Abstract The use of zirconia has significantly enhanced the aesthetic outcomes of implant restorations. However, peri-implantitis remains a challenge to long-term functionality of implants. Unlike the perpendicularly arranged collagen fibers in periodontal tissue, those in peri-implant tissue lie parallel to the abutment surface and contain fewer fibroblasts, making them more prone to inflammation. Studies have shown that microgroove structures on implant abutments could improve surrounding soft tissue structure. However, creating precise microgrooves on zirconia without compromising its mechanical integrity is technically challenging. In this study, we applied inkjet printing, an additive manufacturing technique, to create stable silk fibroin microgroove (SFMG) coatings of various dimensions on zirconia substrates. SFMG significantly improved the hydrophilicity of zirconia and showed good physical and chemical stability. The SFMG with 90 μm interval and 10 μm depth was optimal in promoting the proliferation, alignment, and extracellular matrix production of human gingival fibroblasts (HGFs). Moreover, the in vitro results revealed that SFMG stimulated key glycolytic enzyme gene expression in HGFs via the PI3K-AKT-mTOR pathway. Additionally, the in vivo results of histological staining of peri-abutments soft tissue showed that SFMG promoted the vertical alignment of collagen fibers relative to the abutment surface, improving connective tissue sealing around the zirconia abutment. Our results indicated that SFMG on zirconia can enhance HGF proliferation, migration and collagen synthesis by regulating glycolysis though PI3K-AKT-mTor pathway, thereby improving connective tissue sealing. Keywords: Connective tissue sealing, Zirconia abutments, Microgrooves, Inkjet printing, Glycolysis Graphical abstract [37]Image 1 [38]Open in a new tab Highlights * • Inkjet printing technology could accurately and effectively constructSFMG structures with stable properties on zirconia. * • SFMG exhibited performance in promoting soft tissue healing and enhancing connective tissue sealing ability. * • SFMG affected the biological behavior of HGFs by activating the PI3K-AKT-mTOR pathway to alter glucose metabolism. 1. Introduction Implant-supported restorations are widely favored in clinical settings for their benefits, including the preservation of adjacent teeth and enhanced masticatory efficiency [[39]1]. Nevertheless, implant failure rates caused by peri-implant inflammation was as high as 20 % [[40][2], [41][3], [42][4]], without the influence of implant or abutments materials [[43]5]. The genesis of peri-implantitis, similar to periodontitis, is primarily due to bacterial biofilm formation and subsequent bacterial invasion [[44]6]. However, peri-implantitis progresses more swiftly and causes more extensive tissue damage due to the differences in tissue architecture between natural periodontal tissues and peri-implant tissues [[45]7]. Specifically, peri-implant connective tissue lacks the perforating fibers in natural dental connective tissue, weakening its resistance to the apical migration of epithelial cells, a precursor to peri-implantitis [[46]8]. Additionally, the connective tissue near abutments contains lower concentration of type I and type III collagen, an elevated level of type V collagen, and reduced fibroblasts, which leads to a marked decrease in tissue repair capabilities after inflammation [[47]9]. Given these challenges, it is crucial to develop strategies that bolster the structure and composition of peri-implant connective tissue. Improving the integration and sealing properties of soft tissues around implant abutments and restorations is essential for mitigating the risks associated with peri-implant inflammation. In recent years, the use of zirconia abutments in clinical practice had significantly increased. Zirconia has mechanical properties comparable to titanium, fully supporting the masticatory function of restorations in the mouth [[48]10]; Compared to traditional titanium abutments, zirconia abutments have superior aesthetic performance, significantly reducing the risk of gingival translucency in anterior dental implants [[49]11]; Zirconia abutments are also known for their bacterial resistance, with a biologically inert surface that is less prone to bacterial attachment compared to titanium, reducing the risk of infection in the surrounding tissues [[50]12]. However, the biologically inert surface of zirconia also affects its integration with the surrounding soft tissues. Studies have found that compared to titanium abutments, the sealing ability of connective tissue around zirconia abutments was relatively weaker, which might adversely affect the long-term stability of both soft and hard tissues around the implant [[51]13,[52]14]. The extracellular substrate topography and the geometric patterns of the substrate surfaces influence cellular functions, known as contact guidance [[53]15,[54]16]. Nanotube and microgroove structures on zirconia and titanium surfaces have been found to promote the proliferation of gingival fibroblasts [[55]17]. Compared to the surfaces treated with non-patterned techniques such as acid etching and sandblasting, the surfaces with groove structure were more conducive to enhancing the bioactivity of the abutment [[56]18]. Microgroove structures on titanium abutment surfaces have been shown to encourage the development of vertically oriented fibers [[57]19] and to deter the apical migration of epithelial cells along the abutment [[58]20]. However, existing methods for creating microgrooves on abutment surfaces are predominantly subtractive, significantly reducing the mechanical integrity of zirconia abutments [[59]21], thus constraining the use of grooved structures on zirconia and related research. Moreover, the effectiveness of contact guidance is contingent upon the size of the microgrooves, and there is currently no agreed-upon optimal dimension for maximizing connective tissue integration [[60]17]. Inkjet printing emerged as a versatile patterning method that employs voltage-driven mechanisms to deposit coating materials without compromising the structural and mechanical integrity of the underlying material [[61]22]. This technology facilitated the exact and programmable creation of patterned surfaces, thereby minimizing material waste [[62]23]. The selection of bio-ink is vital for ensuring the biological effectiveness of the coated surface, with recent studies exploring the use of bioactive substances like platelet lysates and fibrinogen to enhance tissue compatibility of implant [[63][24], [64][25], [65][26], [66][27], [67][28]]. Despite their benefits, these materials often presented challenges related to immunogenicity and required extensive separation and purification processes, elevating costs and increasing the risk of infection [[68]29]. Silk fibroin (SF) is a natural bioprotein derived from silkworm silk, composed of a heavy chain of 350 kDa and a light chain of 25 kDa, connected by disulfide bonds [[69]30]. Due to the industrial scalability, excellent biocompatibility, and robust mechanical attributes, SF has been successfully applied in the development of biomaterials for bones [[70]31] and blood vessels [[71]29] regeneration. Besides, SF solution could promote the migration and proliferation of fibroblasts (key effector cells in soft tissue healing) [[72]32] and the healing of skin wounds [[73]30]. Furthermore, the rheological characteristics of SF solutions make them well-suited for use as bio-inks [[74]33]. Recently, cellular metabolism was found may be a key pathway through which surface morphology regulated cell behavior [[75]34,[76]35]. Glyco-metabolism is the primary source of cellular energy and one of the most likely metabolic pathways to impact cell behavior [[77]36]. Ball et al. [[78]37] found that changes in titanium roughness could affect osteoblast glucose metabolism, thereby influencing ALP expression. Different substrate hardness could alter the glycolysis process of fibroblasts, thereby regulating their proliferation and synthesis of collagen fibers [[79]38]. These findings collectively suggested that cellular glyco-metabolism plays a crucial role in the regulation of cell behavior in response to surface morphology. This study aimed to develop silk fibroin microgroove (SFMG) coatings on zirconia substrates utilizing inkjet printing technology, assess the efficacy of SFMG coatings in enhancing connective tissue integration, and investigate the underlying mechanisms. The outcomes of this investigation are expected to contribute new insights and theoretical underpinnings for the advancement of ceramic material surfaces and soft tissue integration with zirconia abutments. 2. Materials and methods 2.1. Preparation of SFMG coatings on zirconia Commercial zirconia discs (33 mm, 15 mm and 3.5 mm in diameter, 0.8 mm in thickness) (Dachuan Ceramics, China) served as the substrates for our in vitro experiments. For the in vivo studies, Zirconia abutment (cubes with 1.5 mm edges, Dachuan Ceramics, China) and titanium implants (Gensray CNC, China) (Supplemental materials 1) were employed. These components were securely bonded using Panavia adhesive (Kuraray, Japan). The zirconia materials used in this study were all yttria-stabilized tetragonal zirconia polycrystals, and the titanium metal was commercial-grade five titanium. Anhydrous sodium carbonate (4.24 g) was dissolved in 2 L ultrapure water at 100 °C to prepare a 0.02 M sodium carbonate solution. Chopped mulberry silkworm cocoons (5 g, Northwest Silkworm Base, China) were added into the boiled sodium carbonate solution for 120 min with stirring to be degummed. After degumming, the solution was removed through a filter, to get the degummed SF, rinsed with ultrapure water 3 times at room temperature. At this stage, the SF appeared as a fibrous clump ([80]Fig. 1a). The agglomerate SF was spread on a clean piece of tin foil and dried in a drying oven at 37 °C overnight ([81]Fig. 1b). The dried SF fibers were added into lithium bromide (LiBr, Xiya Reagent, China) solution (9.3 M) (the volume of LiBr solution required is four times the mass of SF). The solution contained SF fibers was heated in a water bath at 60 °C for 4 h until the SF fibers was completely dissolved. Now the solution became an amber-colored transparent liquid ([82]Fig. 1c). Dialyze the SF solution with a dialysis membrane with a molecular weight cutoff of 14 kDa and ultrapure water, dialysis lasts for 2 days, changing the water 8 times, every 6 h ([83]Fig. 1d). The dialyzed solution was then centrifuged at 4 °C, 8000 rpm for 20 min, the supernatant was centrifuged again under the same conditions to thoroughly remove impurities. The SF solution was stored at 4 °C and used within one month ([84]Fig. 1e). Fig. 1. [85]Fig. 1 [86]Open in a new tab Process diagram for preparing SFMG. SF solution (4 %, w/v) was filled into the printing nozzle (5 μm in diameter). Utilizing a super inkjet printer (SIJ-S150, Japan), SFMG parallel lines with certain intervals were constructed on the surface of zirconia according to the design pattern. These intervals, referred to as 'widths' in our study, represented the spacing between SF ridges rather than the widths of the SF ridges themselves. The widths of the microgrooves were set at 30 μm, 60 μm, and 90 μm, while the depths were set at 5 μm or 10 μm (each group is represented by width/depth). These intervals were controlled by adjusting the printing nozzle intervals. Considering that the SF ridges themselves have a certain width (approximately 30 μm), we set the printing nozzle interval to the required interval plus the width of the SF ridges to construct grooves with specific intervals. For instance, to construct grooves with a spacing of 90 μm, we need to set the printing interval to 120 μm, which includes the 90 μm spacing plus the 30 μm width of the SF ridges. The depth of the coating was regulated by the total number of printing passes: 50 cycles for achieving a 5 μm depth and 100 cycles for a 10 μm depth. The printing was executed under a voltage of 600 V, with a speed set at 20 mm/s and an acceleration of 10 mm/s^2. Plain zirconia and SF-coated discs without grooves (thickness: 5 μm) served as the control groups, labeled as Control and SF, respectively ([87]Fig. 1f). After printing, the samples were submerged in anhydrous ethanol for 1 h and left to dry in the drying oven at 37 °C overnight ([88]Fig. 1g). 2.2. Specimen characterization The rheological properties of the SF solution were evaluated using a rotational rheometer (3790340, HAAKE, Germany). The SF solution's surface tension and dynamic contact angle with the zirconia surface were determined using an optical surface tension meter (Theta Flow, Attention, Sweden). The size and zeta potential of solutes in the SF solution were measured using the nanoparticle tracking analysis (NTA) (Particle Metrix, Zeta View, Germany). A camera (D750, Canon, Japan) was used to capture the macroscopic images of the SFMG on the zirconia surface. The morphologies of the fabricated SFMG coatings were examined using a polarized light microscope (PLM, SANYO, Japan) and a confocal laser scanning microscope (CLSM, LSM700, ZEISS, Germany). To further assess the thickness of the coating, we analyzed the cross-section (perpendicular to the groove direction) of the coating with a scanning electron microscopy (SEM, Phenom-World, the Netherlands). To assess the stability of the coatings, the samples were submerged in artificial saliva for 28 days, mirroring the time required for soft tissue integration around dental abutments [[89]39]. Changes in the coatings' morphology and chemical composition before and after immersion were analyzed using PLM and fourier-transform infrared spectroscopy (FTIR, Nicolet iS20, Thermo, America). 2.3. In vitro experiment 2.3.1. Cell culture and treatment with inhibitors Primary HGFs were isolated using an enzymatic digestion method from gingival connective tissues harvested from patients undergoing third molar extractions (Ethical number: KQEC2019-37). HGFs were then cultured in Dulbecco's modified eagle's medium (DMEM, Gibco, America), supplemented with 10 % (v/v) fetal bovine serum (FBS, Gibco, America) and 1 % penicillin-streptomycin (P/S, Beyotime, China). The cell cultures were maintained in T25 culture flasks at 37 °C in a humidified incubator with 5 % CO[2]. Subculturing was performed once the cell confluence reached approximately 80 %–90 % within the flasks. For the experiments, cells between passages 3 to 6 were employed. To suppress glycolytic activity in HGFs, the cells were treated with a glycolysis inhibitor, 2-Deoxy-d-glucose (2-DG, HY-13996, medchemexpress (MCE), 15 mM/mL), for 24 h. Cells that did not receive the 2-DG treatment served as the control group. Furthermore, HGFs were exposed to 2-Morpholino-8-benzylchromone (LY294002, HY-10108, MCE, 10 μM/mL), a PI3K inhibitor, for 24 h to block the PI3K-AKT pathway. Cells untreated with LY294002 were utilized as the control for the current experiments. 2.3.2. The cell cytoskeleton of HGFs cultured on SFMG coatings HGFs cultured on different zirconia discs within 24-well cell culture plates for 24 h were gently washed three times with phosphate-buffered saline (PBS, Beyotime, China) and subsequently fixed with 4 % paraformaldehyde (PFA, Beyotime, China) for 20 min. Following fixation, the cells were permeabilized using a 0.2 % Triton™ X-100 in PBS solution (Biosharp, China) for 5 min. After permeabilization, the cells were stained with fluorescein isothiocyanate (FITC)-labeled phalloidin (1:50, Beyotime, China) to visualize the actin filaments and counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for nuclear visualization. Representative fluorescence images were captured utilizing a CLSM (LSM980, ZEISS, Germany). 2.3.3. Cell proliferation assay HGFs were seeded onto zirconia samples within 96-well cell culture plates. At a density of 1 × 10^4 cells per well and were cultured for 24 h. Following this, the proliferation of HGFs was assessed using an EdU assay kit (KGA9602-100, KeyGEN Biotech, China). The cells were incubated with a 10 μM EdU solution under constant temperature conditions, fixed with 4 % PFA, and neutralized with glycine (2 mg/mL). Subsequently, cells were permeabilized using a 0.2 % Triton™ X-100 in PBS solution for 5 min. The Click-iT reaction was applied to label EdU-positive cells, while DAPI staining was utilized to label all cells. The proportion of EdU-positive cells relative to the total cell count was determined by capturing images with a CLSM (Axio observer Z1, ZEISS, Germany). CCK-8 (CK04, Dojindo, Japan) was utilized to evaluate the proliferation of HGFs cultured on various zirconia discs. The cells were plated at a density of 5 × 10^3 cells per well on zirconia disks in 96-well cell culture plates. At 1, 3, and 7 days post-seeding, the cells were gently washed three times with PBS. Subsequently, each well received 100 μL of fresh DMEM (without FBS) containing 10 μL of CCK-8 reagent. The cells were then incubated at 37 °C for an additional 2 h. Afterwards, 100 μL of the medium from each well was transferred to a new plate to measure the optical density at 450 nm using a Microplate Reader (800 TS, BioTek, America). The group exhibiting the most significant effect in promoting cell proliferation was selected for further experimentation and denoted as the SFMG group. 2.3.4. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis HGFs were cultured on zirconia discs placed in 6-well cell culture plates, with an initial seeding density of 2 × 10^5 cells per well. For the analysis of collagen type I (COL1A1) and collagen type III (COL3A1) mRNA expression, cells were incubated for 7 days. In contrast, for the assessment of other mRNA types, a 24-h culture period was applied. This study focused on evaluating the cellular glycolytic capacity by measuring mRNA levels of key enzymes: hexokinase 2 (HK2), phosphoglycerate kinase 1 (PGK1), fructose-2,6-bisphosphatase 3 (PFKFB3), pyruvate kinase M2 (PKM2), and lactate dehydrogenase A (LDHA). Additionally, the cellular oxidative phosphorylation capacity was assessed by examining the mRNA expression of pyruvate dehydrogenase E1 component subunit alpha (PDHA1), dihydrolipoamide dehydrogenase (DLD), dihydrolipoamide S-acetyltransferase (DLAT), isocitrate dehydrogenase 1 (IDH1), and citrate synthase (CS). Following the specified culture periods, total RNA was extracted using an RNA rapid extraction kit (RN001, Yishan, China). Each well was treated with 500 μL of lysis buffer, followed by complete lysis and subsequent neutralization with an equal volume of anhydrous ethanol. The mixtures were then subjected to high-speed centrifugation to eliminate impurities. The purified RNA was resuspended in an elution buffer, and its concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, America). Complementary DNA (cDNA) synthesis was performed using the PrimeScript™ RT Reagent Kit (RR036A, TaKaRa, Japan). qRT-PCR was conducted using Hieff® qPCR SYBR Green Master Mix (No Rox) (Yeasen Biotechnology, China), with the prepared cDNA serving as the template. The cycle threshold (CT) values obtained were analyzed, employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control for normalization purposes. Primer sequences for the target genes are listed in [90]Table 1. Table 1. The primer sequences for the target genes. Genes Primers Sequences FN Forward Reverse CGGTGGCTGTCAGTCAAAG AAACCTCGGCTTCCTCCATAA COL1A1 Forward Reverse GAGGGCCAAGACGAAGACATC CAGATCACGTCATCGCACAAC COL3A1 Forward Reverse GGAGCTGGCTACTTCTCGC GGGAACATCCTCCTTCAACAG PDHA1 Forward Reverse TGGTAGCATCCCGTAATTTTGC ATTCGGCGTACAGTCTGCATC DLAT Forward Reverse CGGAACTCCACGAGTGACC CCCCGCCATACCCTGTAGT DLD Forward Reverse CTCATGGCCTACAGGGACTTT GCATGTTCCACCAAGTGTTTCAT CS Forward Reverse TGCTTCCTCCACGAATTTGAAA CCACCATACATCATGTCCACAG IDH1 Forward Reverse TGTGGTAGAGATGCAAGGAGA TTGGTGACTTGGTCGTTGGTG PI3K Forward Reverse GGAGCTGGCTACTTCTCGC GGGAACATCCTCCTTCAACAG HK2 Forward Reverse GAGCCACCACTCACCCTACT CCAGGCATTCGGCAATGTG PKM2 Forward Reverse ATGTCGAAGCCCCATAGTGAA TGGGTGGTGAATCAATGTCCA PGK1 Forward Reverse TGGACGTTAAAGGGAAGCGG GCTCATAAGGACTACCGACTTGG LDHA Forward Reverse ATGGCAACTCTAAAGGATCAGC CCAACCCCAACAACTGTAATCT PFKFB3 Forward Reverse TTGGCGTCCCCACAAAAGT AGTTGTAGGAGCTGTACTGCTT GAPDH Forward Reverse TTGCAGTGGCAAAGTGGAGA GATGGGCTTCCCGTTGATGA [91]Open in a new tab 2.3.5. Western blot analysis HGFs were seeded onto zirconia discs placed in 6-well cell culture plates at a density of 2 × 10^5 cells per well. For the analysis of fibronectin (FN) and proteins related to the PI3K-AKT pathway, cells were cultured for 24 h. For collagen-related proteins, the culture period was extended to 7 days. The cells were lysed using RIPA buffer containing 1 % protease and phosphatase inhibitors, followed by centrifugation at 15,000 g for 20 min at 4 °C. Protein concentrations were determined using the Bicinchoninic Acid (BCA) Protein Assay Kit (XL357479, Thermo Scientific, America). The cell lysates were then mixed with sodium dodecyl sulfate (SDS) sample loading buffer (#20315ES05; Yeasen Biotechnology, China) and heated for 10 min at 95 °C to denature the proteins. These denatured proteins were loaded into pre-made gels (M00657, GenScript, America) and subjected to electrophoresis, followed by transfer to PVDF membranes. The membranes were blocked with 5 % bovine serum albumin (BSA, Beyotime, China) for 1 h at room temperature. After three washes with PBS, the membranes were incubated overnight at 4 °C with primary antibodies targeting FN (ab268020, Abcam, UK), COL1A1 (BA0325, Boster, China), COL3A1 (68320-1-Ig, Proteintech, China), GAPDH (60004-1-Ig, Proteintech, China), PI3K (4257T, Cell Signaling Technology (CST), America), phospho-AKT (P-AKT, 4060T, CST, America), and phospho-mTOR (P-mTOR, 66888-1-Ig, Proteintech, China). Following another set of three PBS washes, the membranes were incubated with respective secondary antibodies (Jackson, America) at room temperature for 1 h. Protein bands were visualized using an electrochemiluminescence plus reagent (Bio-Rad, ChemiDoc, America), and images were captured for analysis. For the assessment of phosphorylated proteins, after the initial exposure, membranes were washed three times with Stripping Buffer (Beyotime, China), re-sealed, and then re-incubated with primary antibodies targeting AKT (4691S, CST, America) and mTOR (66888-1-Ig, Proteintech, China), followed by incubation with respective secondary antibodies and re-exposure for imaging. 2.3.6. Cell migration Transwell assay was utilized to assess the impact of SFMG on the migration of HGFs. In short, HGFs were plated in the upper chambers of a 24-well Transwell plate (Corning, with a pore size of 8 μm) at a density of 2 × 10^4 cells per well. Subsequently, 600 μL of DMEM, containing supernatants collected from various groups in which HGFs had been previously cultured for 24 h, was introduced into the lower chambers. After a culture period of 24 h, non-migrated cells on the upper surfaces of the upper chamber filters were gently removed using a cotton swab. The filters' lower surfaces were then fixed with 4 % PFA and stained with 0.1 % crystal violet for 10 min. Migration was quantified by examining five randomly chosen fields per filter under a microscope (CKX53, OLYMPUS, Japan). 2.3.7. RNA sequencing and data analysis HGFs were cultured on zirconia discs in 6-well cell culture plates at a density of 2 × 10^5 cells per well for 24 h. The cells were lysed using Trizol reagent (Beyotime, China), and the resultant lysates were submitted to Nanjing Persomics Genetech Company Limited for mRNA isolation, construction of Illumina sequencing libraries, synthesis of double-stranded cDNA, bioinformatics analysis, and high-throughput sequencing. Each experimental group was represented by three biological replicates. Differentially expressed genes (DEGs) were defined based on a log2-fold change (FC) > |1| and a p-value <0.01. To understand the biological implications of these DEGs, enrichment analysis was conducted utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify significant signaling pathways impacted by the DEGs (threshold: p-value <0.05). 2.4. In vivo experiment 2.4.1. Implant surgical and histological examination Animal experiments were conducted following approval from the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University (approval no. SYSU-IACUC-2022-001406) and in accordance with the ethical standards established by the Animal Ethical and Welfare Committee of Sun Yat-sen University. Twenty-four male Wistar rats (six weeks old and weighing between 120 and 130 g) were randomly allocated into three groups and maintained according to the animal care guidelines of Sun Yat-sen University. Anesthesia was induced using 5 % pentobarbital sodium (0.3 mL per 100 g body weight). The experimental procedure began with the extraction of the bilateral upper first molars, which was accomplished using minimally invasive extraction forceps and vascular clamps to minimize damage to the soft and hard tissues. 4 weeks after the extraction, once the hard and soft tissues at the extraction sites had healed, the implant surgery was performed as the procedures described before [[92]40]. A full thickness mucoperiosteal flap was carefully elevated to expose the underlying bone at the implant site. Subsequently, custom-made twist drills (diameters 1 mm, 1.5 mm, and 2.0 mm) were used for gradual drilling, during which 50 mL syringes continuously dripped physiological saline to locally cool the area. Finally, a screwdriver was used to place the micro-implants with a zirconia base into these prepared sites. After an additional 4-week healing period, euthanasia was performed on the rats through overdose anesthesia, and the small implants along with the surrounding soft tissues were simultaneously excised. The collected samples were immediately fixed in 4 % PFA (Beyotime, China), then immersed in a decalcifying solution composed of 5 % ethylenediaminetetraacetic acid (EDTA, Beyotime, China) and 4 % sucrose (diluted in 0.01 M PBS (Beyotime, China)), and stored at 4 °C for 4 days for decalcification. The procedure of obtaining soft tissue samples was performed as described before [[93]40]. After decalcification, the buccal gingival tissues were peeled off from the surface of the implants. Because the peeling action may damage the interface between the gingiva and the implants, this part of the tissue was not used. The remaining tissues were gently stirred at 4 °C in the same decalcifying solution for one day, until the implants and soft tissues naturally separated, then the mandibular side soft tissues were taken for further analysis. After the samples were embedded in paraffin, serial sections with a thickness of 5 μm were prepared using a microtome (RM2255, Leica Biosystems, Germany), followed by Masson's trichrome staining. The stained sections were then digitally imaged using a slide scanning system (Aperio AT2, Leica Biosystems, Germany) for detailed examination and analysis. 2.4.2. Immunofluorescence staining The sections were subjected to antigen retrieval by boiling in antigen repair solution (G1203, Servicebio, China) for 15 min. Following this, they were rinsed with PBS and blocked with bovine serum albumin (BSA, [94]GC305010, Servicebio, China) for 20 min to prevent non-specific binding. The sections were then incubated with a primary antibody against FN ([95]GB114491, Servicebio, China) overnight at 4 °C. After washing with PBS, they were incubated with a secondary antibody (111-585003, Jackson, America) at 37 °C for 1.5 h. Subsequent to another PBS rinse, the cell nuclei were counterstained with DAPI (1:100, Beyotime, China). Following the staining process, the sections were dehydrated, cleared with xylene (Sigma-Aldrich, America) for 1 min, air-dried, and finally mounted using neutral resin. Images of the sections were acquired using a CLSM (FV3000, OLYMPUS, Japan) for detailed analysis. 2.4.3. HRP penetration experiment Four weeks post-implantation, rats underwent anesthesia following the protocol outlined in section [96]2.3.1. Cotton threads saturated with a 50 mg/mL horseradish peroxidase (HRP)-infused saline solution were gently positioned around the gingival margins of the implants, ensuring no mechanical stress was applied. An additional saline solution containing HRP was applied to the cotton every 10 min over 1 h. Subsequent to the treatment, the samples were harvested, embedded, and sectioned as detailed in section [97]2.3.1. The obtained sections were then processed using a DAB staining kit (LM80010C, LMAI Bio, China). The extent of HRP penetration within the tissue sections was assessed using a digital slide scanning system (Aperio AT2, Leica, Germany). 2.5. Statistical analysis All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA). All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD). One-way ANOVA was used to assess differences between group means, with Levene's test initially testing for homogeneity of variances. In cases where Levene's test indicated variance inequality, the Brown-Forsythe test was employed. When variances were equal, Tukey's Honestly Significant Difference (HSD) test was used for post hoc comparisons; if variances were unequal, the Games-Howell test was applied. Statistical significance was set at p < 0.05. 3. Results 3.1. Printing performance of SF solution The SF solution demonstrated shear-thinning properties, with its viscosity measured at 3 mPa s in the second Newtonian plateau, indicating its suitability for printing ([98]Fig. 2A). Additionally, the surface tension of the SF solution was recorded at 44.41 ± 0.05 mN/m ([99]Fig. 2B), which falls within the optimal range for inkjet printing. Dynamic contact angle assessments between the SF solution and the zirconia substrate revealed a moderate level of ink-substrate interaction, showcasing the potential for adequate adhesion and spreading of the SF solution on the zirconia surface ([100]Fig. 2C a, b). The NTA results indicated that the particle size of solutes in the SF solution was 128.3 ± 58.2 nm, and the zeta potential was −35.41 ± 1.44 mV, which were consistent with the basic characteristics of proteins and meet the requirements for piezoelectric inkjet printing ([101]Fig. 2D and E). Fig. 2. [102]Fig. 2 [103]Open in a new tab Physicochemical characterization of SF solution. (A) Dynamic viscosity of SF solution. (B) Surface tension of SF solution. (C) Dynamic contact angle between SF solution and zirconia surface. (D) The presented particle distributions of solute molecule in SF solution by NTA. (E) The presented zeta potential distributions of solute molecule in SF solution by NTA. 3.2. Morphology of SFMG-coated zirconia surface Images of camera and PLM confirmed the successful formation of consistent and uniform SFMG coatings on the zirconia surfaces using an inkjet printing method ([104]Fig. 3A–C). Furthermore, three-dimensional reconstructions obtained from CLSM and the images of SEM displayed that the coatings achieved depths of 5 and 10 μm and widths of 30, 60, and 90 μm, respectively ([105]Fig. 3B–D). Fig. 3. [106]Fig. 3 [107]Open in a new tab Morphology characterization of SFMG coatings on zirconia. (A) Macroscopic (Scale bar = 3 mm) and locally magnified images of SFMG (Scale bar = 200 μm) on zirconia surface (B) Cross-sectional SEM Image of SFMG (Scale bar = 10 μm). (C) The morphology of zirconia surface without SF (control group) and with SF uniformly sprayed (SF group) and SFMG coatings of different widths (PLM, Scale bar = 50 μm). (D) Three-dimensional reconstruction of SFMG coatings with different combinations of widths and depths (CLSM, 100 × ). FTIR and PLM analyses were performed to assess the degradation of the SFMG coatings. FTIR spectra maintained the characteristic peaks of SF within the coatings (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O at 1620 cm^−1, N–H at 1515 cm^−1, and C–N at 1235 cm^−1), but the diminished peak intensities suggested partial degradation of the SFMG ([108]Fig. 4A). However, the PLM images did not show a significant difference in the morphology of the SFMG coatings ([109]Fig. 4B), suggesting that the observed chemical changes did not substantially alter the overall structural integrity visible under PLM. Fig. 4. [110]Fig. 4 [111]Open in a new tab Stability of SFMG coatings on zirconia surface. (A) FTIR spectrum of SFMG coatings before and after immersion in artificial saliva. The y-axis is only applicable to the lowest curve, and for clarity, the remaining curve moved vertically. The dashed line represents the main peak. (B) Surface morphology of SFMG coatings before and after immersion in artificial saliva (PLM, Scale bar = 100 μm). 3.3. SFMG guided the arrangement of HGFs CLSM results revealed that HGFs neatly aligned along the microgrooves, with their long axis parallel to the direction of the grooves in the 60/5, 60/10, 90/5, and 90/10 groups. Conversely, HGFs in the control and SF groups displayed a random orientation. The 30/5 and 30/10 groups showed a mixture of cells aligned with the grooves and cells oriented randomly. Additionally, it was observed that cells covered the entire surface area of the specimens in the groups where the groove width was 30 μm. In contrast, for grooves 60 μm and 90 μm in width, cells exhibited a spindle-like morphology and predominantly populated the bottom of the grooves ([112]Fig. 5). Fig. 5. [113]Fig. 5 [114]Open in a new tab The morphology of HGFs cultured on the zirconia surfaces with and without SFMG. The groups were marked as width/depth. Zirconia surface without SF was marked as the Control (Con) group, and Zirconia surface with SF uniformly sprayed coating was marked as the SF group. Representative fluorescence images of the morphology of HGFs cultured on the zirconia surfaces for 24 h. F-actin stained with FITC (green), and cell nuclei stained with DAPI (blue) (Scale bar = 50 μm). (For interpretation of the references to color in this figure legend, the