The cells were cultured in a controlled condition in an incubator with 5% CO₂ at 37°C. The best cell growth can be obtained with a controlled cell culture medium with 10% serum supplement and antibiotics (Penicillin + Streptomycin, Sigma-Aldrich, Taufkirchen, Germany, P078) to prevent bacterial growth. All primary cell isolation and cultures were performed under the approval of the Local Ethics Committee of the University of Wuerzburg (182/10) and informed consent of the patients. The culture steps were also recorded. Cells were monitored regularly under the microscope. All the processes including medium exchange, passaging, assays were performed under sterile conditions using sterile cell specific mediums and supplements inside Biosafety cabinet. The cells were passaged when they have reached 90% confluency.

NIH3T3 (ATCC CRL1658, Germany) is known to be as standard fibroblast cell line, was kindly provided by the “Experimental orthopedic research lab, at University Hospital Magdeburg”. NIH3T3 is an adherent fibroblastic cell line, most commonly used in tissue engineering research. These were cultured starting from passage 2 and cultured in T175 flasks. NIH3T3 fibroblasts are then used in preliminary investigation of 2D fibroblast stimulation assay on a 12 well plate, to investigate particle uptake and RTPCR analysis. Briefly, fibroblast stimulation assay was performed in 12 well plate with NIH3T3 1 × 10⁵ cells per well with 2 mL fibroblast medium. After 24 h cells achieved 80%–90% confluence and were then treated overnight with particle dilutions (1, 10, 100 μg/mL) prepared in fibroblast medium under sterile conditions. CT800 ceramic Al₂O₃ particle (powder) were kindly provided by Experimental Orthopedic department, OvGU Clinic Magdeburg. After 24 h, cells were used in further standard processes of RNA isolation with Tryzol and RTPCR analysis, using lab standardized SOPs. The biological replications were performed using progressive passage numbers of NIH3T3 fibroblasts three times followed by consecutive RTPCR analysis with two technical replications. Similarly, phalloidin and DAPI staining’s were performed on the stimulated cells fixed on coverslips in 48 well plate.

Primary human dermal fibroblasts (pFb) were obtained from Chair of TERM, University Hospital Wuerzburg, Germany. These were isolated and used in culture under the approval of Local Ehical Committee of the University of Wuerzburg (182/10) and informed con-sent of the patients. In the study, isolated primary fibroblasts from a single donor were utilised for all test series, with the objective of eliminating individual differences between multiple donors. The pFbs were defreezed and cultured until passage 6 using standard operating protocols for defreezing and passaging. For setting up the pFb-hydrogel models with stimulated fibroblasts, firstly the fibroblast stimulation was performed in 2D cell culture flasks. The pFbs were cultured for 3 days in T75 adherent cell culture flasks at a density of 5 × 10⁵ and then the flasks were incubated with the respective material particles diluted to a concentration of 10 μg/mL for CT800 and 1 μg/mL for stainless steel and titanium. For the other group of unstimulated fibroblasts in collagen hydrogel, the same patient fibroblasts were used cultured in separate T75 flask without any particle contact.

THP-1 monocyte like cell line is widely used as a model for monocytes in research technologies. The cells are cultured in suspension culture at a density of 3 – 5 × 10⁵ cells/mL or less in a fresh medium. THP-1 cells were initially derived from ECACC and kindly donated by the “Heart & Thoracic research lab at University hospital Magdeburg” (Murkar et al., 2024). The THP-1 cell line is a spontaneously immortalised monocyte-like cell line, derived from a patient with acute monocytic leukaemia, specifically from the peripheral blood of a one-year-old male (Bosshart and Heinzelmann, 2016). THP-1 monocytic cell line follows a suspension culture technique. The cells were cultured in a density of 5 × 10⁵ cells/mL in a T175 cell culture flask and were controlled under microscope every third day. The cell culture can be maintained by the addition of fresh medium or by replacement of the medium. The cells are passaged when the cell confluence reaches almost 90%. The cell concentration is not allowed to seed above 1 × 10⁶ cells/mL. Subsequent cultures can be established with following resuspension at 2 – 4 × 10⁵ viable cells/mL. The media is changed every 2–3 days. While passaging, the cell suspension is centrifuged at 1,000 rpm for 5 min. Freezing protocol for the cells follow the same steps as in for NIH3T3 cell line. Following Table 2 summarizes the cell/cell lines, along with their culture medium composition and necessary order information for transparency.

As described in previous publications, THP1 monocytes were differentiated into M0, M1, and M2 macrophages (Smith et al., 2015; Murkar et al., 2024). Briefly, cells in a density of 1 × 10⁵ were seeded in each well of a 12-well plate with coverslips and incubated for 1 hour with a THP-1 medium consisting of RPMI-1640 with 2 mM of L-glutamine (Gibco, Darmstadt, Germany, 21875034) + 10% FCS (Bio&Sell, Feucht bei Nürnberg, Germany, FBS.S0615) + 1% PenStrep (Sigma, Taufkirchen, Germany, P0781). Phorbol-12-myristat-13-acetat (PMA, Sigma-Aldrich, Taufkirchen, Germany, P1585) with a working concentration of 50 ng/mL was added and incubated for 24 h. After 24 h, the PMA-containing medium was removed and the cells were washed with the THP-1 medium three times. Then, the THP-1 medium was added and incubated for 1 h. For differentiation in the M0, M1, and M2 macrophages, different mediums were used as follows: M0 (THP-1 medium), M1 (THP-1 medium + IFN-γ (50 ng/mL), Sigma-Aldrich, SRP3058) and M2 [THP-1 medium + IL-4 (Sigma-Aldrich, SRP4137) + IL-13 (Sigma-Aldrich, SRP3274) (each 25 ng/mL)]. Differentiation in the cells was achieved after 48 h (Smith et al., 2015).

Analysis was performed using Super Vision 2 HRP Kit (DCS, Hamburg, Germany, PD000KIT) according to manufacturer protocols. The tissue sections were stained against actin, procollagen, fibronectin, collage1, Ki-67 in optimized concentrations as summarized in the table below along with corresponding negative controls Mouse IgG serum (Sigma-Aldrich, Taufkirchen, Germany, I8765) and rabbit IgG serum (Sigma-Aldrich, Taufkirchen, Germany, I8140). Previously established standard operating protocol for immunehistology was used (Murkar et al., 2024).

Staining was performed according to the previously published protocols (Murkar et al., 2024) for macrophage characterization using the respective CD markers for M0, M1, and M2 macrophages, as shown in Table 2, as well as for cellular tissue characterization on the fixed in vitro tissue models. THP1 derived M0, M1, M2 macrophage populations were characterized using selected macrophage specific cell surface markers. The cell populations were differentiated in 12 well plate separately as explained in the methods section above and characterized using immunofluorescence staining according to the standardized protocols (20). Briefly, the cells were gently washed with PBS- and fixed in Paraformaldehyde (PanReac AppliChem, Darmstadt, Germany, 256462) for 10 min, which was followed by permeabilization (0.5% Tween20 in PBS-) and blocking steps with intermittent washing with PBS-. Following 1 h of blocking at room temperature, cells were coated with the respective primary antibodies (see Table 3) and counterstained with host-specific fluorescently labelled secondary antibodies and DAPI nuclei staining. Fluorescence imaging was performed using a ZEISS Axio Observer fluorescence microscope (Keyence GmbH, Neu-Isenburg, Germany).

The tissue sections were also stained against primary antibodies specifically fibronectin, Procollagen, Ki-67 along with DAPI nuclear counterstain in concentrations as summarized in the table below. According to the standardized protocols (Murkar et al., 2024), briefly, after blocking using 5% FCS + 1% BSA in PBS- for 4 h, these primary antibodies (for incubation time, refer to Table 3) were counterstained using host-specific fluorescently labelled secondary antibodies. Finally, nuclei were stained blue using DAPI.

Supernatants from 3D co-cultures were collected and analyzed first using Sandwich ELISA for IL-6 and TNF-α. The measurement of cytokines IL-6 and TNF-α is a practicability item for the valuation of foreign body tissue response. These cytokines have a key role in the inflammatory response and are essential mediators of the immune response to implants. A multiplex enzyme-linked immunosorbent assay (ELISA) was conducted using the LEGENDplex essential immunoresponse panel to quantify the levels of nine cytokine markers (Supplementary Table S1). Supernatants were collected from stimulated primary human fibroblasts cultured in 3D collagen hydrogels in co-culture with M1 and M2 macrophages on days 3, 7, and 10 for ceramics, as well as on day 14 after stimulation with steel and titanium. All samples were collected under sterile conditions. Subsequently, the samples were stored at −80°C in a sterile state until measurement was undertaken using the multiplex ELISA method. The multiplex ELISA sub-panel mix-and-match kit permitted the measurement of the expression of IL-1β, IL-2, IL-4 IL-6, IL-8, IL-10, IL-12, TNF-α and TGF-β1 in a multiplex setting using the flow cytometer FACS CantoII FACS machine. The results were subsequently analysed using the BioLEGEND software, which is available online from the panel manufacturer.

Image processing was done using the Image Processing Toolbox™ (The MathWorks, Inc. (2022). Image Processing Toolbox version: 11.5 (R2022a), n.d.) in MATLAB R2022a. Images were processed using a semi-supervised algorithm where the raw RGB images were converted into HSV images (Supplementary Figure S2). Segmentation of the area corresponding to the tissue was done on the hue channel of the HSV image by using an automated global threshold using Otsu’s method (Otsu, 1979). Identification of the area within the tissue which was positively stained, was done using a fixed threshold on the saturation channel of the HSV image. The output images were individually inspected to ensure a successful segmentation and identification process. The percentage of tissue stained positively was then defined as follows: %   o f   I H C   p o s i t i v e   t i s s u e = I H C   p o s i t i v e   a r e a   w i t h i n   t h e   t i s s u e   P i x e l s A r e a   o f   t h e   t i s s u e   P i x e l s × 100

In the case of 2D fibroblast culture of NIH3T3 fibroblasts stimulated with CT800 ceramic particles, RNA isolation approach was followed using the trizol lysing method according to the standard operating protocol for “RNA isolation”. Henceforth followed by reverse transcription to obtain copy DNA for PCR analysis of specific target genes, namely, GAPDH, TNF-α, TGF-ß1, IL-1ß, FGF, collagen-1 (Col-1), fibronectin (FN), IL-6. Table 4 shows gene primer sequences for selected genes. RTPCR analysis was repeated for three biological replications of NIH3T3 ceramic particle stimulation, along with each three technical replicates.

Quantitative RT-PCR data was collected from three experimental repetitions and three technical replicates for each model. Average of quantity mean (q-mean) was obtained for each replicates and normalized against home gene GAPDH. Fold change was then calculated using the values from control samples where no CT800 stimulation was performed against those stimulated with controlled concentrations of CT800, namely, 1, 10, 100 μg/mL. Finally, the fold change data obtained for each experimental replicates is averaged and depicted in the supplementary section of this article. Additional statistical analysis was performed using OriginPro 2019 software, by pooling the raw data for RTPCR. Briefly, Friedman ANOVA test was performed on the calculated normalized q mean values and p values were calculated. To analyse the results of the ELISA measurements, three series of tests were conducted. In each independent test series, one sample was taken from each material, sampling time point and macrophage population in the co-culture. Each sample was analysed twice for the respective cytokine concentration, and the resulting values were averaged to form a mean value. This mean value was then combined with the respective mean values of the other two test series to form a general mean value. In this way, we achieved a test size of n = 6 for each test group. The Microsoft Excel programme was utilised for this process, as well as for the graphical representation of the mean value (Supplementary Figures S3A–F, S5A–F). The results were visualised in bar charts showing the measured concentration (pg/mL) of the different cytokines over time from day 3 to day 10 (A-B) or day 14 (Supplementary Figures S3A–F, S5C–F).

The measurements for the prominent cytokines, TNF-α and IL-6, were performed using a sandwich enzyme-linked immunosorbent assay (ELISA). TNF-α concentrations exhibited minimal variation in the majority of cultures, reaching negligible levels. In each stimulated and unstimulated co-cultures, including M1 and M2 macrophages, TNF-α demonstrated a consistent concentration (Supplementary Figures S3A–F). The absolute values of TNF-α were found to be lower in the CT800 (Supplementary Figures S4A,B) concentration than in the un-stimulated or metal particle-stimulated cultures (Supplementary Figures S3A–F). Irrespective of the stimulation employed, the TNF-α level remained at a lower level than in the unstimulated culture and exhibited no discernible trend. As shown in Supplementary Figures S4A,B the cultures stimulated with ceramic exhibited an increase in IL-6 concentration over time up to day 10, with the concentration in the M2 culture being considerably higher than in the M1 culture [cM1 (ceramic) = 139 pg/mL; cM2 (ceramic) = 1,344.0 pg/mL]. Notably, distinct variations were evident in the IL-6 concentration of cultures subjected to stimulation with stain-less steel or titanium particles. In the cultures that had not been stimulated previously and which contained both M1 and M2 macrophages, no measurable concentration of IL-6 was observed in the supernatant, with the exception of the M1 cultures on day 14 (Supplementary Figures S3A,B). Following stimulation with stainless steel, both the M1 and M2 cultures exhibited an almost identical concentration between days 3 and 7. However, this increased on day 14 [cM1 (steel) = 48.1 pg/mL; cM2 (steel) = 63.1 pg/mL]. In contrast, the IL-6 concentration after stimulation with titanium particles exhibited an increase from day 3, with the initial concentrations being higher with titanium as with stainless steel [cM1 (steel) = 3.8 pg/mL < cM1 (titanium) = 23.7 pg/mL; cM2 (steel) = 20.5 pg/mL < cM2 (titanium) = 39.1 pg/mL]. Following stimulation with titanium particles, a difference of >20 pg/mL was noticed in the final measurement (day 14) [cM1 (titanium) = 49.4 pg/ml/cM2 (titanium) = 73.6 pg/mL]. According-ly, the M1 and M2 co-cultures on day 14 exhibited notable discrepancies in IL-6 concentration for both metal types (Supplementary Figures S3C–F).

A comparative analysis of cytokine concentrations was performed by multiplex ELISA after stimulation with CT800 (Supplementary Figures S4A,B), titanium (Supplementary Figures S4C,D) and stainless steel (Supplementary Figures S4E,F), in each case using M1 or M2 macrophages as a co-culture. For the stimulation with CT800, the analysis was conducted over a period of 10 days to determine the cytokines IL-10, TNF-α, IL-1β, IL-8 and TGF-β1. Following stimulation with metal particles, the period was extended to 14 days to measure IL-4, IL-2, IL-1β, IL-12 and TGF-β1. Significant differences were observed between M1 and M2 co-cultures following stimulation with CT800, particularly in the case of TGF-β1. The concentration of TGF-β1 in M1 macrophages exhibited a decline from day 3 to day 10 (Supplementary Figure S4A), whereas in the M2 culture, the TGF-β1 concentration in the cell supernatant demonstrated a marked increase (c3d(CT800) = 7.7 pg/mL; c10d(CT800) = 1,372 pg/mL). The concentration of IL-8 in the M1 culture had a continuous increase, with a doubling of the concentration was observed between the 3rd and 10th day. In contrast, the M2 co-culture exhibited a markedly elevated initial concentration of IL-8 on day 3, which declined by a factor of 17 by day 7 and subsequently rebounded. However, the final concentration did not reach the initial level. No discernible difference or trend was identified in the concentration of the cytokines TNF-α, IL-1β and IL-10 Following stimulation with metal particles, a discernible pattern was observed in the production of TGF-β1. Upon stimulation with titanium in co-culture with M2 macrophages, a continuous increase in TGF-β1 concentration over time was observed (Supplementary Figure S4D), whereas in the M1 culture, the maximum concentration was reached on day 7 (Supplementary Figure S4C). Following stimulation with stainless steel, the TGF-β1 concentration in both co-cultures (M1 and M2) exhibited a comparable trajectory to that observed after titanium stimulation in the M1 culture, with no discernible continuous increase between the third and fourteenth days. Furthermore, the concentration of IL-4 demonstrated disparities between M1 and M2 macrophages. The expression of IL-4 was significantly higher in the M2 culture than in the M1 culture at the outset of the experiment (on day 3 and 7) following stimulation with both titanium and stainless steel. On day 3, IL-4 expression was approximately five-fold higher in the M2 culture following titanium stimulation, and on day 7, it was approximately tenfold higher than in the M1 culture. A comparable, albeit less pronounced, discrepancy was noted following stainless steel stimulation. Subsequently, IL-4 concentrations declined following both titanium and steel stimulation. No discernible trends in cytokine concentration were observed over the course of the analysis for IL-2, IL-1β, and IL-12.

Differentiation of THP1 monocytes into M0, M1, and M2 macrophages was successfully achieved and characterized by specific markers. It is well established that macrophages exhibit distinct phenotypic and functional alterations in response to environmental stimuli, such as cytokines, pathogens, foreign particles, and tissue damage (Stout et al., 2005; Haschak et al., 2021). Consequently, it is imperative to refrain from the utilisation of donor macrophages within an in vitro systems, thereby ensuring the exclusion of any potential stimulation that may have been induced by as yet unidentified factors. The THP1 cells utilised in this study are influenced by stimulation and the various cytokines present in the microenvironment, undergoing a process of polarisation that subsequently leads to the differentiation of these cells into a variety of functional forms (Jannasch et al., 2017b) (3). Resting M0 macrophages can get polarized towards classic pro-inflammatory phenotype M1 macrophages by the stimulation with either microbial factors, like LPS, or using proinflammatory cytokines, such as IFN-γ. On the contrary, M0 can polarize towards M2 subset when exposed to cytokines such as IL-4, or IL-13 (38). The established SOP “THP1 - Differentiation” proved to be a reliable differentiation measure into the different macro-phage types (M1, M2, M0) and could be verified by immunofluorescence. The antibodies used against CD-markers served to monitor the success of differentiation. Anti-CD80 (Zhou et al., 2017) was used for M1 macrophages, anti-CD163 (Hu et al., 2017) for M2 macrophages and anti-CD68 (Wang et al., 2017) for M0 macrophages. Co-culture experiments demonstrated that the macrophage phenotype profoundly affects fibroblast behavior. M1 macrophages, typically associated with pro-inflammatory responses, induced higher levels of fibroblast activation and cytokine secretion compared to M2 macrophages, which are linked to anti-inflammatory and wound healing responses. This suggests that macrophage polarization can be a strategic target to modulate FBR and improve implant biocompatibility. We decided in favour of the cell line of THP1 cells for co-stimulation and against primary immune cells in order to exclude prior activation of the macrophages before removal from the donor organism, as this could have negative consequences on the usability of our measurements (Hörner et al., 2004; Borsini et al., 2020; Migulina et al., 2024).

A substantial body of evidence from numerous studies has demonstrated that M2 macrophages exert a profound impact on collagen production, particularly through their capacity to modulate processes such as defect healing and the three-dimensional structure of collagen (Knipper et al., 2015). This is achieved by modulating the activity of fibroblasts, which are stimulated to form the extracellular matrix (ECM) via the TGF-β1 pathway (Yang et al., 2023). The objective of our study was to further investigate the capacity of these cells to regulate collagen. Initially, we focused on procollagen formation up to day 10 following CT800 stimulation. Subsequent experiments involving metal particles shifted the focus to collagen I production, extending the analysis to a 14-day period. It is well documented that procollagen, the precursor to collagen, is produced in large quantities at the onset of tissue regeneration or wound healing. Typically, production decreases over time as healing progresses (Birk, 2001; Gelse et al., 2003). In this con-text, the 3D collagen hydrogel model provided a physiologically relevant environment in which to study fibroblast behaviour over time. The embedded fibroblasts exhibited dynamic alterations in proliferation, procollagen and collagen I expression, which mirror their pivotal function in matrix remodelling. The results of the time-course analysis demonstrated that the responses of the fibroblasts varied significantly in accordance with the stimulation by particles. This underscores the importance of temporal studies in order to gain a deeper understanding of the long-term tissue integration processes and the potential for the mitigation of fibrosis. These insights are of critical importance for the design of biomaterials that not only promote favourable tissue responses but also enhance the longevity of implants. The results demonstrated that fibroblasts produced collagen I, which was used to construct the extracellular matrix, regardless of whether they were stimulated or not, and even in the absence of external signals. The production of collagen remained largely unaltered across different macrophage cultures, whether in the absence of stimuli or following stimulation with steel. However, following stimulation with titanium, notable differences were observed. The M2 macrophage culture demonstrated a considerable increase in collagen production, reaching approximately 150% of the baseline level over time, whereas the M1 culture exhibited a significant decline, with collagen production decreasing by approximately 50%. In conclusion, it can be stated that both procollagen and collagen I are suitable for the detection of ECM synthesis by fibroblasts and for the demonstration of the connection with different types of macrophages. Given that pro-collagen is strongly expressed at the initial stage of the FBR, with collagen I forming several days later, we propose measuring the proportion of procollagen in short-term cultures and collagen I in long-term cultures (>10 days).

The regulation of FBR in the human body and in in vitro biosystems is a highly intricate and multifaceted set of rules (Anderson et al., 2008; Mariani et al., 2019). In this context, the primary objective of our experimental configuration was to ascertain consistent concentration patterns of cytokines that can be uniformly expressed, irrespective of the material composition of the individual stimulants. Concurrently, the number of pro- and anti-inflammatory cytokines investigated was limited, as the high number of different cytokines (Holdsworth and Gan, 2015) would not be suitable for standardising the measurement principle. This methodological approach facilitates a more precise and standardised analysis of immune responses, as the variability cannot be distorted by different materials or physical properties of the stimuli. Cytokines are optimal markers for the study of immune responses, as they can reflect the activation and modulation of immune cells in real time. A significant benefit of utilising ELISA for cytokine measurement as opposed to conventional methods, such as PCR or immunohistochemistry, is its capacity for non-invasive assessments (Jalalypour et al., 2016). Conventional methods necessitate the extraction of cells from their culture environment and subsequent fixation, a process that results in the cessation of culture and the determination of endpoint measurements. Additionally, there is a possibility that the RNA quantity determined by PCR does not accurately reflect the protein level (Amsen et al., 2009). Consequently, the continuous monitoring of reactions over time is hindered by PCR and IHC, as they necessitate the creation of multiple cultures. In contrast, cytokine measurements by ELISA can be performed directly from the culture medium without disturbing or terminating the cell culture. Furthermore, the samples can be analysed without complex pre-treatment (Sakamoto et al., 2018). This enables simple, continuous and dynamic monitoring of the immune response over time, facilitating a more precise assessment of alterations in cytokine concentrations and, consequently, the biological response to implants and other stimuli. This facilitates the early detection of changes in the inflammation and regeneration process. These advantages make measurement by ELISA an invaluable tool for monitoring cell responses in in vitro models, as it allows detailed and continuous observation of process dynamics without the need to interrupt the culture phase.

Both sandwich ELISA and multiplex ELISA were used for the qualitative and quantitative determination of cytokines. In principle, both methods are suitable for the accurate determination of cytokine concentrations in cell culture supernatants (Leng et al., 2008). In our experimental setup, we found that the available sample volume was quickly reached if the supernatant was collected regularly (on days 3, 7, 10, 14). In our work, we have aimed to identify a cytokine pattern that can give an indication of the tissue compatibility of a foreign material. For this reason, we believe that the multiplex ELISA method will be more practical in the future, as it requires less sample material for the determination of different cytokines and they can be determined simultaneously. In conclusion, the higher time and material efficiency of the multiplex method compared to the sandwich ELISA is an important factor for the future evaluation of clinical feasibility.

The cytokine profile measurements indicate that IL-6 and TGF-β1 are instrumental in the expression of the tissue response (Meléndez et al., 2010; Gingery et al., 2014; Sheikh et al., 2015). It is evident that the unstimulated culture demonstrates a lack of IL-6 expression, indicating that the cells require an external stimulus (FBR) to initiate the production of IL-6. In order to establish a connection between IL-6 formation and foreign body stimulation, a variety of materials were employed. The elevation in IL-6 levels over the 14-day observation period was evident across all materials tested. This demonstrated that IL-6 is an appropriate marker for examining whether a tissue model has been stimulated with a foreign body or whether a non-stimulated culture is present. As has been previously documented in the scientific literature, interleukin (IL-6) plays a role not only in the inflammatory response of the immune system, but also in the modulation of anti-inflammatory processes within the body (Fu et al., 2017; Borsini et al., 2020; Yan et al., 2023). This could explain why IL-6 is produced in greater quantities after stimulation with titanium than with steel, given that titanium is more biocompatible and therefore presumably elicits a stronger anti-inflammatory response (Hanawa, 2022). This theory is supported by the observation that the concentration of IL-6 is higher in the co-culture with the anti-inflammatory M2 macrophages. Furthermore, the increasing concentration of TGF-β1 over time (day 3 to day 14) following stimulation with titanium particles lends additional support to this hypothesis. Recent findings have demonstrated that IL-6 stimulates the production of TGF-β1 (Fu et al., 2017). Given that TGF-β1 is crucial for the promotion of tissue proliferation, the elevated concentration of this cytokine in the titanium-stimulated culture relative to the steel culture suggests enhanced tissue adaptation (Wahl et al., 1988). The concentration of IL-4 is higher in M2 macrophages than in M1 macrophages at the outset of the culture period. This is due to the fact that M2 macrophages are activated in an anti-inflammatory environment and produce IL-4, which is an important signaling substance that promotes repair processes and tissue healing (Mosser and Edwards, 2008). These cells initially demonstrate an augmented response to foreign body stimulation, as they utilise elevated IL-4 production during the initial phase to modulate inflammation and tissue repair (Dunster, 2016). However, as the culture progresses, there is a reduction in IL-4 levels as macrophages adapt to microenvironmental stimulation. Furthermore, the production of pro-inflammatory mediators decreases as tissue repair progresses. Therefore, the greatest degree of clarity was observed in the measured values of IL-4, IL-6 and TGF-β1. Conversely, no discernible differences or similarities were identified in the patterns of the other cytokines with regard to concentration level or tendency.

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is liberated during the early stages of inflammation, stimulated the recruitment of immune cells and the activation of inflammatory processes (Parameswaran and Patial, 2010). Macrophages that have been stimulated with particles favor to produce pro-inflammatory cytokines following interaction (Zhang and An, 2007). IL-6 has categorised as a classic pro-inflammatory cytokine for long time. However, newer studies have demonstrated that the concentration of IL-6 has an influence on the pro-inflammatory or anti-inflammatory function (Borsini et al., 2020). It has been demonstrated that an elevated concentration of IL-6 is conducive to an anti-inflammatory response and can inhibits the activity of M1 (Fu et al., 2017). Elevated levels of pro-inflammatory cytokines (IL-6, TNF-α) were observed in cultures with M1 macrophages, while M2 macrophages promoted anti-inflammatory cytokine production (IL-10, TGF-β1). These cytokine profiles offer predictive markers for the type of tissue response (inflammatory vs. regenerative) elicited by different implant materials, aiding in the design of biomaterials that promote favorable outcomes.