Functionalization and characterization of collagen sponges with TiO2 nanoparticles as potential use for the preservation of the alveolar ridge

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Guerrero-Benítez, Israel Alfonso Núñez-Tapia, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8673478/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Collagen sponges are widely used as hemostatic agents in oral surgery due to their biocompatibility and ability to promote clotting. However, their inherent mechanical fragility in wet environments limits their performance. This study proposes the functionalization of commercial collagen sponges with titanium dioxide nanoparticles (TiO₂-NPs) using polycaprolactone (PCL) as a dispersant vehicle, aiming to enhance mechanical integrity while maintaining biocompatibility and hemostatic function. Spongostan™ collagen sponges were functionalized with a PCL solution containing 0.03% TiO₂-NPs (< 25 nm). The resulting composite (Col-PCL-TiO₂) and control sponges (Col) were characterized using FTIR, XRD, and SEM. The mechanical strength was assessed using compression testing. Biocompatibility was evaluated using human fetal osteoblasts (hFOB) through WST-1 viability assays on days 3, 7, 10, and 14, and cell-material interactions were analyzed via SEM and fluorescence microscopy (DAPI/CellTracker). FTIR and XRD confirmed the successful incorporation of TiO₂-NPs and PCL without altering the chemical structure of collagen. SEM revealed a homogeneous coating that preserved the porous architecture. A tenfold increase in compressive strength was observed in the Col-PCL-TiO₂ group (434.69 ± 92.34 kPa) compared to control (41.11 ± 5.39 kPa). Cell viability remained high and comparable to that of the control at all time points, with no significant differences (p > 0.05). Fluorescence and SEM imaging revealed robust cell adhesion, proliferation, and extracellular matrix formation with both materials. These findings demonstrate that the functionalization of collagen sponges with TiO₂-NPs using PCL significantly enhances their mechanical strength while preserving their porous microstructure and excellent biocompatibility, making this composite a promising candidate for alveolar ridge preservation post-extraction. Collagen Sponge TiO₂ nanoparticles Mechanical strength Alveolar ridge preservation Biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Biodegradable collagen sponges are hemostatic agents widely used in oral surgery because of their biocompatibility, ability to promote platelet adhesion and rapid clot formation, and degradation in accordance with the tissue healing process. In in vivo models, these sponges have demonstrated reduced hemostasis times, favorable interactions with blood cells, and biodegradability compliant with ISO 10993, confirming their clinical value as devices for bleeding control and pro-regenerative scaffolding [ 1 – 3 ]. Recent reviews on hemostatic sponges emphasize that porous architecture, wettability, and blood-material interactions determine rapid hemostasis, placing collagen among the most effective and clinically accepted substrates [ 4 ]. However, native collagen sponges have inherent limitations, such as relative mechanical fragility in moist environments [ 5 ]. To overcome these limitations, a promising strategy is to functionalize these matrices with nanoparticles that confer enhanced mechanical properties [ 6 ]. In this context, titanium dioxide nanoparticles (TiO₂-NPs) have attracted considerable interest in biomedicine and dentistry. This material is characterized by its high stability, biocompatibility, photocatalytic properties, and antibacterial properties [ 7 , 8 ]. TiO₂-NPs have proven to be effective mechanical reinforcements when incorporated into polymeric matrices, enhancing the tensile strength and elastic modulus of various composite materials [ 9 ]. In dentistry, they have been successfully incorporated into composite resins, cements, and implant coatings to improve their mechanical performance, durability, and biological profile [ 10 , 11 ], providing a basis for their translation into collagen sponges [ 12 ]. On surfaces and implants, TiO₂-NPs can modulate oxidative stress pathways related to bone remodeling, with size-, dose-, and microenvironment-dependent effects on gingival fibroblasts. The TiO₂-NPs influence cell viability and migration compared to microparticles at certain concentrations, highlighting the importance of the therapeutic window and particle size [ 9 ]. It is important to achieve a homogeneous dispersion of nanoparticles within the polymeric matrix and avoid their agglomeration to prevent the creation of mechanical failure points and negatively affect biocompatibility [ 13 ]. To address this, polycaprolactone (PCL) has emerged as a widely used dispersing vehicle [ 14 ]. This biodegradable aliphatic polymer, which is soluble in volatile organic solvents, enables the preparation of stable nanoparticle suspensions. Its hydrophobic nature facilitates the impregnation and anchoring of the nanoparticle suspension within the porous matrix through a simple absorption coating process, forming a thin composite film after solvent evaporation [ 15 , 16 ], making it an accessible and low-cost procedure [ 17 ]. Therefore, this study proposes the incorporation of TiO₂-NPs into collagen sponges intended for hemostatic control after dental extraction, using PCL as a dispersing vehicle. The main objective of this study was to enhance the mechanical integrity of the collagen matrix while maintaining or improving the biocompatibility and rapid hemostatic activation inherent to collagen. 2 Materials and Methods 2.1 Materials Spongostan™ brand collagen sponges, TiO 2 -NPs (< 25 nm particle size), and PCL (mw = 192,000 g/mol) were purchased from Sigma-Aldrich. 2.2 Preparation of the composite Thirty samples were prepared and divided into two groups: collagen sponge (Col) and collagen sponge with TiO 2 -NPs dispersed in PCL (Col-PCL-TiO₂). For the preparation of the Col-PCL-TiO₂ group, TiO₂-NPs were dispersed at a concentration of 0.03% (w/w) in a 6% PCL solution (w/v) using PCL granules dissolved in a mixture of chloroform and acetone at a 3:1 ratio with magnetic stirring for 4 h. Subsequently, the collagen sponges were immersed in the solution for functionalization by absorption. Once loaded, the sponges were placed on a support to dry for 24 h (Fig. 1 ). 2.3 Characterization 2.3.1 Fourier transform infrared (FTIR) Fourier-transform infrared (FTIR) spectra of the scale samples were obtained using a Thermo Scientific Nicolet 6700 spectrometer. It was equipped with a ZnSe attenuated total reflectance (ATR) accessory. The samples were scanned in the 4000–500 cm − 1 spectral range, with 32 scans performed at a resolution of 4 cm − 1 . 2.3.2 X-Ray diffraction A D5000 diffractometer was used with–Bragg Brentano geometry, and a Cu-ϰα and λ = 1,5419 Å lamp at 34 kV and 25 mA in a measurement interval of 5 to 60 2θ degrees. The crystallographic phase identification was based on the International Centre for Diffraction Data (ICDD) database. 2.3.3 Morphological characterization The structure and morphology of the nanoparticles and sponges were examined using a scanning electron microscope (JEOL JSM-6700F; SEM). The samples were sputter-coated with a 5-nm-thick gold layer. 2.3.4 Compressive strength test The compression test was performed on an INSTRON 5567 universal testing machine at 20°C and 50% relative humidity. The collagen sponges were measured using a Mitutoyo Corp. micrometer (Kawasaki, Japan; Model No. CD-6 CSX) at a crosshead speed of 1 mm/min. Fifteen samples from each group were tested using a 5000 N load cell. The initial separation of each sample (Lo) was measured. The compressive strength was calculated using the stress-strain curves. 2.4 Cell viability 2.4.1 Cell culture Human fetal osteoblast cells (hFOB, 1.19, ATCC: CRL-11372) were used to evaluate the biological responses. hFOB cells were cultured in 75 cm 2 cell culture flasks containing a 1:1 mixture of Ham’s F12 medium and Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS, Biosciences) and 2.5 nM L-glutamine and antibiotic solution (streptomycin 100 g/mL and penicillin 100 U/mL, Sigma-Aldrich). The cell cultures were incubated in a 100% humidified environment at 37°C in 95% air and 5% CO 2 . hFOB cells in passages 2–6 were used for all experimental procedures. 2.4.2 Resazurin assay Before the biological assays, the Col and Col-PCL-TiO₂ groups were sterilized using UV light for 10 min. To evaluate the cell viability of hFOB in the Col and Col-PCL-TiO₂ groups, cells were seeded at 1 × 10 4 cells/mL and analyzed after 24 and 48 h, and after 3, 7, 10, and 14 days of culture. Cell viability was assessed using a resazurin colorimetric assay after the prescribed time. Then, 20 µL of resazurin solution (BioReagent R7017, CAS number 62758-13-8) was added to the samples and incubated for 4 h at 37°C. Then, 200 µL of the supernatant was removed, and the absorbance was quantified by spectrophotometry at 545 nm using a ChroMate plate reader (Awareness Technology, MN, USA). 2.5 Evaluation of material-cell interaction 2.5.1 Scanning electron microscopy The Col and Col-PCL-TiO₂ groups with cells were fixed with 0.2% glutaraldehyde for 1 h at 4°C. For SEM analysis, after 48 h of incubation, scaffolds were washed three times with PBS, fixed with 2% glutaraldehyde, dehydrated with a graded series of ethanol (25–100%), and air-dried. Next, the samples were sputter-coated with a thin layer of gold and examined using SEM. 2.5.2 Fluorescence assay The colonization and spreading interactions of hFOB cells seeded at 1 × 10 4 cells/mL onto the Col and Col-PCL-TiO₂ groups were examined after 24h using fluorescence microscopy. For fluorescence observation, before seeding the hFOB 1.19 cells onto the Col and Col-PCL-TiO₂ group, cells were incubated with CellTracker™ Green (CMFDA, 5-chloromethylfluorescein diacetate) in phenol red-free medium at 37°C for 30 minutes. Subsequently, the cells were washed with PBS and incubated for 1 h in complete medium. DAPI (4', 6-diamidino-2-phenylindole) solution staining was used to identify cell nuclei. hFOB 1.19 cells were trypsinized and counted to obtain the desired cell concentration (1 × 10 4 cells/mL), incubated for 24 h, and examined under an epifluorescence inverted microscope (AE31E, MOTIC). All experiments were conducted in triplicate and repeated at least three times. 2.6 Statistical analysis The Shapiro–Wilk test was applied to verify data normality, and subsequently, the ANOVA statistical test was used with GraphPad Prism 8 software. For the mechanical tests, Student's t-test was applied. Statistical significance was set at p ≤ 0.05. 3 Results 3.1 Fourier transform infrared Figure 2 shows the FTIR spectra of the Col and Col-PCL-TiO₂ groups. In the spectrum of Col, the characteristic vibrations of this compound can be observed, corresponding to amide A at 3270 cm − 1 , amide I at 1630 cm − 1 , amide II at 1550 cm − 1 , and amide III at 1240 cm − 1 . The Col-PCL-TiO₂ spectrum retains the vibrational modes of amide A (3280 cm − 1 ), amide I (1625 cm − 1 ), and amide II (1549 cm − 1 ), present in collagen [ 18 ], while for PCL, the vibrational modes corresponding to -CH 2 asymmetric stretching, C = O carbonyl stretching, and asymmetric C–O–C stretching were found at the wavenumbers 2946 cm − 1 , 1725 cm − 1 , and 1240 cm − 1 , respectively [ 19 ]; and the vibrational modes found in the range 580 cm − 1 – 480 cm − 1 is associated with Ti-O-Ti interaction [ 20 ]. The Col-PCL-TiO₂ spectrum contains the same vibrational modes found in electrospun membranes with the same components [ 21 ]. 3.2 X-ray diffraction In the X-ray diffraction pattern associated with the Col-PCL-TiO₂ composite (Fig. 3 ), the contribution of the unordered collagen (COL) components can be observed in the range of 2Θ = 5°–30° [ 21 ]. The signals at 2Θ = 21.5° and 2Θ = 23.8° are associated with the (110) and (200) planes of PCL (ICDD 00-062-1286), respectively. The signals at 2Θ = 25.8°, 2Θ = 48.2°, and 2Θ = 54.1 are associated with the presence of TiO 2 (ICDD 01-089-491) [ 22 ]. 3.3 Morphological characterization Figure 4 A shows the TiO 2 -NPs forming agglomerates; their size ranged from 7 to 21 nm. Figure 4 B shows a micrograph of the unmodified collagen sponge. The structure appears highly porous with thin walls, displaying a heterogeneous morphology, with some regions in which the pores seem to be collapsed or partially closed. Figure 4 C shows the sponge coated with TiO 2 -NPs. It was observed that PCL formed a thin layer around the collagen matrix, achieving proper integration and homogeneous distribution of TiO 2 -NPs. However, it was also observed that the pores decreased in size, but without being completely covered, so as not to alter their properties. 3.4 Compressive strength test Figure 5 shows the compressive strengths of the Col and Col-PCL-TiO₂ groups. The Col group exhibited 41.11 kPa, whereas the Col-PCL-TiO₂ group reached 434.69 kPa. 3.5 Cell viability Regarding the cellular response (Fig. 6 ) at the early evaluation times of 24 and 48 h, a decrease in metabolic activity was observed at 48 h with significant differences; therefore, it was decided to extend the experimentation time to the long term: 3, 7, 10, and 14 days. On the seventh day, both groups showed an increase in cell viability; on the tenth day, a decrease in cell viability was observed in both sponges, but no statistically significant differences were observed between the groups. No decrease in the metabolic rate was observed on day 14. Therefore, cell viability was maintained throughout the experimental period. 3.6 Evaluation of material-cell interaction Figure 7 A shows the cells on the surface of the Col group; the extracellular matrix generated by the cells covering the material and some of the sponge pores can be seen. Figure 7 B shows the cells in the Col-PCL-TiO₂ group, where virtually the entire surface was covered with cells. The TiO₂-NPs were not visible because the surface was coated with the extracellular matrix generated by the cells. Figure 8 , obtained using a fluorescence microscope after 24 h of cell culture, shows that hFOB adhered to and grew on the surfaces of Col and Col-PCL-TiO₂. The cells appeared to attach to the surface and cover the entire surface, showing a cell affinity with isolated cells or with cells in small, dispersed groups over the surfaces. 4 Discussion The results of this preliminary study demonstrated the feasibility of functionalizing commercial collagen sponges with TiO₂-NPs embedded in a PCL matrix to develop a composite material that maintains the inherent hemostatic properties of collagen while enhancing its mechanical properties and evaluating its biocompatibility. Structural characterization by FTIR confirmed the chemical integrity of the collagen after the functionalization process, as evidenced by the preservation of the characteristic vibrational modes of the amide groups (A, I, and II). The appearance of bands associated with carbonyl (C = O) stretching at 1725 cm⁻¹ and the C–O–C group at 1240 cm⁻¹ is indicative of the presence of PCL, while signals in the range of 480–580 cm⁻¹ suggest the formation of Ti–O–Ti bonds, confirming the successful incorporation of TiO₂-NPs into the sponge [ 18 ]. The XRD analysis corroborated the presence of TiO₂ (signals at 25.8°, 48.2°, and 54.1°), as well as the semi-crystalline nature of PCL and the amorphous structure of collagen [ 21 , 23 ]. The absence of significant shifts in the peaks suggests that the interactions between the components are mainly physical, without major alterations in their individual structures. Morphological characterization by SEM revealed that coating with the PCL/TiO₂-NPs solution resulted in the formation of a thin and homogeneous layer over the porous collagen matrix, integrating the nanoparticles. The interconnection between the observed pores indicates that the sponge has a suitable architecture for fluid diffusion and cell migration [ 24 ]. Although a reduction in pore size was observed in sponges with TiO₂-NPs due to the formation of this thin layer, the pores remained open and interconnected, allowing for fluid permeability, cell infiltration, and angiogenesis [ 25 , 26 ]. This preserved microarchitecture is essential for the hemostatic function and scaffolding properties of the material. The compressive strength test showed a significant improvement with the modification of the material, recording values of 41.11 ± 5.39 kPa (Col) and 434.69 ± 92.34 kPa (Col-PCL-TiO 2 ), an increase of 10 times (Fig. 5 ). This significant mechanical reinforcement in the composite with incorporated PCL-TiO 2 (p < 0.001 compared to the Col control) is likely attributed to the optimal dispersion of TiO 2 nanoparticles and the resulting increase in crosslinking density between Col and PCL [ 27 , 28 ]. Cell viability assays with hFOB using the resazurin assay showed that the Col-PCL-TiO₂ sponge maintained biocompatibility comparable to that of the Col sponge at all evaluated time points (days 3, 7, 10, and 14). Although a slightly lower trend was observed in the viability of the experimental group on days 3 and 7, attributed to the reduction in pore size [ 29 ]. These differences were not significant. This demonstrates that the incorporation of TiO₂-NPs and PCL does not exert cytotoxicity under the tested conditions. Previous studies have reported that the biological effects of TiO₂-NPs are highly dependent on dose, particle size, and cell type [ 30 , 31 ], indicating that the size and concentration of TiO₂-NPs used in this study did not alter the biological properties of the sponge. The evaluation of cell interactions with the sponges using SEM and fluorescence assays (DAPI and CellTracker) provided visual evidence of the biocompatibility of the material. The images show a confluent layer of cells and an abundant extracellular matrix completely covering the surface of both the control and functionalized sponges. This demonstrates robust cell adhesion, proliferation, and metabolic activity on the material [ 32 , 33 ]. Fluorescent staining corroborated the high cell density in both groups, confirming that functionalization did not inhibit cell colonization of the scaffold. An important consideration arises from the functionalization strategy using the PCL coating. Although this polymer acts as an effective vehicle for the dispersion and fixation of TiO₂-NPs and has proven to be biocompatible, its hydrophobic character does not appear to have interfered with the properties of the material [ 14 ]. These results support the use of this material as an alternative treatment for the preservation of the alveolar ridge due to its improved mechanical properties without affecting cell viability compared to the control sponge. 5 Conclusions Collagen sponges were successfully coated with TiO 2 -NPs using PCL as a dispersant, as demonstrated by the FTIR, XRD, and SEM results. The porous microarchitecture of the sponge was preserved, exhibiting increased compressive strength and cell viability similar to that of the collagen sponge over 14 days. Furthermore, the hFOB cells used showed adequate cell-material interaction by enabling colonization within the sponges. These results suggest that this composite could be proposed for possible use in the preservation of the alveolar ridge. Declarations Acknowledgments The authors are grateful for the financial support provided by the DGAPA-UNAM-PAPIIT-IN208324 and IA204325 projects. Author contribution: Original draft writing, Methodology, Formal analysis – Febe Carolina Vázquez-Vázquez; Material preparation, data collection, and analysis – Víctor I. Guerrero-Benítez, Israel Alfonso; Conception, data collection, writing and editing manuscript, verification, and supervision – Rafael Álvarez-Chimal and Osmar Alejandro Chanes-Cuevas. Funding: Authors Febe Carolina Vázquez-Vázquez and Osmar Alejandro Chanes-Cuevas are thankful for financial support through the research projects DGAPA-UNAM-PAPIIT-IN208324 and IA204325. Data Availability: Research data are not shared. 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Influence of titanium nanoparticles on cytotoxicity and inflammatory cytokines expression in gingival fibroblasts - An in vitro study. F1000Res , 13 , 1117. https://doi.org/10.12688/f1000research.150936.1 Carvalho, M. S., Silva, J. C., Cabral, J. M. S., Silva, C. L., & Vashishth, D. (2019). Cultured cell-derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells. Journal Of Tissue Engineering And Regenerative Medicine , 13 , 1544–1558. https://doi.org/10.1002/term.2907 Wang, F., Cai, X., Shen, Y., & Meng, L. (2023). Cell–scaffold interactions in tissue engineering for oral and craniofacial reconstruction. Bioact Mater , 23 , 16–44. https://doi.org/10.1016/j.bioactmat.2022.10.029 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Apr, 2026 Reviews received at journal 07 Apr, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers agreed at journal 10 Feb, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 27 Jan, 2026 Submission checks completed at journal 26 Jan, 2026 First submitted to journal 22 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8673478","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581267937,"identity":"281cd43e-265f-4dd3-8069-d310362a6b0d","order_by":0,"name":"Febe Carolina Vázquez-Vázquez","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Febe","middleName":"Carolina","lastName":"Vázquez-Vázquez","suffix":""},{"id":581267938,"identity":"41098ebf-8d1f-4203-9767-abbd35662cdb","order_by":1,"name":"Víctor I. Guerrero-Benítez","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Víctor","middleName":"I.","lastName":"Guerrero-Benítez","suffix":""},{"id":581267939,"identity":"0613fbaa-59ed-4064-95e7-7ba13062742f","order_by":2,"name":"Israel Alfonso Núñez-Tapia","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Israel","middleName":"Alfonso","lastName":"Núñez-Tapia","suffix":""},{"id":581267940,"identity":"4c2e987e-99fe-407b-9ad1-dfbfb1fee855","order_by":3,"name":"Rafael Álvarez-Chimal","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Álvarez-Chimal","suffix":""},{"id":581267941,"identity":"530911ec-bcea-4c30-8df5-26a051940745","order_by":4,"name":"Osmar Alejandro Chanes-Cuevas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYHACNjDJz3wAziZSi2RbAoTNQ7QWg2PEauFnYH724OcOG3vjY0CXfSizybNnb2CTLqjZxqA7IwGrFskGNnPD3jNpiduOsSUwzjiXVszDc4BNesax2wxmZw5g1WJwgIdNgrftcILZ/R4DZiAjsUcigU2ahw2o5XgDTi2Sf9v+2xu38SBr+QfUchi7X0BapHnbDjBuYEPWwtuG2xbJZjYzadm25MQZQL8cBPolsefMwWZr3r7bPLj8ws/e/EzybZudPX8b88EHwBBLbG9vPnib59ttObMb2EOMgRmJDTWVEewgYqJ0FIyCUTAKRgEOAABBSFWVaFpGIQAAAABJRU5ErkJggg==","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":true,"prefix":"","firstName":"Osmar","middleName":"Alejandro","lastName":"Chanes-Cuevas","suffix":""}],"badges":[],"createdAt":"2026-01-22 22:38:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8673478/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8673478/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101439934,"identity":"7ae87e51-15f7-4a48-93d6-1d2a07e8f9f5","added_by":"auto","created_at":"2026-01-29 16:51:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":181648,"visible":true,"origin":"","legend":"\u003cp\u003eFunctionalization of collagen sponges with TiO\u003csub\u003e2\u003c/sub\u003e-NPs\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/7a92f08345a6ebee2c74fdd5.png"},{"id":101439930,"identity":"3ff24fb5-46ed-4524-9078-6ea63ce884c0","added_by":"auto","created_at":"2026-01-29 16:51:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":439212,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of Col and Col-PCL-TiO₂ groups\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/019b96f80cbec9ae50749483.png"},{"id":101439932,"identity":"b08bf814-8595-4dd7-8a2f-91f9a8b49df6","added_by":"auto","created_at":"2026-01-29 16:51:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95615,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray pattern diffractograms of Col and Col-PCL-TiO₂ groups\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/c92519690fb693eb145196f6.png"},{"id":101751676,"identity":"ce9e0285-913c-4cc5-8a75-de881ae3e2a2","added_by":"auto","created_at":"2026-02-03 10:22:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":532706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e The size and shape of TiO2-NPs can be observed. \u003cstrong\u003eB\u003c/strong\u003e Unmodified collagen sponge showing a highly porous structure. \u003cstrong\u003eC\u003c/strong\u003eCollagen sponge with TiO2-NPs; TiO2-NPs can be seen distributed within the sponge\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/0f425514de8ca40aae1db7f4.png"},{"id":101751459,"identity":"b3431a58-d9e8-42c7-a4ca-13ac8fc9916d","added_by":"auto","created_at":"2026-02-03 10:20:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47130,"visible":true,"origin":"","legend":"\u003cp\u003eCompressive strength of the collagen sponge (Col) and that functionalized with PCL-TiO₂ (Col-PCL-TiO₂). \u003cem\u003ep≤\u003c/em\u003e0.05\u003cem\u003e (*)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/4ad26f4d625eadff20168bd0.png"},{"id":101751596,"identity":"cb131a4a-9002-4728-b09d-4c8dec62dcf6","added_by":"auto","created_at":"2026-02-03 10:21:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":255550,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the cell viability study using a resazurin assay kit for the Col and Col-PCL-TiO₂ groups. \u003cstrong\u003eA\u003c/strong\u003eEvaluation at 24 and 48 h. \u003cstrong\u003eB\u003c/strong\u003eEvaluation at days 3, 7, 10, and 14\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/4c6b626f5b7eca1bc4e5b4cd.png"},{"id":101439937,"identity":"c6b0e25a-5e0b-488c-8842-dddc73a17e3a","added_by":"auto","created_at":"2026-01-29 16:51:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":387311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Col sponge colonized by the cells; the extracellular matrix generated by the cells can be seen deposited on the surface of the sponge. \u003cstrong\u003eB\u003c/strong\u003e Some cells were observed on Col-PCL-TiO₂ sponge surface, and the extracellular matrix was deposited on the surface of the functionalized sponge\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/f0196d36342a3facbad0ecb4.png"},{"id":101439935,"identity":"b57f4966-522e-4ae9-bf94-2ed314a72f63","added_by":"auto","created_at":"2026-01-29 16:51:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":811595,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent micrographs of the cell morphology on Col and Col-PCL-TiO₂ and cell distribution with DAPI and CellTracker\u003csup\u003eTM\u003c/sup\u003e after 24 h of cell culture\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/fdf4e89d3208621df2e5498e.png"},{"id":101755136,"identity":"51e46219-0576-4e99-b753-fb837c63801e","added_by":"auto","created_at":"2026-02-03 10:49:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3743854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8673478/v1/0d4ed1c9-6045-4fa3-a062-ed89f5353af5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eFunctionalization and characterization of collagen sponges with TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e nanoparticles as potential use for the preservation of the alveolar ridge\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBiodegradable collagen sponges are hemostatic agents widely used in oral surgery because of their biocompatibility, ability to promote platelet adhesion and rapid clot formation, and degradation in accordance with the tissue healing process. In in vivo models, these sponges have demonstrated reduced hemostasis times, favorable interactions with blood cells, and biodegradability compliant with ISO 10993, confirming their clinical value as devices for bleeding control and pro-regenerative scaffolding [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recent reviews on hemostatic sponges emphasize that porous architecture, wettability, and blood-material interactions determine rapid hemostasis, placing collagen among the most effective and clinically accepted substrates [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, native collagen sponges have inherent limitations, such as relative mechanical fragility in moist environments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To overcome these limitations, a promising strategy is to functionalize these matrices with nanoparticles that confer enhanced mechanical properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, titanium dioxide nanoparticles (TiO₂-NPs) have attracted considerable interest in biomedicine and dentistry. This material is characterized by its high stability, biocompatibility, photocatalytic properties, and antibacterial properties [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. TiO₂-NPs have proven to be effective mechanical reinforcements when incorporated into polymeric matrices, enhancing the tensile strength and elastic modulus of various composite materials [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In dentistry, they have been successfully incorporated into composite resins, cements, and implant coatings to improve their mechanical performance, durability, and biological profile [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], providing a basis for their translation into collagen sponges [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn surfaces and implants, TiO₂-NPs can modulate oxidative stress pathways related to bone remodeling, with size-, dose-, and microenvironment-dependent effects on gingival fibroblasts. The TiO₂-NPs influence cell viability and migration compared to microparticles at certain concentrations, highlighting the importance of the therapeutic window and particle size [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is important to achieve a homogeneous dispersion of nanoparticles within the polymeric matrix and avoid their agglomeration to prevent the creation of mechanical failure points and negatively affect biocompatibility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To address this, polycaprolactone (PCL) has emerged as a widely used dispersing vehicle [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This biodegradable aliphatic polymer, which is soluble in volatile organic solvents, enables the preparation of stable nanoparticle suspensions. Its hydrophobic nature facilitates the impregnation and anchoring of the nanoparticle suspension within the porous matrix through a simple absorption coating process, forming a thin composite film after solvent evaporation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], making it an accessible and low-cost procedure [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, this study proposes the incorporation of TiO₂-NPs into collagen sponges intended for hemostatic control after dental extraction, using PCL as a dispersing vehicle. The main objective of this study was to enhance the mechanical integrity of the collagen matrix while maintaining or improving the biocompatibility and rapid hemostatic activation inherent to collagen.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eSpongostan\u0026trade; brand collagen sponges, TiO\u003csub\u003e2\u003c/sub\u003e-NPs (\u0026lt;\u0026thinsp;25 nm particle size), and PCL (mw\u0026thinsp;=\u0026thinsp;192,000 g/mol) were purchased from Sigma-Aldrich.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of the composite\u003c/h2\u003e \u003cp\u003eThirty samples were prepared and divided into two groups: collagen sponge (Col) and collagen sponge with TiO\u003csub\u003e2\u003c/sub\u003e-NPs dispersed in PCL (Col-PCL-TiO₂). For the preparation of the Col-PCL-TiO₂ group, TiO₂-NPs were dispersed at a concentration of 0.03% (w/w) in a 6% PCL solution (w/v) using PCL granules dissolved in a mixture of chloroform and acetone at a 3:1 ratio with magnetic stirring for 4 h. Subsequently, the collagen sponges were immersed in the solution for functionalization by absorption. Once loaded, the sponges were placed on a support to dry for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Fourier transform infrared (FTIR)\u003c/h2\u003e \u003cp\u003eFourier-transform infrared (FTIR) spectra of the scale samples were obtained using a Thermo Scientific Nicolet 6700 spectrometer. It was equipped with a ZnSe attenuated total reflectance (ATR) accessory. The samples were scanned in the 4000\u0026ndash;500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e spectral range, with 32 scans performed at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 X-Ray diffraction\u003c/h2\u003e \u003cp\u003eA D5000 diffractometer was used with\u0026ndash;Bragg Brentano geometry, and a Cu-ϰα and λ\u0026thinsp;=\u0026thinsp;1,5419 \u0026Aring; lamp at 34 kV and 25 mA in a measurement interval of 5 to 60 2θ degrees. The crystallographic phase identification was based on the International Centre for Diffraction Data (ICDD) database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Morphological characterization\u003c/h2\u003e \u003cp\u003eThe structure and morphology of the nanoparticles and sponges were examined using a scanning electron microscope (JEOL JSM-6700F; SEM). The samples were sputter-coated with a 5-nm-thick gold layer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Compressive strength test\u003c/h2\u003e \u003cp\u003eThe compression test was performed on an INSTRON 5567 universal testing machine at 20\u0026deg;C and 50% relative humidity. The collagen sponges were measured using a Mitutoyo Corp. micrometer (Kawasaki, Japan; Model No. CD-6 CSX) at a crosshead speed of 1 mm/min. Fifteen samples from each group were tested using a 5000 N load cell. The initial separation of each sample (Lo) was measured. The compressive strength was calculated using the stress-strain curves.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell viability\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Cell culture\u003c/h2\u003e \u003cp\u003eHuman fetal osteoblast cells (hFOB, 1.19, ATCC: CRL-11372) were used to evaluate the biological responses. hFOB cells were cultured in 75 cm\u003csup\u003e2\u003c/sup\u003e cell culture flasks containing a 1:1 mixture of Ham\u0026rsquo;s F12 medium and Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS, Biosciences) and 2.5 nM L-glutamine and antibiotic solution (streptomycin 100 g/mL and penicillin 100 U/mL, Sigma-Aldrich). The cell cultures were incubated in a 100% humidified environment at 37\u0026deg;C in 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e. hFOB cells in passages 2\u0026ndash;6 were used for all experimental procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Resazurin assay\u003c/h2\u003e \u003cp\u003eBefore the biological assays, the Col and Col-PCL-TiO₂ groups were sterilized using UV light for 10 min. To evaluate the cell viability of hFOB in the Col and Col-PCL-TiO₂ groups, cells were seeded at 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL and analyzed after 24 and 48 h, and after 3, 7, 10, and 14 days of culture. Cell viability was assessed using a resazurin colorimetric assay after the prescribed time. Then, 20 \u0026micro;L of resazurin solution (BioReagent R7017, CAS number 62758-13-8) was added to the samples and incubated for 4 h at 37\u0026deg;C. Then, 200 \u0026micro;L of the supernatant was removed, and the absorbance was quantified by spectrophotometry at 545 nm using a ChroMate plate reader (Awareness Technology, MN, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Evaluation of material-cell interaction\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Scanning electron microscopy\u003c/h2\u003e \u003cp\u003eThe Col and Col-PCL-TiO₂ groups with cells were fixed with 0.2% glutaraldehyde for 1 h at 4\u0026deg;C. For SEM analysis, after 48 h of incubation, scaffolds were washed three times with PBS, fixed with 2% glutaraldehyde, dehydrated with a graded series of ethanol (25\u0026ndash;100%), and air-dried. Next, the samples were sputter-coated with a thin layer of gold and examined using SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Fluorescence assay\u003c/h2\u003e \u003cp\u003eThe colonization and spreading interactions of hFOB cells seeded at 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL onto the Col and Col-PCL-TiO₂ groups were examined after 24h using fluorescence microscopy. For fluorescence observation, before seeding the hFOB 1.19 cells onto the Col and Col-PCL-TiO₂ group, cells were incubated with CellTracker\u0026trade; Green (CMFDA, 5-chloromethylfluorescein diacetate) in phenol red-free medium at 37\u0026deg;C for 30 minutes. Subsequently, the cells were washed with PBS and incubated for 1 h in complete medium. DAPI (4', 6-diamidino-2-phenylindole) solution staining was used to identify cell nuclei. hFOB 1.19 cells were trypsinized and counted to obtain the desired cell concentration (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL), incubated for 24 h, and examined under an epifluorescence inverted microscope (AE31E, MOTIC). All experiments were conducted in triplicate and repeated at least three times.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe Shapiro\u0026ndash;Wilk test was applied to verify data normality, and subsequently, the ANOVA statistical test was used with GraphPad Prism 8 software. For the mechanical tests, Student's t-test was applied. Statistical significance was set at p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Fourier transform infrared\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the FTIR spectra of the Col and Col-PCL-TiO₂ groups. In the spectrum of Col, the characteristic vibrations of this compound can be observed, corresponding to amide A at 3270 cm\u003csup\u003e− 1\u003c/sup\u003e, amide I at 1630 cm\u003csup\u003e− 1\u003c/sup\u003e, amide II at 1550 cm\u003csup\u003e− 1\u003c/sup\u003e, and amide III at 1240 cm\u003csup\u003e− 1\u003c/sup\u003e. The Col-PCL-TiO₂ spectrum retains the vibrational modes of amide A (3280 cm\u003csup\u003e− 1\u003c/sup\u003e), amide I (1625 cm\u003csup\u003e− 1\u003c/sup\u003e), and amide II (1549 cm\u003csup\u003e− 1\u003c/sup\u003e), present in collagen [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while for PCL, the vibrational modes corresponding to -CH\u003csub\u003e2\u003c/sub\u003e asymmetric stretching, C = O carbonyl stretching, and asymmetric C–O–C stretching were found at the wavenumbers 2946 cm\u003csup\u003e− 1\u003c/sup\u003e, 1725 cm\u003csup\u003e− 1\u003c/sup\u003e, and 1240 cm\u003csup\u003e− 1\u003c/sup\u003e, respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]; and the vibrational modes found in the range 580 cm\u003csup\u003e− 1\u003c/sup\u003e – 480 cm\u003csup\u003e− 1\u003c/sup\u003e is associated with Ti-O-Ti interaction [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The Col-PCL-TiO₂ spectrum contains the same vibrational modes found in electrospun membranes with the same components [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 X-ray diffraction\u003c/h2\u003e \u003cp\u003eIn the X-ray diffraction pattern associated with the Col-PCL-TiO₂ composite (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the contribution of the unordered collagen (COL) components can be observed in the range of 2Θ = 5°–30° [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The signals at 2Θ = 21.5° and 2Θ = 23.8° are associated with the (110) and (200) planes of PCL (ICDD 00-062-1286), respectively. The signals at 2Θ = 25.8°, 2Θ = 48.2°, and 2Θ = 54.1 are associated with the presence of TiO\u003csub\u003e2\u003c/sub\u003e (ICDD 01-089-491) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Morphological characterization\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows the TiO\u003csub\u003e2\u003c/sub\u003e-NPs forming agglomerates; their size ranged from 7 to 21 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows a micrograph of the unmodified collagen sponge. The structure appears highly porous with thin walls, displaying a heterogeneous morphology, with some regions in which the pores seem to be collapsed or partially closed. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows the sponge coated with TiO\u003csub\u003e2\u003c/sub\u003e-NPs. It was observed that PCL formed a thin layer around the collagen matrix, achieving proper integration and homogeneous distribution of TiO\u003csub\u003e2\u003c/sub\u003e-NPs. However, it was also observed that the pores decreased in size, but without being completely covered, so as not to alter their properties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Compressive strength test\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the compressive strengths of the Col and Col-PCL-TiO₂ groups. The Col group exhibited 41.11 kPa, whereas the Col-PCL-TiO₂ group reached 434.69 kPa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Cell viability\u003c/h2\u003e \u003cp\u003eRegarding the cellular response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) at the early evaluation times of 24 and 48 h, a decrease in metabolic activity was observed at 48 h with significant differences; therefore, it was decided to extend the experimentation time to the long term: 3, 7, 10, and 14 days. On the seventh day, both groups showed an increase in cell viability; on the tenth day, a decrease in cell viability was observed in both sponges, but no statistically significant differences were observed between the groups. No decrease in the metabolic rate was observed on day 14. Therefore, cell viability was maintained throughout the experimental period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Evaluation of material-cell interaction\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows the cells on the surface of the Col group; the extracellular matrix generated by the cells covering the material and some of the sponge pores can be seen. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB shows the cells in the Col-PCL-TiO₂ group, where virtually the entire surface was covered with cells. The TiO₂-NPs were not visible because the surface was coated with the extracellular matrix generated by the cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, obtained using a fluorescence microscope after 24 h of cell culture, shows that hFOB adhered to and grew on the surfaces of Col and Col-PCL-TiO₂. The cells appeared to attach to the surface and cover the entire surface, showing a cell affinity with isolated cells or with cells in small, dispersed groups over the surfaces.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe results of this preliminary study demonstrated the feasibility of functionalizing commercial collagen sponges with TiO₂-NPs embedded in a PCL matrix to develop a composite material that maintains the inherent hemostatic properties of collagen while enhancing its mechanical properties and evaluating its biocompatibility.\u003c/p\u003e\u003cp\u003eStructural characterization by FTIR confirmed the chemical integrity of the collagen after the functionalization process, as evidenced by the preservation of the characteristic vibrational modes of the amide groups (A, I, and II). The appearance of bands associated with carbonyl (C = O) stretching at 1725 cm⁻¹ and the C–O–C group at 1240 cm⁻¹ is indicative of the presence of PCL, while signals in the range of 480–580 cm⁻¹ suggest the formation of Ti–O–Ti bonds, confirming the successful incorporation of TiO₂-NPs into the sponge [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe XRD analysis corroborated the presence of TiO₂ (signals at 25.8°, 48.2°, and 54.1°), as well as the semi-crystalline nature of PCL and the amorphous structure of collagen [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The absence of significant shifts in the peaks suggests that the interactions between the components are mainly physical, without major alterations in their individual structures.\u003c/p\u003e\u003cp\u003eMorphological characterization by SEM revealed that coating with the PCL/TiO₂-NPs solution resulted in the formation of a thin and homogeneous layer over the porous collagen matrix, integrating the nanoparticles. The interconnection between the observed pores indicates that the sponge has a suitable architecture for fluid diffusion and cell migration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although a reduction in pore size was observed in sponges with TiO₂-NPs due to the formation of this thin layer, the pores remained open and interconnected, allowing for fluid permeability, cell infiltration, and angiogenesis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This preserved microarchitecture is essential for the hemostatic function and scaffolding properties of the material.\u003c/p\u003e\u003cp\u003eThe compressive strength test showed a significant improvement with the modification of the material, recording values of 41.11 ± 5.39 kPa (Col) and 434.69 ± 92.34 kPa (Col-PCL-TiO\u003csub\u003e2\u003c/sub\u003e), an increase of 10 times (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This significant mechanical reinforcement in the composite with incorporated PCL-TiO\u003csub\u003e2\u003c/sub\u003e (p \u0026lt; 0.001 compared to the Col control) is likely attributed to the optimal dispersion of TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles and the resulting increase in crosslinking density between Col and PCL [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCell viability assays with hFOB using the resazurin assay showed that the Col-PCL-TiO₂ sponge maintained biocompatibility comparable to that of the Col sponge at all evaluated time points (days 3, 7, 10, and 14). Although a slightly lower trend was observed in the viability of the experimental group on days 3 and 7, attributed to the reduction in pore size [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These differences were not significant. This demonstrates that the incorporation of TiO₂-NPs and PCL does not exert cytotoxicity under the tested conditions. Previous studies have reported that the biological effects of TiO₂-NPs are highly dependent on dose, particle size, and cell type [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], indicating that the size and concentration of TiO₂-NPs used in this study did not alter the biological properties of the sponge.\u003c/p\u003e\u003cp\u003eThe evaluation of cell interactions with the sponges using SEM and fluorescence assays (DAPI and CellTracker) provided visual evidence of the biocompatibility of the material. The images show a confluent layer of cells and an abundant extracellular matrix completely covering the surface of both the control and functionalized sponges. This demonstrates robust cell adhesion, proliferation, and metabolic activity on the material [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Fluorescent staining corroborated the high cell density in both groups, confirming that functionalization did not inhibit cell colonization of the scaffold.\u003c/p\u003e\u003cp\u003eAn important consideration arises from the functionalization strategy using the PCL coating. Although this polymer acts as an effective vehicle for the dispersion and fixation of TiO₂-NPs and has proven to be biocompatible, its hydrophobic character does not appear to have interfered with the properties of the material [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese results support the use of this material as an alternative treatment for the preservation of the alveolar ridge due to its improved mechanical properties without affecting cell viability compared to the control sponge.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eCollagen sponges were successfully coated with TiO\u003csub\u003e2\u003c/sub\u003e-NPs using PCL as a dispersant, as demonstrated by the FTIR, XRD, and SEM results. The porous microarchitecture of the sponge was preserved, exhibiting increased compressive strength and cell viability similar to that of the collagen sponge over 14 days. Furthermore, the hFOB cells used showed adequate cell-material interaction by enabling colonization within the sponges. These results suggest that this composite could be proposed for possible use in the preservation of the alveolar ridge.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful for the financial support provided by the DGAPA-UNAM-PAPIIT-IN208324 and IA204325 projects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOriginal draft writing, Methodology, Formal analysis \u0026ndash; Febe Carolina V\u0026aacute;zquez-V\u0026aacute;zquez; Material preparation, data collection, and analysis \u0026ndash; V\u0026iacute;ctor I. Guerrero-Ben\u0026iacute;tez, Israel Alfonso; Conception, data collection, writing and editing manuscript, verification, and supervision \u0026ndash; Rafael \u0026Aacute;lvarez-Chimal and Osmar Alejandro Chanes-Cuevas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Authors Febe Carolina V\u0026aacute;zquez-V\u0026aacute;zquez and Osmar Alejandro Chanes-Cuevas are thankful for financial support through the research projects DGAPA-UNAM-PAPIIT-IN208324 and IA204325.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e Research data are not shared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJiang, X., Wang, Y., Fan, D., Zhu, C., Liu, L., \u0026amp; Duan, Z. 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Cell\u0026ndash;scaffold interactions in tissue engineering for oral and craniofacial reconstruction. \u003cem\u003eBioact Mater\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e, 16\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bioactmat.2022.10.029\u003c/span\u003e\u003cspan address=\"10.1016/j.bioactmat.2022.10.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Collagen Sponge, TiO₂ nanoparticles, Mechanical strength, Alveolar ridge preservation, Biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-8673478/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8673478/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCollagen sponges are widely used as hemostatic agents in oral surgery due to their biocompatibility and ability to promote clotting. However, their inherent mechanical fragility in wet environments limits their performance. This study proposes the functionalization of commercial collagen sponges with titanium dioxide nanoparticles (TiO₂-NPs) using polycaprolactone (PCL) as a dispersant vehicle, aiming to enhance mechanical integrity while maintaining biocompatibility and hemostatic function. Spongostan\u0026trade; collagen sponges were functionalized with a PCL solution containing 0.03% TiO₂-NPs (\u0026lt;\u0026thinsp;25 nm). The resulting composite (Col-PCL-TiO₂) and control sponges (Col) were characterized using FTIR, XRD, and SEM. The mechanical strength was assessed using compression testing. Biocompatibility was evaluated using human fetal osteoblasts (hFOB) through WST-1 viability assays on days 3, 7, 10, and 14, and cell-material interactions were analyzed via SEM and fluorescence microscopy (DAPI/CellTracker). FTIR and XRD confirmed the successful incorporation of TiO₂-NPs and PCL without altering the chemical structure of collagen. SEM revealed a homogeneous coating that preserved the porous architecture. A tenfold increase in compressive strength was observed in the Col-PCL-TiO₂ group (434.69\u0026thinsp;\u0026plusmn;\u0026thinsp;92.34 kPa) compared to control (41.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39 kPa). Cell viability remained high and comparable to that of the control at all time points, with no significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Fluorescence and SEM imaging revealed robust cell adhesion, proliferation, and extracellular matrix formation with both materials. These findings demonstrate that the functionalization of collagen sponges with TiO₂-NPs using PCL significantly enhances their mechanical strength while preserving their porous microstructure and excellent biocompatibility, making this composite a promising candidate for alveolar ridge preservation post-extraction.\u003c/p\u003e","manuscriptTitle":"Functionalization and characterization of collagen sponges with TiO2 nanoparticles as potential use for the preservation of the alveolar ridge","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 16:50:40","doi":"10.21203/rs.3.rs-8673478/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-08T07:15:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T14:52:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150298019731856242069168671122848983631","date":"2026-03-26T11:07:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T04:02:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167696304802798196674663734123487679653","date":"2026-02-25T01:39:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44898221886613327641061121080177832356","date":"2026-02-23T08:24:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313866444601431266455543973895367208974","date":"2026-02-10T09:46:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T12:04:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-27T12:00:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-27T04:09:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2026-01-22T22:30:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"85ff6e28-e858-489a-8bd4-76560ebbe90d","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T08:57:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 16:50:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8673478","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8673478","identity":"rs-8673478","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}