an essential role in the sensitivity of innate immune cells to surface topography and wettability on Ti implants. Future approaches involving modulation of CD4⁺ and CD8⁺ T cells or their effector subsets may be an effective therapeutic strategy to promote accelerated healing and improve osseointegration post-implantation.

Statement of Significance.

CD4⁺ and CD8⁺ T cells are essential in modulating the peri-implant microenvironment during the inflammatory response to biomaterial implantation. This study shows that deficiency of either CD4⁺ or CD8⁺ T cell subsets altered macrophage polarization and reduced MSC recruitment and proliferation at the implantation site.