Amorphous titanium oxide (aTiO2) thin films biofunctionalized with CAP-p15 induce mineralized-like differentiation of human oral mucosal stem cells (hOMSCs)

Insufficient osseointegration of titanium-based implants is a factor conditioning their long-term success. Therefore, different surface modifications, such as multifunctional oxide coatings, calcium phosphates, and the addition of molecules such as peptides, have been developed to improve the bioactivity of titanium-based biomaterials. In this work, we investigate the behavior of human oral mucosal stem cells (hOMSCs) cultured on amorphous titanium oxide (aTiO2), surfaces designed to simulate titanium (Ti) surfaces, biofunctionalized with a novel sequence derived from cementum attachment protein (CAP-p15), exploring its impact on guiding hOMSCs towards an osteogenic phenotype. We carried out cell attachment and viability assays. Next, hOMSCs differentiation was assessed by red alizarin stain, ALP activity, and western blot analysis by evaluating the expression of RUNX2, BSP, BMP2, and OCN at the protein level. Our results showed that functionalized surfaces with CAP-p15 (1 µg ml−1) displayed a synergistic effect increasing cell proliferation and cell attachment, ALP activity, and expression of osteogenic-related markers. These data demonstrate that CAP-p15 and its interaction with aTiO2 surfaces promote osteoblastic differentiation and enhanced mineralization of hOMSCs when compared to pristine samples. Therefore, CAP-p15 shows the potential to be used as a therapeutical molecule capable of inducing mineralized tissue regeneration onto titanium-based implants.


Introduction
One of the biggest challenges in the biomedical area is the development of implants that exert a robust osseointegration process.Titanium-based biomaterials have gained importance, particularly within the orthopedic and dental domains, owing to their biocompatibility, mechanical properties, and remarkable corrosion resistance, attributed mainly to the protective titanium oxide (TiO 2 ) layer naturally formed onto their surface upon exposure to the ambient [1].
The surface characteristics of biomaterials profoundly influence the intricate interactions occurring at the interface between the implant and the adjacent tissues.Thus, diverse methodologies to tailor the surface properties of titanium-based biomaterials have been explored.Surface properties modifications are mainly aimed at tailoring the chemical composition [2,3], the surface topography [4][5][6], and the roughness [7].All these properties play pivotal roles in shaping cellular behavior by influencing cell attachment, proliferation, and differentiation.
A novel strategy for enhancing osseointegration involves immobilizing organic molecules on the surface of biomaterials to provide the cells with the appropriate signals for attachment and differentiation.These chemical surface modifications immobilize proteins from the extracellular matrix (ECM) on the biomaterial surface to influence cell migration, attachment, proliferation, and differentiation processes.Among these proteins, we can find proteins such as vitronectin and fibronectin [8], biomineralization-related proteins such as bone morphogenetic proteins (BMP 2, 7) [2], Bone Sialoprotein [9], Elastin [10], Alkaline Phosphatase [11], and antimicrobial proteins like Histatin [12].Although these proteins demonstrate improvement in osseointegration while offering versatility due to their high affinity for specific receptors and abundance of binding sites, their use still presents limitations.Natural proteins are difficult to isolate or produce, and their immobilization onto different biomaterial surfaces might alter their conformation, triggering the risk of undesirable immune reactions.The use of small peptide sequences from these proteins is a promising alternative since peptides are more chemically stable to environmental conditions (pH and temperature) variations [13][14][15], and larger yields can be obtained in comparison to proteins, thus considerably reducing the costs.
In a recent paper, we reported the immobilization by physical adsorption of a novel peptide sequence, CAP-p15, onto an amorphous titanium oxide layer (aTiO 2 ) that simulates the surface of a Tibased implant [16].This sequence of 15 amino acid residues is derived from the C-terminal region of the cementum attachment protein (CAP) and possesses acidic and hydrophobic properties.More importantly, CAP-p15 exhibited the ability to facilitate carbonate hydroxyapatite crystal formation, enhancing cell attachment, influencing cytoskeleton organization, and improving cell proliferation and migration of human periodontal ligament cells in vitro [16][17][18].
Numerous studies employing diverse peptides as a surface functionalization method have consistently shown that it induces cell differentiation toward mineralized phenotypes when using mesenchymal stem cells (MSC).In this regard, the oral cavity is a source of different MSCs populations, for example, dental pulp-derived stem cells [19], periodontal ligamentderived stem cells [20], or dental follicle-derived stem cells [21].Particularly, the lamina propria of the oral mucosa possess a primitive neural crest stem cell population with high clonogenicity, self-renewal, and proliferation capabilities, capable of differentiating into tissue cells of the three germ layers and expressing embryonic stem cell markers such as Sox2 Oct3/4 [22][23][24][25].
Considering these promising findings, the present study focuses on evaluating the ability of CAP-p15functionalized aTiO 2 surfaces to induce the differentiation of stem cells derived from the lamina propria as a model system.Through this study, we aim to unravel the extent to which CAP-p15 can effectively induce the differentiation of stem cells towards the osteoblastic phenotype, contributing to the further understanding of the potential applications of this peptide to enhance implant osseointegration.

Peptide synthesis
The synthesis of CAP-p15 (VSFPSCCFSIAVIFM) was carried out by FMOC solid phase synthesis (New England Peptide, NEP).After synthesis, the synthetic peptide was purified using C-18 reverse-phase liquid chromatography to a >95% purity level (New England Peptide, Ipswich, MA, USA).The lyophilized peptide was dissolved in distilled deionized water, filtered (0.22 µm filter), and stored at 4 • C before use.

Amorphous titanium oxide surfaces
Amorphous titanium oxide (aTiO 2 ) thin films were deposited using a radio frequency magnetron sputtering system that has been thoroughly previously described [26].Briefly, Si (100) 1 cm × 1 cm squares samples were cut using a diamond pencil from Si (100) wafers of 100 mm in diameter.Si (100) samples were then coated using a radio frequency magnetron sputtering system attached to a high-vacuum chamber (base pressure 1.33 × 10 4 Pa), using a 4-inchdiameter high-purity titanium cathode with 99.99% pure, attached to a balanced magnetron.The deposits were carried out at 20 mTorr of pressure in a combined atmosphere of argon and oxygen as sputtering gasses (8/4.6 standard cubic centimeters per minute) and 200 W radio frequency power.The time to obtain the coatings was 45 min.Then, the aTiO 2 -coated Si (100) samples were removed from the vacuum chamber and washed in an ultrasonic bath using three independent different solutions: acetone, isopropyl alcohol, and deionized water; each washing was carried out for 25 min.Finally, samples were air-dried and stored.Before cell culture studies, samples were sterilized for 1 h under UV light.

Peptide functionalization of aTiO 2 surfaces
CAP-p15 functionalization of aTiO 2 -coated samples was performed by peptide physical adsorption as previously described [16].Briefly, the lyophilized peptide was resuspended at a concentration of 1 µg ml −1 in purified deionized water, then, aTiO 2 -coated samples were individually placed in a 24-well plate and covered with 300 µl of the peptide dissolution, carefully distributed over the whole surface of the aTiO 2 -coated samples.Finally, samples were left to dry overnight in a vacuum chamber at room temperature (RT).Study groups were divided as native or untreated surfaces, aTiO 2 , (aTiO 2 -coated Si (100) samples) and treated surfaces, aTiO 2 -CAP-p15, (aTiO 2 -coated Si (100) samples treated with the CAP-p15 peptide sequence).

Cells isolation and expansion
The protocol for collection and use of human tissue samples from the oral cavity for the isolation and culturing of human oral mucosal-derived stem cells (hOMSCs) from the lamina propria followed the principles of the Declarations of Helsinki.It was approved by the Ethics Committee of the Faculty of Dentistry of the National Autonomous University of Mexico (CIE/0303/02/2018).All participants gave written informed consent to participate in the study.The protocol used has been thoroughly described elsewhere [24].In brief, tissue explants were obtained from edentulous gingiva and alveolar mucosa, mechanically disaggregated into smaller samples and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 µg ml −1 streptomycin and 100 UI ml −1 penicillin: Sigma Chemical CO) at 37 • C, 100% humidity, 95% air, and 5% CO2 in 75 cm 3 plates (Nunc, Life Technologies, MX).When the cultures reached confluence, cells were harvested with 0.25% trypsin, passaged, and expanded in the same, but fresh, culture medium that will be referred to hereafter as expansion medium.Early passages (2nd-3rd passages) cells were used for the biological assays in the present study.Concerning cell characterization, the hOMSCs used in this study were previously characterized in our laboratory by flow cytometry, corroborating the positive expression of MSC markers such as CD73, CD90, CD105, and Stro-1 [22,24].

Cell attachment
To assess the effect of CAP-p15 on cell attachment, hOMSCs were plated at 2 × 10 3 cell density per sample and cultured onto aTiO 2 -CAP-p15 samples; aTiO 2 -fibronectin (FN) coated samples (5 µg ml −1 ) were used as positive controls and pristine aTiO 2 samples as negative controls.Samples were incubated with 2 mg ml −1 bovine serum albumin in a serum-free medium (DMEM) to block possible nonspecific attachment.Cells were plated at the density described above and incubated for 24 h in DMEM serum-free medium.After this time, the medium was collected and discarded, and samples were washed three times with PBS (1X) to remove unattached cells.To carry out the quantification of cells attached to the substrates, after attached-cells fixation, samples were incubated in crystal violet staining for 4 h and carefully washed with PBS (1X) to remove the excess non-specifically bonded colorant.Cells-specifically bonded colorant was dissolved using 500 µl of SDS (1%), and 100 µl aliquots of dissolution were transferred to 96/well plates for absorbance reading.Absorbance was measured at 605 nm in a spectrophotometer Filter Max F5 Multimode Microplate Reader, Molecular Devices.For scanning electron microscopy (SEM) analysis, attached cells were fixed with 4% paraformaldehyde followed by dehydration in a series of ethanol gradients (20%-100%).Then, the samples were coated with gold for subsequent analysis using a DSM 950 Digital scanning microscope (West Germany, ZEISS).Images obtained were analyzed with ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).

Cell proliferation
To determine the effect of CAP-p15 functionalization on cell proliferation, hOMSCs were plated at a density of 2 × 10 3 on different groups samples and incubated in 24-well plates (Thermo Fisher ScientificTM) with DMEM medium (Gibco-BRL, Rockville, MD, USA).The samples groups were as follows: cells seeded on aTiO 2 with DMEM supplemented with 0.2% or 10% FBS, for negative and positive control, respectively; and a third group corresponding to cells seeded on aTiO 2 -CAP-p15 samples and cultured in DMEM supplemented with 0.2% FBS (Thermo Fisher ScientificTM, Waltham, MA, USA).All groups were incubated in an atmosphere of 95% air and 5% CO2.Cultures were maintained for 0, 24, 48, and 72 h.After each period, 10 µl of MTT (0.5 mg ml −1 ) was added to the medium and incubated for 3 h; then, the medium was removed, and 100 µl of dimethylsulfoxide (DMSO) (Sigma, Saint Louis, MO, USA) were added and incubated for 1 h at 37 • C. Absorbance was read at 570 nm in a plate reader (Filter Max F5 Multimode Microplate Reader, Molecular Devices).The experiment was performed by triplicate and repeated twice.

Red alizarin staining (ARS)
The identification of the mineralized matrix was conducted using the alizarin red assay.The hOMSCs were plated on the samples at 2 × 10 4 density in 24-well plates and allowed to attach overnight; the following group samples were used to plate the cells on them: peptide-free surfaces (aTiO 2 ) as a control and surfaces functionalized with CAP-p15 (aTiO 2 -CAP-p15).Cell cultures were maintained for 3, 7, and 14 d in DMEM medium supplemented with 10% FBS.In the present work, osteogenesis induction factors were not included in the culture medium to distinguish the specific effect of the surface features on stem cell differentiation.At the end of each incubation period, the medium was removed, and the cells were fixed with 4% paraformaldehyde and subsequently washed with PBS (1X).Calcium nodules were stained with alizarin red solution (Sigma Chemical Co.St. Louis, MO) at pH 4.2 for 10 min.Excess dye was removed by washing with PBS for 5 min.Finally, samples were dried at RT. Three visual fields were chosen randomly for each sample group, calcium nodules were identified under a stereoscopy microscope, and photographs were taken.The images obtained were analyzed using ImageJ (National Institutes of Health (NIH), Bethesda, MD, USA).Quantitative analysis of the presence of calcium nodules was assessed with the 10% cetylpyrimidine chloride (CPC) method, as previously reported elsewhere [27,28].Briefly, red alizarin-stained samples were incubated with 500 µl of CPC for 1 h for alizarin red dissolution, and then the dissolution's absorbance was read in a plate reader (Filter Max F5 Multimode Microplate Reader, Molecular Devices).The analysis was performed in triplicate.
The specific activity was expressed as micrograms of p-NPP per minute per milligram of protein, where hydrolysis of p-NPP was proportional to the ALP activity.The absorbance was read at 405 nm in a spectrophotometer (Filter Max F5 Multimode Microplate Reader, Molecular Devices), and the assays were performed in triplicate.

Western blot analysis
Protein expression of osteogenic differentiation markers was assessed by western blotting.hOM-SCs were plated at a density of 2 × 10 4 cells in aTiO 2 -CAP-p15 and pristine aTiO 2 surfaces in 24well plates and cultured during 3, 7, and 14 d under the culture conditions described elsewhere [28].At the end of each specified culture period, cells were scrapped and dissolved in a lysis buffer solution containing 1% SDS and a protease inhibitor cocktail.Western blots were performed with polyclonal antibodies against human RUNX2, BMP-2, OCN, and GAPDH, and monoclonal antibodies against BSP.Sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) was performed using 12% crosslinked slab gels, charging equal content of proteins (20 mg/lane), and electroblotted onto a PVDF membrane (Amersham Hybond, GE Healthcare, Germany).Membranes were blocked with 5% non-fat milk for 1 h, incubated with 1:1000 diluted antibodies overnight, and washed with PBS/Tween three times.After that, membranes were incubated with 1:1000 diluted in HRP (horseradish peroxidase) conjugated goat anti-rabbit, and goat anti-mouse IgG secondary antibodies (Santa Cruz Biotech, USA) for 2 h.Membranes were analyzed using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).The relative level of each protein was evaluated by measuring the integrated intensity of all pixels in each band, excluding the local background.Results are expressed as percentages of protein intensity.

Statistical analysis
All the trials were performed in triplicate.Two-way ANOVA was used to analyze overall differences in both groups being compared, and Tukey´s post hoc test to search for statistical differences between data sets.p < 0.05 was considered as statistically significant.Statistical analysis was performed with GraphPad Prism 8 software.

Cell attachment
Analysis of SEM images corresponding to hOM-SCs cells cultured for 24 h on the different sample groups revealed that cells plated on the control, that is TiO 2 pristine surfaces (figure 1(A)), adopted a rounded morphology with few cytoplasmic prolongations, unlike cells cultured on CAP-p15-functionalized surfaces (figure 1(B)), which presented an elongated and even cuboidal morphology with numerous cytoplasmic extensions.Quantitative analysis exhibited that surfaces treated with CAP-p15 showed a significant increase of 3.4-fold in cell attachment compared to the pristine TiO 2 surfaces; (figure 1(D)).

Cell proliferation
In this study, hOMSCs were cultured in aTiO 2 and aTiO 2 -CAP-15 in a DMEM medium supplemented with 0.2% FBS.This allowed us to know the effect of functionalization with CAP-p15 in a medium with the minimum conditions necessary to promote cell proliferation (0. 2% FBS).Besides, cell proliferation on pristine aTiO 2 surfaces was also evaluated using a positive control (10% FBS), which is an ideal medium to promote cell proliferation because FBS is a supplement composed of proteins and growth factors that induce the activation of the MAPK signaling and increase cell proliferation (29).The results indicated that aTiO 2 -CAP-p15 functionalized surfaces did not affect hOMSCs viability and cell proliferation on these surfaces.Nevertheless, the number of cells on aTiO 2 -CAP-p15 surfaces at 48 and 72 h of culture increased 2.8 and 2.4-fold when compared to cells cultured onto pristine aTiO 2 surfaces with DMEM supplemented with 10% FBS (positive control), indicating that the proliferation phenomena was induced by CAP-p15 on the functionalized surfaces in the presence of 0.2% FBS (figure 2) Two-way ANOVA (p < 0.0001).

Mineralization of hOMSCs
The alizarin red staining (figure 3) demonstrated that after 3 d of cell culture, both control and experimental groups showed an incipient production of mineralized matrix characterized by the presence of small nodules.Quantification of the mineralization showed that CAP-p15 exerted a 1.4-fold increase compared with the control at 3 d of culture.However, from culture day 7 and up to day 14, only the samples functionalized with CAP-p15 revealed an increase of 1.3 and 2.1-fold compared to the control (p < 0.0001).These results indicate that while titanium oxide can promote differentiation towards a mineralizing phenotype, the immobilization of the CAP-p15 peptide on the aTiO 2 -treated surfaces appears to synergistically affect mineralizing matrix production, sustaining this process for a longer period.

ALP activity assay
ALP is a well-known early marker of the biomineralization process, the assessment of ALP activity results an essential assay to determine the differentiation of stem cells towards a mineralizing phenotype.Figure 4 shows that hOMSCs cultured on aTiO 2 -CAP-p15 exhibited a significant increase (p < 0.05) of 0.2 and 1.2-fold in ALPspecific activity at day culture 3 and 14, respectively, when compared with the aTiO 2 untreated surfaces.

Western blot
The evaluation at the protein level of the differentiation process of the hOMSCs cells toward mineralized-like tissue cells was conducted through Western blot analysis (figure 5).Notably, cells cultured on aTiO 2 -CAP-p15 surfaces exhibited an elevated expression of RUNX2 throughout the cell culture period, 4-fold higher than upon culture on control surfaces.Additionally, BSP maximum protein expression occurred at 3 d of cell culture, followed by a decrease at 7 d for cell culture on both surfaces, control and aTiO 2 -CAP-p15 surfaces.However, even at 7 d of culture, the BSP expression remained higher for cells cultured on the CAP-p15 treated surfaces than that on control surfaces; with an increase of 1.9-fold, 1.2-fold, and 105-fold at 3, 7, and 14 d of cell culture, respectively, when compared to control surfaces.A similar trend was evident for BMP2 expression, revealing a 3.9-fold, Figure 2. Effect of functionalization of aTiO2 surfaces with CAP-p15 peptide in hOMSCs proliferation, at 24 h there were no differences between the three groups; at 48 and 72 h it was observed an increase of 2.8-and 2.4-fold in the number of cells on the surface treated with CAP-p15 sequence, compared with control surfaces.Two-way ANOVA p < 0.0001, ns: no significant.1.2-fold, and 1.4-fold increase when compared cells culture on CAP-p15 treated surfaces in comparison to the control pristine aTiO 2 surfaces.The protein expression of OCN showed 1.9-fold, 1.3-fold, and 1.8-fold at 3, 7, and 14 d, respectively, when comparing cells culture on aTiO 2 -CAP-p15 surfaces to TiO 2 pristine surfaces.These findings revealed a positive impact of the surfaces treated with CAP-p15 peptide, promoting an elevated cell expression of molecular markers associated with mineralized tissue.

Discussion
Surface functionalization represents a novel approach for improving the bioactivity of implantable materials, allowing for a successful osseointegration process.It is well known that the amorphous native oxide layer formed onto titanium implants allows better protein adsorption and cell-surface interactions, besides reducing the ions release [3,29].In this study, we carried out the synthesis of amorphous titanium oxide (aTiO 2 ) thin films onto smooth silicon substrates to simulate the native oxide layer formed onto titanium implants under physiological conditions, and subsequently the surfaces were functionalized with a peptide derived from CAP-p15.Previous works conducted by Almaguer-Flores [30] and Silva-Bermudez [26] demonstrated by XPS and XRD studies, the stoichiometric chemical composition, and the amorphous nature of the TiO 2 thin films deposited on Si (100) substrates under the conditions used to deposit the TiO 2 thin films used in the present study.In addition, antimicrobial and osteogenic differentiation was probed, where pristine aTiO 2 displayed lower bacterial adhesion, better cell adhesion, and osteogenic differentiation of MSC compared with pristine crystalline titanium oxide (cTiO 2 ) surfaces.
The present work demonstrates that the physical immobilization of CAP-p15 onto amorphous titanium oxide (aTiO 2 ) surfaces promotes cell attachment of hOMSCs, which could be associated with the hydrophilic nature of these biofunctionalized surfaces.It is acknowledged that hydrophilic surfaces enhance the early stages of cell attachment, proliferation, and differentiation more than hydrophobic surfaces [31].This agreed with Silva-Bermudez et al [26] where amorphous titanium oxide (aTiO 2 ) substrates displayed better cell attachment than their counterpart crystalline titanium oxide (cTiO 2 ).The  authors attributed these results to the nano topography and the basic nature of aTiO 2 .
In a recent investigation [16] we evaluated the water contact angle of bare and functionalized aTiO 2 surfaces with CAP-p15, demonstrating that aTiO 2 -CAP-p15 surfaces kept their hydrophilic nature.This could be related to the charge and polar amino acids exposed on the surface, creating hydrogen bonds with water droplets.Moreover, the regulation of cell attachment is governed by electrostatic forces within proximity of 2-5 nm from the surface, wherein electron donor sites on the cell surface interact with electron acceptor sites on the TiO 2 surface, thereby promoting cell attachment [32].
SEM image analysis shows that cells adhered to CAP-p15-functionalized surfaces are broadly spread and exhibited a stellate morphology with the presence of cytoplasmic structures such as filopodia (figure 1).
Similar observations have been reported in studies involving biofunctional peptides such as DN3 [33] or Ln2-P3 [34], these functionalized surfaces displayed increased cell attachment within the first 24 h.Furthermore, our findings demonstrate that the immobilization of CAP-p15 promotes the proliferation and, more importantly, triggers the differentiation of hOMSCs towards a mineralizing phenotype.Previous studies have established that the incorporation of homogeneous TiO 2 layers [35] or amorphous TiO 2 nanotubes alone can induce osteogenic differentiation [36].
Notably, recent research has illustrated that the addition, whether by physical adsorption or covalent bonding of peptides or proteins to TiO 2 surfaces, improves the osteogenic differentiation state of mesenchymal cells [8,33,35,37].However, the addition of osteogenic inducers (β-glycerophosphate and ascorbic acid) is necessary to induce mineralization [38].Here, we emphasize that the presence of CAP-p15 immobilized on aTiO 2 surfaces without osteogenic inducers was enough to promote the osteogenic differentiation of hOMSCs, as evidenced by the increased number of calcium nodules in cell culture from alizarin red staining, ALP-specific activity, and mineralizing-related protein expression analyses.
Particularly, ARS technique was employed to illustrate that surfaces treated with CAP-p15 exhibited a robust presence of calcium-rich nodules primarily distributed across the surface, starting at day 3 of cell culture and progressively increasing until 14 d of culture.This ARS assay is considered a pivotal technique for evaluating MSC differentiation by the International Society for Cellular Therapy [39].
Our results demonstrate that surfaces functionalized with CAP-p15 upregulated osteoblastic differentiation and enhanced mineralization of hOM-SCs.This is shown by a significant increase in ALPspecific activity, which is a crucial and early differentiation marker in the biomineralization process since this enzyme hydrolyzes organic phosphoric acid and releases inorganic phosphorus necessary for the assembly of the mineralizing matrix [40].The activity of this enzyme acts as an indicator of osteoblast maturation that is normally maintained for 1-2 weeks in cell culture [41].Moreover, the hOM-SCs cultured onto surfaces functionalized with CAP-p15 overexpressed runt-related transcription factor 2 (RUNX2), essential for stem cell differentiation, promoting expression of key mineralization-related molecules.It has been shown that RUNX2 overexpression significantly upregulates osteoblastic differentiation and mineralization of bone marrow stromal cells in vitro and in vivo [42].After commitment into the osteoblastic lineage, the osteoblasts express bone matrix proteins at various expression levels depending on the maturation level of the cells [41][42][43][44][45].
In this sense, the BSP and BMP2 proteins are considered early osteogenic markers expressed in the early stages of the differentiation process.BSP is crucial for starting the mineralization process by nucleating hydroxyapatite crystals, while BMP2 regulates the expression of genes involved in the mineralization process.OCN is a molecule of late-stage differentiation of the mineralization process and regulates the growth and maturation of HA crystals.Here, we observed higher expression of BSP and BMP2 at the initial stages of mineralization.Meanwhile, OCN was highly expressed at the medium and late stages of the mineralization process.This expression pattern could be related to the gradual increase of mineralized matrix observed with red alizarin stain assay, the increase in ALP activity, and OCN expression on day 14, which is necessary to start the maturation phase.These findings align with other studies that obtained similar patterns using MSCs onto surfaces functionalized with peptides such as MP144 or ESQES, allowing preosteoblast differentiation [37,[46][47][48].
Surface functionalization with peptides can result in the recognition of specific transmembrane receptors that activate intracellular mechanisms like cell attachment, cell differentiation, and mineralization of the ECM.For instance, peptides like DN3 (PPFEGCIWN) or GFOGER have been demonstrated to induce signals through the interaction with transmembrane receptors α3β1 and α2β1, respectively [33,49].Other sequences as FN9-10ELP [8] have been demonstrated to increase the expression of TAZ, a transcriptional coactivator that promotes the expression of other mineralization-related genes such as OCN and RUNX2.
So far, the exact mechanism by which CAP-p15 exerts its effects remains unclear.Therefore, further studies, encompassing additional in vitro and future in vivo models, are necessary to unravel the intricacies of CAP-p15's activity.This ongoing research will contribute valuable insights into the mechanisms underlying the observed effects and potentially uncover new avenues for enhancing osteogenic differentiation.

Conclusions
This work was aimed to assess the effect of CAP-p15 peptide physically adsorbed onto aTiO 2 surfaces.Our results indicate that CAP-p15 possesses a synergistic effect promoting cell attachment and cell proliferation.Moreover, CAP-p15 increased ALPspecific activity, deposition of calcium nodules, and the expression of osteogenic-related proteins such as RUNX2, BMP2, BSP, and OCN, inducing hOM-SCs differentiation toward a mineralized-like phenotype in vitro.As the peptide was physically adsorbed to amorphous titanium oxide surfaces, studies are required that allow us to elucidate the mechanisms by which this sequence interacts with the surface in conjunction with peptide release and corrosion tests.Although the synergistic effect of CAP-p15 on osteogenic differentiation was demonstrated, more studies are required to understand the mechanisms associated with intracellular recognition and activation.

Data availability statement
The data cannot be made publicly available upon publication due to legal restrictions preventing unrestricted public distribution.The data that support the findings of this study are available upon reasonable request from the authors.

Figure 1 .
Figure 1.hOMSCs adhered to different surfaces after 24 h of culture.(A) A rounded morphology with a scarce presence of prolongations was observed for hOMSCs cells cultured on pristine surfaces; (B) hOMSCs cultured on surfaces functionalized with CAP-p15 displayed elongated and cuboidal morphology with the presence of cytoplasmic prolongations (white arrows); (C) FN-treated surfaces (positive control) showed cells with numerous cytoplasmic extensions 100 and 500x magnifications.(D) Surfaces treated with CAP-p15 significantly increased 3.4-fold more than the pristine surfaces group.T-student p < 0.0001.

Figure 3 .
Figure 3. Red alizarin staining in pristine and functionalized aTiO2 surfaces at 3, 7 and 14 d of cell (hOMSCs) culture.Incipient mineralized matrix is present in both groups at 3 d of culture.aTiO2 treated with CAP-p15 sequence shows an increase in calcium nodules at 7 and 14 d compared with the pristine aTiO2.Two-way ANOVA * * * p < 0.001, * * * * p < 0.0001.Images at 4X.