Linezolid combined Strontium substituted hydroxyapatite-Bi polymeric composite for Osteomyelitis affected bone regeneration analysis

Linezolid combined Strontium substituted hydroxyapatite-Bi polymeric composite for Osteomyelitis affected bone regeneration analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Linezolid combined Strontium substituted hydroxyapatite-Bi polymeric composite for Osteomyelitis affected bone regeneration analysis Hua Li, Qi Du, Pei-Yu Guo, Yong-Tao Yi, Suresh Mickymaray, Anbarasan Balu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5280375/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The primary objective of this investigation is to rectify bacterial infections in bone (osteomyelitis) and bone regeneration by utilizing an antibiotic-loaded hydroxyapatite polymer composite. In this regard, strontium (Sr)-substituted hydroxyapatite (mHAP)-reinforced polymeric composites with linezolid (LNZ) were utilized for osteomyelitis-affected bone repair. The brittle nature of the mHAP ceramic was overcome by adding with polymers such as polyvinyl pyrrolidone (PVP) and poly(sodium 4-styrene sulfonate) (PSSS). The composite formation, crystallinity, surface morphology, and zeta potential were investigated by Fourier Transform Infrared (FTIR), x-ray diffraction (XRD), scanning electron microscopy with Energy dispersive X-ray spectroscopy (SEM-EDX), high resolution - transmission electron microscopy (HR-TEM), and Zeta potential and particle size analysis techniques. The particle size and zeta potential were noted, and the zeta potential values of mHAP/PVP-PSSS and mHAP/PVP-PSSS/LNZ composites were found to be − 14.8 mV and − 40.3 mV, respectively. The bioactive results with SBF favored apatite formation and confirmed the composite’s biocompatibility with new bone formation. The cell viability of human bone marrow mesenchymal stem cells (hBMSCs) and the gene expression analysis confirmed the osteogenic potential of the prepared materials. Because the prepared composite obtained promising results, these studies confirm that the prepared composite can release the antibiotic for the treatment of osteomyelitis-affected bone repair. Biomedical Hydroxyapatite Linezolid Osteomyelitis Polyvinyl pyrrolidone Poly(sodium 4-styrene sulfonate) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The risk that an artificial implant will fail increases if the implant site becomes infected by any foreign pathogen (Narayan Subramanyam et al., 2023 ; Sumathra and Rajan, 2019 ; Ihtisham Ul Haq et al., 2024 ). Implant materials coated with antibiotics have been available to treat bone tissue infected during the surgical process (Chen et al., 2023; Juodzbalys et al., 2019 ). Today, scientists still believe that osteomyelitis generally needs antibiotic drugs and surgical treatment (Kuo et al., 2023 ; Jha et al., 2022). In the last decades, several controlled-release drug delivery systems have been manufactured with different forms for the inhibition of the osteomyelitis pathogen. In particular, Hassani Besheli et al. reported vancomycin-loaded silk fibroin nanoparticles (SFNPs) with two different pHs (4.5 and 7.4), which affected the release profiles of vancomycin (Hassani Besheli et al., 2017 ). More recently, calcium alginate nanoparticles crosslinked with phosphorylated polyallylamine were produced to allow the controlled release of clindamycin for osteomyelitis treatment and was reported to support rapid osteomyelitis-affected bone recovery without any destructive effect (Momodu and Savaliya, 2023 ; Gowri et al., 2021 ). However, these reports focused on the delivery of antibiotic drugs and did not significantly address the regeneration of the affected bone. As a bio-ceramic, hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) (HAP) has been employed in recent years for various purposes like delivery of therapeutics into a targeted area and dental or bone applications due to its biocompatibility and resemblance to the bones’ inorganic constituents (Kuo et al., 2023 ). HAP is a brittle ceramic; 70–80% of implants are made of various combinations of biocompatible ceramic materials that meet a wide range of human bone requirements (Amarnath Praphakar et al., 2019 ). Recently, the bioactivity of HAP ceramics has been enhanced by the substitution of minerals such as strontium, titanium, zinc, etc. (Zahra et al., 2013 ; El-Wassefy et al., 2017 ; Prabakaran et al., 2021 ). The mineral substitution on the HAP ceramic was first carried out by Wei et al. ( 1999 ) using the thermal spring methods. Later, antibiotics or active antimicrobial minerals were substituted on the HAP composite (El-Wassefy et al., 2017 ). Sr 2+ is the principal component for the activation of strontium ranelate (SR). SR promotes alkaline phosphatase (ALP) activity, expression of osteoblast markers, increased osteogenesis, and collagen synthesis (Kern et al., 2019 ). Sr 2+ -substituted HAP enhances the bone regeneration process. The polymers used for the composite material for coating metal implantation are generally biodegradable (Dziadek et al., 2017 ). Previously, several polymers were used in implantation, such as poly(sodium 4-styrene sulfonate), polyvinyl alcohol (PVA), silk, collagen, gelatine, polymethyl methacrylate, polyvinyl pyrrolidone (PVP), etc. (Li et al., 2020 ). Natural polymers serve as binding agents in a HAP ceramic materials (Xu et al., 2021 ). Polymer reinforcement with calcium-ceramic materials improves mechanical strength with improved biological interactions (Rafikova et al., 2023 ). Poly(sodium 4-styrene sulfonate) (PSSS) is a water-soluble polymer. The sulfonic groups in PSSS enhance the cell connection (Yazdimamaghani et al., 2015 ). Here, for the first time, the spheres of poly(sodium 4-styrene sulfonate) (PSSS)altered hydroxyapatite (HAP) were prepared by adding the HAP ceramic to the polymer solution used by Babaei et al. ( 2019 ). A recent report on poly(3,4-ethylene dioxythiophene) and poly(sodium 4-styrene sulfonate) suggested that bioactive composites containing these polymers could increase cellular conductivity due to the conductive nature of these polymers (Yazdimamaghani et al., 2015 ). Currently, the PVP polymer has been in medical applications because of its exceedingly promising properties like non-toxicity, biocompatibility, swelling properties, non-carcinogenicity, and bioadhesive nature (Mostafa et al., 2011 ). Many antibacterial drugs are currently used to treat infections: gentamicin, ciprofloxacin, linezolid, amoxicillin, vancomycin, etc. (Yazdimamaghani et al., 2015 ; McNally et al., 2016 ; Xiong et al., 2017 ; Wang et al., 2018 ; Babaei et al., 2019 ). Linezolid (LNZ) is an antimicrobial agent in the family of oxazolidinones and acts against Gram-positive pathogens (Shinto et al., 1992 ). In 1978, oxazolidinones were first promoted for their usefulness in controlling plant diseases (Shinto et al., 1992 ; Stevens et al., 2004 ). Their antibacterial individualities were predicted after 6 years, with meaningfully enhanced antibacterial activity compared to their progenitor compounds (Stevens et al., 2004 ). Moreover, oxazolidinones were treated as a promising innovative class of antibiotics presently used in clinics and allowed by the U.S. FDA in 2000 (Stevens et al., 2004 ). Antibacterial drugs have been combined with other materials and applied to various bacteria-infected diseases. For example, a Zein/PSS-modified HAP composite and a hydroxyapatite-silver nanocomposite have been used to deliver and enhance the inhibitory activity of vancomycin (Yazdimamaghani et al., 2015 ; Wang et al., 2018 ). Silver nanoparticles combined with ciprofloxacin in HAP nanowires to treat antibacterial infections in bone were successfully prepared by Mostafa et al., in 2011 (Mostafa et al., 2011 ). Here, we propose LNZ-loaded antibacterial active strontium mineral-substituted HAP (mHAP) for use in osteomyelitis-affected bone repair. To improve the strength of mHAP, we added bipolymers such as PSSS and PVP to the mHAP ceramic to form the mHAP/PSSS-PVP composite. PSSS and PVP are anionic polymers, and the presence of the charged ions enables them to interact with oppositely charged species in the environment. The negative charge of these anionic polymer-based composites can bind and release positively charged drugs or interact with positively charged proteins to modulate drug release (Hashemian et al., 2018 ; Rafikova et al., 2023 ). The proposed LNZ-loaded mHAP/PSSS-PVP composite is a new and novel material for osteomyelitis-affected bone regeneration. The synergistic antibacterial effects of strontium and linezolid have good inhibition effects, and the ceramic-polymer system has a good ability to adhere the drug to the newly formed bone. This is the first work on anionic polymers with the ability to release ions on bone surfaces and osteoconductive materials made of hydroxyapatite-based composites for bacterial-affected bone regeneration. 2. Experimental section 2.1 Materials and methods Strontium nitrate (Sr) (99.0%, MW-211.63), calcium nitrate tetrahydrate (99.0%, MW: 236.15), ammonia solution (MW-17.03), diammonium hydrogen phosphate (MW: 132.06), poly(sodium 4-styrene sulfonate) (≥ 90%, MW: 206.19), polyvinyl pyrrolidone (K 60, 45% in H 2 O, AMW: 10,000), linezolid (≥ 98%, MW: 337.35), and ethanol (MW: 46.07) were purchased from Sigma Aldrich (Poole, United Kingdom). Calcium chloride (≥ 97%, MW: 110.98), sodium chloride (≥ 99.0%, MW: 58.44), hydrogen chloride, di-potassium hydrogen phosphate anhydrous (99.99%), potassium chloride (≥ 99.0%, MW: 74.55), magnesium chloride (≥ 98%, MW: 95.21), sodium bicarbonate (99.7%, MW: 84.01), and Tris buffer were purchased from Sigma Aldrich (Poole, United Kingdom) for the SBF solution preparation. Double-distilled (DD) water was used throughout the experiments for washing during material preparation. 2.2. Preparation of strontium-substituted hydroxyapatite (mHAP) The preparation of the hydrothermal method followed a previous report (Ghosal and Kaushik, 2020 ). Briefly, 50 mL of 0.45 M of Ca (NO 3 ) 2 .4H 2 O solution was taken into an RB flask and stirred under a magnetic stirrer. Similarly, 50 mL of 0.05 M of Sr(NO 3 ) 2 solution was prepared and slowly added to the Ca solution through a burette. The pH 11.0 was maintained using a 0.1 M ammonia solution. The reaction continued for 30 min, after which 50 mL of 0.33 M (NH 4 ) 2 HPO 4 solution was added slowly dropwise. The mixture was maintained under a stirrer for 24 h, then filtered and washed with DD water, and the substance was dried using a hot air oven at 75°C for 5 h. The dried mHAP was grained into small particles by a porcelain pestle and was calcined at 450°C in a muffle furnace for 4–5 h to eliminate unwanted impurities. Finally, the substituted ceramic was collected and stored in vials. A similar method was followed to prepare HAP without the Sr mineral. 2.3 Preparation of the mHAP/PSSS composite Initially, 2.0% PSSS polymer in 10 mL of an aqueous solution was refluxed for 30 min. After 30 min, a mixture of 0.5 g mHAP in 25 mL water was added to the PSSS solution. The PSSS and mHAP mixture was maintained at 80°C for 24 h under constant stirring. After 24 h, the reaction component was centrifuged using centrifuge tubes. The pellet was collected in a watch glass and dried in a hot air oven at 45°C. The obtained mHAP/PSSS composite was grained as small particles by using a porcelain pestle, collected, and stored in vials (Yazdimamaghani et al., 2015 ). 2.4 Preparation of the mHAP/PVP composite Initially, a 2.0% solution of PVP polymer was prepared in 10 mL of water and stirred for 30 min under a magnetic stirrer. After 30 min, 0.5 g of mHAP dispersed in 25 mL of the aqueous solution was added to the PVP solution. The reaction was maintained for 24 h at RT under constant stirring. After 24 h, the reaction mixture was centrifuged using centrifuge tubes. The pellet was collected in a watch glass and dried in a hot air oven at 45°C. After the composite was grained, the mHAP/PVP composite was collected and stored in vials (Lu et al., 2012 ). 2.5 Preparation of the mHAP/PSSS-PVP composite Initially, a mixture of 2.0% PSSS polymer and 10 mL of aqueous solution was refluxed for 30 min. After 30 min, approximately 1 g of mHAP dispersed in 50 mL of water was mixed into the PSSS solution. The reaction was maintained at 80°C for 24 h under constant stirring. After that, 2.0% PVP was added and stirred for 30 min. After 24 h of reaction time at 80°C, the reaction mixture was centrifuged using centrifuge tubes. The pellet was collected in a watch glass and dried in a hot air oven. After the composite was grained, the mHAP/PSSS-PVP composite was collected and stored in vials. 2.6 Preparation of the LNZ drug-loaded mHAP/PSSS-PVP composite The final composite of mHAP/PSSS-PVP/LNZ was synthesized by adding 0.25 g of LNZ drug dispersed in 25 mL of ethanol to the above solution before centrifuging it. After adding the drug, the reaction was maintained at 95°C for 24 h. Then, the solution was collected in a watch glass and dried in a hot air oven. After the composite was grained, the mHAP/PSSS-PVP/LNZ composite was collected and stored on vials. Scheme 1 describes the possible interactions between the mHAP/PSSS-PVP/LNZ composite components. 2.7 Preparation of the SBF solution A 300 mL aliquot of DD water was placed in a 500 mL plastic beaker and stirred with a magnetic stirrer. NaCl (2.4 g) was weighed, added to the beaker, and stirred well to dissolve it completely. Then, 0.11 g of NaHCO 3 , 0.07 g of KCl, 0.07 g of KHPO 4 , 0.09 g of MgCl 2 , 0.09 g of CaCl 2 , 0.022 g of Na 2 SO 4 , 1.8 g of Tris buffer, and 12 mL of 1M HCl were separately added to the water solution after each salt was completely dissolved. The pH of the reaction was maintained at neutral pH (7.0) by adding either HCl or Tris buffer. The SBF solution was stored in a refrigerator (Isikli et al., 2012 ). (El-Ghannam and Ducheyne, 2011 ). 2.8 Physicochemical characterizations The synthesized composites were studied for their physical properties by Fourier transform infrared (FTIR), x-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS), HR-TEM, EDX, and Zeta potential and particle size analysis instrumentation. Details of the procedure are given below. 2.8.1 FTIR analysis The functionality of the synthesized composites was performed to confirm all the distinct components. The FTIR characterization was performed using the KBr pellet method with IRTRACER-100 Infra-Red absorption spectroscopy (Shimadzu). The spectra were recorded within the region of 4,000 cm − 1 to 400 cm − 1 and at a resolution of 4 cm − 1 . 2.8.2 XRD analysis A diffractometer system (ECO D8 ADVANCE) was used to identify the phase of all prepared composites and crystallinity with Cu K α incident radiation over the 2θ range from 10° to 80°, working at 40 kV and 30 mA and a scanning rate (2θ) of 0.02°. 2.8.3 Morphology analysis by SEM and HR-TEM techniques Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) (TESCAN, VEGA3 SBH) were utilized to determine the surface morphology of the minerals present in the prepared composites. A glass plate coated with acetone-dispersed composites was used for the SEM morphological analysis. A FEI Techni G220 S-TWIN high-resolution transmission electron microscope (HR-TEM) was employed to evaluate the microstructures of all prepared composites. Selected area electron diffraction (SAED) was used to classify the constituent phases. HR-TEM was performed on a copper grid coated with acetone-dispersed composites. 2.8.4 Zeta potential and particle size analysis The composite particle size and surface charge were assessed using dynamic light scattering (DLS) using the Delsa analyzer instrument (Beckman colter) at 25°C with water as a dispersant. 2.8.5 Bioactivity analysis in SBF solution An SBF solution with an ionic concentration comparable to blood plasma was prepared using the procedure mentioned above (Section 2.6 ). Three samples of the final composite were placed in a sealed bottle with the SBF solution and maintained for 1 day, 3 days, and 7 days at room temperature. The solution was refreshed every day. The bioactivity and hydroxyapatite-formation ability of the composites were analyzed using SEM and XRD techniques. 2.9 Biological studies 2.9.1 In-vitro cell culture The American type culture collection (ATCC PCS-500–012) was the source of the human bone marrow mesenchymal stem cells (hBMSCs), which were then maintained in 24-well tissue culture plates with the addition of 10% fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM, GIBCO), and minimal essential media (Hi-Media Laboratories). To prevent bacterial infection, 100 UmL − 1 of streptomycin and 100 U mL − 1 of penicillin were administered for 48 h. Next, the culture environment was adjusted to a 37°C humidified atmosphere with 95% air and 5% CO 2 (Szymon et al., 2019 ). 2.9.2 hBMSC attachment and proliferation All testing samples were added to DMEM/F12 (50:50 ratio) containing 10% FBS and the bare minimum of necessary media for 150 min prior to cell seeding. Prior to this immersion, all composites were sterilized with 75% alcohol and washed three times with PBS solution. Untreated cells were included as a control group, and the cells were detached at 24 h, 36 h, and 48 h. To assess cell attachment and proliferation, they were seeded into new 96-well cell plates at a density of 2×10 4 and 1×10 4 cells per well, respectively, and left for 24 h. Next, 2 mL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each sample in serum-free medium, and the samples were developed for 4 h at 37°C in a humidified atmosphere with 5% CO 2 . A DMSO solution (10%) was added to dissolve the formazan crystals. The composites’ capacity for cell proliferation was assessed by their optical density (OD) values at 570 nm on the spectrophotometric microplate (Neo et al., 2023 ) using the following equation. This experiment was repeated in triplicate. 2.9.3 Osteogenic differentiation analysis Real-time polymerase chain reaction (RT-PCR) was used to measure the mRNA levels of the osteogenic marker genes such as osteocalcin (OCN), runt-related transcription factor (RUNx), and vascular endothelial growth factor (VEGF), in order to conduct the osteogenic differentiation study. After 24 h, 36 h, and 48 h of culture, the culture plates were washed with PBS and suspended in 1 mL of cold TRIzol Reagent (Life Technologies Co.). Each sample’s total RNA was extracted using the standard TRIzol protocol, and the extract was then resuspended in 50 µL of RNase-free water. Next, cDNA was produced using the transcriptase reaction mix method (SuperScript III First-Strand Synthesis System, Life Technologies), and the produced cDNA was kept frozen at − 20°C. Using a power SYBR green RT-PCR kit (Life Technologies), quantitative PCR analysis was carried out (n = 3). The transcripts of the other relative gene were defined using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous housekeeping gene (Liao et al., 2023 ). 2.10 Statistical analysis The results were examined statistically using one-way ANOVA in Origin Pro 8.5 software. A significance threshold of p < 0.05 was established. 3. Results and discussion 3.1 FTIR characterizations The prepared HAP, mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composite formation and the interactions between the components in the composites were determined by FTIR spectroscopy. Initially, the HAP formation was confirmed by the FTIR spectrum presented in Fig. 1 A. The phosphate ions (PO 4 3− ) are the main constituents of HAP and appear as absorbance peaks between 1,200 cm − 1 and 550 cm − 1 . The HAP ceramic featured peaks at 431 cm − 1 , 542 cm − 1 , 726 cm − 1 , 936 cm − 1 , 1028 cm − 1 , and 1,121 cm − 1 (ν 3 to ν 6 ), corresponding to PO 4 3− stretching vibrations, and a peak at 599 cm − 1 corresponding to PO 4 3− deformation vibrations. As with different key peaks, -OH ions were found to correspond to the wide-ranging band from approximately 3,700 cm − 1 to 2,500 cm − 1 . The intensity of the peak was about 3,472 cm − 1 (ν 1 ), a typical peak of the stretching frequency of -OH ions (Wang et al., 2021 ). Characteristic vibrations of PSSS, PVP polymers, mHAP ceramics, and LNZ were noted in the FTIR spectra for mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites and shown in Figs. 1 B–F. The absorption peaks at 431 cm − 1 , 542 cm − 1 , 735 cm − 1 , 932 cm − 1 , 1,060 cm − 1 , and 1,130 cm − 1 indicated the presence of the PO 4 3− ions of mHAP within the mHAP/PSSS-PVP composite. The characteristic peaks of PVP were identified as a large -OH absorption stretching band at 3,097 cm − 1 , which represented the polymeric affiliation of the unfastened hydroxyl organizations and bonded -OH stretching vibration (Hao et al., 2015 ). The characteristic peaks of the LNZ-loaded mHAP/PSSS-PVP composite were obtained, indicating the interactions between the mHAP, PVP, PSSS, and LNZ components. The FTIR spectrum of the mHAP/PSSS-PVP/LNZ composite showed the functional peaks of LNZ in Fig. 1 F. These were 1769 cm − 1 for -the C = O stretching, 1,226 cm − 1 and 1,350 cm − 1 for the -C-O stretching, 2,722 cm − 1 for the asymmetric vibration, 1,072 cm − 1 for the -C-N stretching of the amine group, 3,298 cm − 1 for the -N–H stretching in the presence of the 2° amine, and 1,075 cm − 1 for the -C-F stretching frequency (Bigi et al., 2007 ). The bands located at 425 cm − 1 , 737 cm − 1 , 936 cm − 1 , 1059 cm − 1 , and 1,115 cm − 1 were attributed to the PO 4 3− ions in the matrix. The mHAP/PSSS-PVP/LNZ composite revealed a specific broad absorbance at 3,090 cm − 1 for -OH stretching and another at 2,721 cm − 1 for alkyl C–H stretching. The C–O and C–C stretching appeared as a sharp peak at 1,225 cm − 1 . The absorption peak at 1,447 cm − 1 represented the symmetric bending mode of the -CH 2 group of the LNZ and PSSS-PVP groups. The FTIR peaks that appeared in the expected regions indicated the formation of the desired LNZ-loaded mHAP/PSSS-PVP composite. Shattering peaks in the range of 1,000–1,100 cm − 1 suggested phosphate group of the HAP and it may have produced a weaker crystallization after substituting Sr and the PSSS and PVP polymers (Atul and Shitalkumar, 2017 ). 3.2 XRD investigations XRD evaluation was utilized to analyze the phase and crystallinity characteristics of the pure HAP, mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites. The spectra are shown in Fig. 2 . The diffraction peak in Fig. 2 A corresponds to pure HAP ceramic. The peaks at 2θ values of 25.8°, 29.6°, 31.1°, 32.3°, 39.8°, 47.2°, and 48.6° correspond to the 201, 217, 211, 300, 310, 222, and 213 planes, respectively. The perceived diffraction peaks are recognized by the preferred JCPDS (File no.09-0432) report and suggest a crystalline shape of HAP (Chen et al., 2012 ). The XRD spectrum of the mHAP matrix is displayed in Fig. 2 B and represents the Sr substitution in HAP, showing the crystalline pattern. After substituting Sr in HAP, the crystalline behavior was found to be less intense and broader than pure HAP. This confirms the reduced crystallinity and size of the HAP crystallites due to the partial substitution of the Ca ion by a Sr ion (Pushpalatha et al., 2023 ). The new peak of the mHAP at 2θ value 27.3°, corresponding to the 002 plane, appeared due to the presence of mineral Sr in the HAP lattice (Guo et al., 2005 ). The XRD spectra of the mHAP/PSSS, mHAP/PVP, and mHAP/PSSS-PVP matrix are shown in Figs. 2 C–F. The Sr-substituted HAP ceramics lose their crystalline behavior after the addition of polymers such as PSSS and PVP due to the dilution of the ceramics by the organic compounds. The mHAP crystalline peaks are retained in a reduced form, and these observations confirm the interactions of the polymers with the mHAP ceramics (Guo et al., 2005 ). In Fig. 2 F, mHAP/PSSS-PVP/LNZ peaks at 2θ values of 27.3° and 32.3° are observed. These diffraction peaks confirm the formation of an mHAP/PSSS-PVP/LNZ composite with a reduced crystalline structure. The mHAP/PSSS-PVP/LNZ composite result showed shifted and wider distinctive peaks of HAP, including the low-intensity phases of the PSSS and PVP polymers. It confirmed the development of the composites with the interaction of mHAP, PSSS, PVP, and LNZ (Lei et al., 2017 ). 3.3 Surface morphology and elemental mapping analysis A microstructural evaluation of the composites HAP, mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ was performed, and the outcomes are given in Figs. 3 A–F. The HAP crystal exhibited a flaky particle-like morphology under the calcination process due to the influence of the sintering temperature. In the case of Sr-substituted HAP, a highly aggregated particle-like morphology can be observed for the synthesized mHAP particles in Fig. 3 B. The mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites also exhibited particle-like morphology and are shown in Figs. 3 C–F. The double polymer and LNZ loading played a crucial role in the morphological changes in these composites. The aggregated particles jointly formed a connected network of particles in the mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ composites (Figs. 3 D, E). The EDX spectra are shown in Fig. 3 G. The EDX examination established that the substituted mineral (Sr) was present with the Ca, P, and C. The mHAP/PSSS-PVP/LNZ composite was formed with a Ca-to-P atomic ratio of 1.81, as evidenced by EDX and elemental mapping studies (Fig. 4 ). In medical applications, a small amount of porosity is required for the development of bone cells to create a successful implant attachment (Rajesh and Ravichandran, 2015 ). In other words, because of its increased capacity to bond to bio-metallic devices, porosity is beneficial as it improves osseointegration. However, a substantial porosity volume may accumulate body fluid and be dangerous. Increasing the amount of porous structure also harms the mechanical properties, particularly the binding strength, which encourages fracturing (Hammood et al., 2020 ). Therefore, it is suggested that the prepared material’s porous ratio be well controlled to ensure optimal physicochemical and biochemical efficiency. The obtained mHAP and composites are aggregated particles that jointly form a connected network of particles with a porous nature. 3.4 Morphological analysis by HR-TEM The microstructures of the mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ composites were further estimated by HR-TEM analysis and the results were presented in Fig. 5 . This showed that the two polymers and the LNZ drug particles were connected in some places by a rod-like morphology (Fig. 5 A and Fig. 5 B). This is additional evidence of the interaction between these two polymers, the drug, and the ceramic composite. The morphological changes of the aggregated particles are the positive result of the LNZ drugs adjusting the crystal morphology of the mHAP/PSSS-PVP matrix. The SAED pattern of the mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ composites is shown in Fig. 5 C & D. The SAED outcomes support the concept of the influence of the LNZ drug on the crystallinity of the mHAP/PSSS-PVP matrix. 3.5 Zeta potential and particle size determination The modulation of physicochemical properties by the LNZ drug in the mHAP/PSSS-PVP/LNZ composite will similarly modify the biological activity of the composite to favor more rapid bone healing than the mHAP/PSSS-PVP composite. The best indicator of bioactivity is the formation of apatite crystals on the composite surface. A negatively charged surface is more conducive to the development of apatite crystals than a positively charged surface (Chen et al., 2012 ). The measurement of the composite’s surface charge will support the determination of the bioactivity of the implants. A lower zeta potential value was observed for the mHAP/PSSS-PVP/LNZ composite than for the mHAP/PSSS-PVP composite. This lower zeta potential value may have been caused by the incorporation of the LNZ drug into the mHAP/PSSS-PVP/LNZ composite because linezolid has functional groups such as nitrogen, oxygen, and fluorine that carry a negative charge. The surface charges of mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ are found to be − 14.8 mV and– 40.3 mV, respectively. The zeta potential results proved that the mHAP/PSSS-PVP/LNZ composite would function as a superior bioactive material in terms of the interactions of the biological molecules. The mobility distribution curve is shown in Fig. 6 (A and B). A particle size distribution analysis was also carried out. The particle size increased from ~ 156.7 nm to ~ 207.8 nm after loading the LNZ composite onto the mHAP/PSSS-PVP composite. This extra organic moiety has influenced the size of the composite, as evidenced by this analysis. The particle size distribution curve is displayed in Figs. 6 C, D. 3.6 Bioactivity of the composite in SBF solution The bioactive character of the composite will be demonstrated by its capacity to form bone chemical bonds. This is a major factor in determining the success of an in-vivo implantation. The generated apatite particles will facilitate the osseointegration process, aiding in the formation of new bone. After soaking the mHAP/PSSS-PVP/LNZ composite implant in SBF solution for 1 day, 3 days, and 7 days, the development of apatite crystals was determined by analyzing the bone-bonding characteristics of the implant. Figures 7 A–C show the morphological study by SEM analysis that verified the apatite production. The apatite crystals on the mHAP/PSSS-PVP/LNZ composite appeared to be taller when the period increased and white particles became visible. The apatite crystals took the form of a particle structure after 7 days of immersion. This outcome validates the bioactive behavior of the implant, confirming its safety for additional in-vivo implantation. The XRD diffraction pattern obtained after immersing the mHAP/PSSS-PVP/LNZ composite in SBF solution demonstrated that the apatite crystals that were generated were weakly crystalline (Fig. 7 D). As seen in Fig. 7 D, apatite development on the mHAP/PSSS-PVP/LNZ composite caused the peak at 32° to rise after the first day of immersion. These results are favorable for apatite formation and confirm that the composite has bone regeneration ability. The rapid growth of biomaterials poses significant issues when designing and preparing composite materials to repair and facilitate the regeneration of damaged or injured tissues (Gu et al., 2019 ). It is anticipated that a suitable biomaterial for bone tissue regeneration would not only be bioactive but also biocompatible and would ultimately be substituted by freshly formed tissue (Metcalfe and Ferguson, 2007 ). When embedded in a body, the bone-like apatite-forming potential is a significant prerequisite for a medicinal substance to possess bone-bonding characteristics. 3.7 Biological studies 3.7.1 In-vitro cell viability and live cells The biocompatibility of the prepared mHAP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites was assessed using human bone marrow mesenchymal stem cells (hBMSCs). Throughout the entire examination process, untreated cells were used as a control. The optical microscopic pictures of hBMSCs following varied composite treatments for varying durations of time are shown in Fig. 8 . Compared to the control and other treatment days, the cells looked more compact and multiplied at 7 days and 14 days of incubation, especially after receiving mHAP/PSSS-PVP/LNZ combination treatment. In addition, there were comparatively fewer dead cells than in other instances. Figure 8 shows that the mHAP/PSSS-PVP/LNZ composite is more biocompatible and has more potential to promote osteogenesis than the mHAP and mHAP/PSSS-PVP composites. This might be because the mHAP/PSSS-PVP and LNZ components work together, and the inclusion of LNZ does not change or lessen the mHAP/PSSS-PVP composite’s capacity to be biocompatible. The current testing and analysis did not reveal any toxic effects on bone regeneration. The mHAP/PSSS-PVP/LNZ composite showed greater cell viability (Fig. 9 A) than the other composites. The results above are further validated by a quantitative MTT assay. After 24 h of treatment, the cells treated with the mHAP/PSSS-PVP/LNZ composite had the greatest proportion of viable cells (96%) observed. Comparing this percentage to control cells and the other two composites, mHAP and mHAP/PSSS-PVP reveals a considerable increase. After 24 h of incubation, only 85% of the cells on the mHAP ceramic were still alive. Following the preparation of the composite using PSSS-PVP polymers, the viability was significantly enhanced, reaching 91% (Fig. 9 A). This finding implies that these polymers are not hazardous. Subsequently, the antibiotic LNZ compound exhibited no cytotoxic effect but promoted cell proliferation. Finally, osteogenic cell proliferation is not adversely affected by adding extra ingredients to the mHAP matrix, such as polymers and antibacterial drugs. The mHAP/PSSS-PVP/LNZ composite that has been created is ideal for applications involving the regeneration of bone tissue. 3.7.2 Analysis of cell differentiation by RT-PCR analysis OCN, VEGF, and RUNx2 gene expression are among the tests that are performed using real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Figure 09 displays the RUNx2 (core-binding factor alpha 1) activities of hBMSCs on a range of testing samples throughout varying culture times, including 24 h. In each sample, the mHAP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites boosted RUNx2, OCN, and VEGF activity. According to an in-vitro cell viability measurement, good cell viability was obtained at 24 h after treatment with the mHAP/PSSS/PVP/LNZ composite because of the coexistence of strontium ions and LNZ compounds. This may be explained by the fact that the mHAP/PSSS-PVP/LNZ composite’s osteoconductive properties promote increased RUNx2, OCN, and VEGF synthesis. Furthermore, as the final composite for RUNx2, OCN, and VEGF production, the blot PCR analysis on RUNx2 and OCN also demonstrated the most enhanced production of osteogenesis genes (Fig. 9 ). The activity on the mHAP/PSSS-PVP/LNZ composite was significantly higher than on the mHAP and mHAP/PSSS-PVP composite treatments, including the control groups. These results imply that hBMSCs treated with the mHAP/PSSS-PVP/LNZ composite achieved a good amount of cell differentiation in addition to cell proliferation. The RT-PCR analysis of OCN, VEGF, and RUNx2 gene expression provided evidence that the materials are biocompatible and can promote osteogenesis. The process of creating the apatite layer on the implant’s surface is already well-known (Diomede et al., 2020 ; Prabakaran and Rajan, 2021 ). First, a positively charged substrate ion is produced by the calcium ions in the SBF solution electrostatically bonding with the hydroxyl substrate ion in the HAP. Second, the apatite layer is formed as a result of phosphate deposition on the cationic Ca 2+ layer, which positively influences the formation of the solid mineral surface of the bone. The hydroxyl ion group functions as an additional nucleation site for biomineralization in the mHAP/PSSS-PVP combination. The apatite layer’s mineralization activity has been enhanced by recent research by combining PSSS and PVA polymers. 4. Conclusion LNZ was successfully combined with strontium (Sr)-substituted HAP as an mHAP matrix and PSSS- and PVP-reinforced composites in this work. In-vitro experiments, such as hBMSC proliferation and differentiation, revealed that the synthesized mHAP/PSSS-PVP/LNZ composite shows superior bioactive osteogenic potential. This could be described as the synergistic osteogenic potential of strontium ions and LNZ compounds in the mHAP/PSSS-PVP/LNZ composite. The bioactivity in the SBF solution and the characterization of those samples suggest that it would be a better implant composite. The highest apatite formation was observed on the mHAP/PSSS-PVP/LNZ composite due to its more electronegative nature inducing calcium ion nucleation, and the negative charge of the materials was confirmed by zeta potential analysis such as − 14.8 mV and − 40.3 mV. The composite has good viability and can enhance osteogenic gene expression. Therefore, the current work validates that the LNZ compound with an mHAP-reinforced polymeric matrix is worthy of orthopedic application. Moreover, the divalent strontium ions also encourage osteoblast proliferation and support bone regeneration. Declarations Data availability statement Data is provided within the manuscript. Author contributions HL: data curation, formal analysis, investigation, methodology, visualization, writing–original draft. QD: investigation, methodology, resources, validation, writing–original draft. P-YG: data curation, formal analysis, investigation, resources, writing–review and editing. Y-TY: data curation, formal analysis, investigation, writing–original draft. SM- data curation, formal analysis, investigation, writing–reviewing and editing. AB- investigation, writing–reviewing, and editing. KS- data curation, formal analysis, investigation, writing–reviewing and editing. XL: conceptualization, funding acquisition, investigation, methodology, project administration, writing–original draft, writing–review and editing. Funding The author(s) declare that financial support was received for the research, authorship, and/or publication of this article from the Priority Union Foundation of Yunnan Provincial Science and Technology Department and Kunming Medical University (202401AY070001-235). The author would like to thank Deanship of Scientific Research at Majmaah University for supporting this work under project number R-2024-1110. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. References Amarnath Praphakar, R., Sumathra, M., Sam Ebenezer, R., Vignesh, S., Shakila, H., and Rajan, M. (2019). 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Med. 26 (12), 274–311. 10.1007/s10856-015-5599-8. Zahra,N., Fayyaz,M., Iqbal,W., Irfan,M., and Alam,S. (2013). A process for the development of strontium hydroxyapatite. Int. Symposium Adv. Material 60, 012056. 10.1088/1757-899x/60/1/012056. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme.1.png Scheme. 1. The probable interactions between the components present in mHAP/PSSS-PVP/LNZ composite. Cite Share Download PDF Status: Posted Version 1 posted 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-5280375","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376321821,"identity":"b5eb5d70-83b1-4007-b9c5-4c716ae11657","order_by":0,"name":"Hua Li","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Li","suffix":""},{"id":376321825,"identity":"782ddf5e-ed67-42d0-aefd-1f359a03afa6","order_by":1,"name":"Qi Du","email":"","orcid":"","institution":"Kunming Medical 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Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACPgbmAwcSeGzkGJiJ1cLGwJb44INMmjEpWniMDWfYHE5sINphbBIJZtI8OYfT57fzHvzAUGMTTVgLz4E0aZ4z6bkbDvMlSzAcS8slaB0be8Mxad4e69wNzDwGEowNh4nQwszYJs37jzldvpnH+AdxWtibmQ1n8DgnMBzmMSPSFp5jjA8+8KQZbgBqsUggxi/8EvkfQFEpL99/xvjGhxobwlpQQQJpykfBKBgFo2AU4AIAJ143XeflIz4AAAAASUVORK5CYII=","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xi","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-10-17 07:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5280375/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5280375/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68728905,"identity":"e42106d1-db57-42f1-9d0d-3114ae844ec4","added_by":"auto","created_at":"2024-11-11 12:03:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126227,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrum of (a) Pure HAP, (b) mHAP, (c) mHAP/PSSS, (d) mHAP/PVP, (e) mHAP/PSSS-PVP, and (f) mHAP/PSSS-PVP/LNZ composite.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/f380815936050e00abc90dee.png"},{"id":68729865,"identity":"cd7af944-4cfa-4045-9359-7cb18d079a6f","added_by":"auto","created_at":"2024-11-11 12:11:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109769,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of a) Pure HAP, b) mHAP, c) mHAP/PSSS, d) mHAP/PVP, e) mHAP/PSSS-PVP, and f) mHAP/PSSS-PVP/LNZ composite.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/3e2519b3ac9f33a73009d30b.png"},{"id":68728910,"identity":"54fd6728-c0f1-4ba5-9bfe-c1aaf5394d59","added_by":"auto","created_at":"2024-11-11 12:03:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":465623,"visible":true,"origin":"","legend":"\u003cp\u003eSurface morphology of the prepared (A) pure HAP, (B) mHAP, (C) mHAP/PVP (D) mHAP/PSSS-PVP, (E \u0026amp; F) mHAP/PSSS-PVP/LNZ composite by the SEM analysis and (G) EDX analysis of the elements present in the mHAP/PSSS-PVP/LNZ composite.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/53f264ad29ccf0c0092da800.png"},{"id":68729868,"identity":"bc730223-111c-43cd-8bb6-d6588fc0bf21","added_by":"auto","created_at":"2024-11-11 12:11:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":232912,"visible":true,"origin":"","legend":"\u003cp\u003eMapping analysis of (A) mHAP/PSSS-PVP/LNZ composite (B) Oxygen, (C) Strontium, (D) Phosphorous, and (E) Calcium.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/352641d45208981be207e7fd.png"},{"id":68729866,"identity":"71ef9863-389f-4f87-a337-0cc43c9fdd48","added_by":"auto","created_at":"2024-11-11 12:11:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":537879,"visible":true,"origin":"","legend":"\u003cp\u003eHR-TEM morphology of (A) mHAP/PSSS-PVP and (B) mHAP/PSSS-PVP/LNZ composite. and SAED patterns of (C) mHAP/PSSS-PVP \u0026amp; (D) mHAP/PSSS-PVP/LNZ composite.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/0cf2ae715459093da54a92ba.png"},{"id":68728911,"identity":"f5b1c273-8692-4c79-868d-6ff27909aa32","added_by":"auto","created_at":"2024-11-11 12:03:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":395343,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential analysis of (A) mHAP/PSSS-PVP composite, (B) mHAP/PSSS-PVP/LNZ composite, and Particle size distribution of (C) mHAP/PSSS-PVP composite, (D) mHAP/PSSS-PVP/LNZ composite.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/f406525e9dc241367c5ab848.png"},{"id":68728913,"identity":"c07a9c85-cd62-4a61-9ea0-795532dc3f5a","added_by":"auto","created_at":"2024-11-11 12:03:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":628320,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM image of the SBF immersed mHAP/PSSS-PVP/LNZ composite for (A) 1 day (B) 3 days (C) 7 days, and (D) XRD of the SBF immersed composite.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/c2aa555a673112bc9a57556e.png"},{"id":68730171,"identity":"dd101ca5-83c4-480a-b0e6-76485bb1f537","added_by":"auto","created_at":"2024-11-11 12:19:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":253932,"visible":true,"origin":"","legend":"\u003cp\u003eOptical microscopic images of hBMSCs viable on mHAP, mHAP/PSSS-PVP composite, and mHAP/PSSS-PVP/LNZ composite treatment at 24, 36 and 48 h.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/6cc399eff30851a787eb1118.png"},{"id":68729869,"identity":"4e3e5e44-a5b5-4d3b-9d3e-20d7cb548688","added_by":"auto","created_at":"2024-11-11 12:11:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":293112,"visible":true,"origin":"","legend":"\u003cp\u003eThe quantitative MTT cell viability analysis of mHAP/PSSS-PVP/LNZ composites treated on hBMSCs and RT-PCR cell differentiation analysis on the specific markers including RUNx2, OCN, VEGF. *Comparison of the indicated group with control cells within the same set. *p \u0026lt; 0.05. ** Comparison of the indicated group with mHAP ceramic within the same set. **p \u0026lt; 0.05. *** Comparision of the indicated group with mHAP/PSSS-PVP composite within the same set. ***p \u0026lt; 0.05. b. PCR images of RUNx2 and OCN genes.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/dd8bb43c87bb53f7abbade49.png"},{"id":71962394,"identity":"1e81ea35-7110-4082-93d6-c30455b2935f","added_by":"auto","created_at":"2024-12-20 07:02:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3697683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/10b85ca7-d18f-4a2e-92da-5b3fd8c7d48d.pdf"},{"id":68728907,"identity":"a426bfb2-81a4-424c-911e-dcfa3e7f4484","added_by":"auto","created_at":"2024-11-11 12:03:04","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":187344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme. 1\u003c/strong\u003e. The probable interactions between the components present in mHAP/PSSS-PVP/LNZ composite.\u003c/p\u003e","description":"","filename":"Scheme.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5280375/v1/47dec296aaec10e245252ef9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Linezolid combined Strontium substituted hydroxyapatite-Bi polymeric composite for Osteomyelitis affected bone regeneration analysis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe risk that an artificial implant will fail increases if the implant site becomes infected by any foreign pathogen (Narayan Subramanyam et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sumathra and Rajan, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ihtisham Ul Haq et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Implant materials coated with antibiotics have been available to treat bone tissue infected during the surgical process (Chen et al., 2023; Juodzbalys et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Today, scientists still believe that osteomyelitis generally needs antibiotic drugs and surgical treatment (Kuo et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jha et al., 2022). In the last decades, several controlled-release drug delivery systems have been manufactured with different forms for the inhibition of the osteomyelitis pathogen. In particular, Hassani Besheli et al. reported vancomycin-loaded silk fibroin nanoparticles (SFNPs) with two different pHs (4.5 and 7.4), which affected the release profiles of vancomycin (Hassani Besheli et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). More recently, calcium alginate nanoparticles crosslinked with phosphorylated polyallylamine were produced to allow the controlled release of clindamycin for osteomyelitis treatment and was reported to support rapid osteomyelitis-affected bone recovery without any destructive effect (Momodu and Savaliya, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gowri et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, these reports focused on the delivery of antibiotic drugs and did not significantly address the regeneration of the affected bone.\u003c/p\u003e \u003cp\u003eAs a bio-ceramic, hydroxyapatite (Ca\u003csub\u003e10\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e) (HAP) has been employed in recent years for various purposes like delivery of therapeutics into a targeted area and dental or bone applications due to its biocompatibility and resemblance to the bones\u0026rsquo; inorganic constituents (Kuo et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). HAP is a brittle ceramic; 70\u0026ndash;80% of implants are made of various combinations of biocompatible ceramic materials that meet a wide range of human bone requirements (Amarnath Praphakar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recently, the bioactivity of HAP ceramics has been enhanced by the substitution of minerals such as strontium, titanium, zinc, etc. (Zahra et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; El-Wassefy et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Prabakaran et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The mineral substitution on the HAP ceramic was first carried out by Wei et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) using the thermal spring methods. Later, antibiotics or active antimicrobial minerals were substituted on the HAP composite (El-Wassefy et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Sr\u003csup\u003e2+\u003c/sup\u003e is the principal component for the activation of strontium ranelate (SR). SR promotes alkaline phosphatase (ALP) activity, expression of osteoblast markers, increased osteogenesis, and collagen synthesis (Kern et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Sr\u003csup\u003e2+\u003c/sup\u003e-substituted HAP enhances the bone regeneration process.\u003c/p\u003e \u003cp\u003eThe polymers used for the composite material for coating metal implantation are generally biodegradable (Dziadek et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Previously, several polymers were used in implantation, such as poly(sodium 4-styrene sulfonate), polyvinyl alcohol (PVA), silk, collagen, gelatine, polymethyl methacrylate, polyvinyl pyrrolidone (PVP), etc. (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Natural polymers serve as binding agents in a HAP ceramic materials (Xu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Polymer reinforcement with calcium-ceramic materials improves mechanical strength with improved biological interactions (Rafikova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Poly(sodium 4-styrene sulfonate) (PSSS) is a water-soluble polymer. The sulfonic groups in PSSS enhance the cell connection (Yazdimamaghani et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Here, for the first time, the spheres of poly(sodium 4-styrene sulfonate) (PSSS)altered hydroxyapatite (HAP) were prepared by adding the HAP ceramic to the polymer solution used by Babaei et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A recent report on poly(3,4-ethylene dioxythiophene) and poly(sodium 4-styrene sulfonate) suggested that bioactive composites containing these polymers could increase cellular conductivity due to the conductive nature of these polymers (Yazdimamaghani et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Currently, the PVP polymer has been in medical applications because of its exceedingly promising properties like non-toxicity, biocompatibility, swelling properties, non-carcinogenicity, and bioadhesive nature (Mostafa et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany antibacterial drugs are currently used to treat infections: gentamicin, ciprofloxacin, linezolid, amoxicillin, vancomycin, etc. (Yazdimamaghani et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; McNally et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Babaei et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Linezolid (LNZ) is an antimicrobial agent in the family of oxazolidinones and acts against Gram-positive pathogens (Shinto et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). In 1978, oxazolidinones were first promoted for their usefulness in controlling plant diseases (Shinto et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Stevens et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Their antibacterial individualities were predicted after 6 years, with meaningfully enhanced antibacterial activity compared to their progenitor compounds (Stevens et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Moreover, oxazolidinones were treated as a promising innovative class of antibiotics presently used in clinics and allowed by the U.S. FDA in 2000 (Stevens et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Antibacterial drugs have been combined with other materials and applied to various bacteria-infected diseases. For example, a Zein/PSS-modified HAP composite and a hydroxyapatite-silver nanocomposite have been used to deliver and enhance the inhibitory activity of vancomycin (Yazdimamaghani et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Silver nanoparticles combined with ciprofloxacin in HAP nanowires to treat antibacterial infections in bone were successfully prepared by Mostafa et al., in 2011 (Mostafa et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we propose LNZ-loaded antibacterial active strontium mineral-substituted HAP (mHAP) for use in osteomyelitis-affected bone repair. To improve the strength of mHAP, we added bipolymers such as PSSS and PVP to the mHAP ceramic to form the mHAP/PSSS-PVP composite. PSSS and PVP are anionic polymers, and the presence of the charged ions enables them to interact with oppositely charged species in the environment. The negative charge of these anionic polymer-based composites can bind and release positively charged drugs or interact with positively charged proteins to modulate drug release (Hashemian et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rafikova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The proposed LNZ-loaded mHAP/PSSS-PVP composite is a new and novel material for osteomyelitis-affected bone regeneration. The synergistic antibacterial effects of strontium and linezolid have good inhibition effects, and the ceramic-polymer system has a good ability to adhere the drug to the newly formed bone. This is the first work on anionic polymers with the ability to release ions on bone surfaces and osteoconductive materials made of hydroxyapatite-based composites for bacterial-affected bone regeneration.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Materials and methods\u003c/h2\u003e\n \u003cp\u003eStrontium nitrate (Sr) (99.0%, MW-211.63), calcium nitrate tetrahydrate (99.0%, MW: 236.15), ammonia solution (MW-17.03), diammonium hydrogen phosphate (MW: 132.06), poly(sodium 4-styrene sulfonate) (\u0026ge;\u0026thinsp;90%, MW: 206.19), polyvinyl pyrrolidone (K 60, 45% in H\u003csub\u003e2\u003c/sub\u003eO, AMW: 10,000), linezolid (\u0026ge;\u0026thinsp;98%, MW: 337.35), and ethanol (MW: 46.07) were purchased from Sigma Aldrich (Poole, United Kingdom). Calcium chloride (\u0026ge;\u0026thinsp;97%, MW: 110.98), sodium chloride (\u0026ge;\u0026thinsp;99.0%, MW: 58.44), hydrogen chloride, di-potassium hydrogen phosphate anhydrous (99.99%), potassium chloride (\u0026ge;\u0026thinsp;99.0%, MW: 74.55), magnesium chloride (\u0026ge;\u0026thinsp;98%, MW: 95.21), sodium bicarbonate (99.7%, MW: 84.01), and Tris buffer were purchased from Sigma Aldrich (Poole, United Kingdom) for the SBF solution preparation. Double-distilled (DD) water was used throughout the experiments for washing during material preparation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Preparation of strontium-substituted hydroxyapatite (mHAP)\u003c/h2\u003e\n \u003cp\u003eThe preparation of the hydrothermal method followed a previous report (Ghosal and Kaushik, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, 50 mL of 0.45 M of Ca (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO solution was taken into an RB flask and stirred under a magnetic stirrer. Similarly, 50 mL of 0.05 M of Sr(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e solution was prepared and slowly added to the Ca solution through a burette. The pH 11.0 was maintained using a 0.1 M ammonia solution. The reaction continued for 30 min, after which 50 mL of 0.33 M (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e solution was added slowly dropwise. The mixture was maintained under a stirrer for 24 h, then filtered and washed with DD water, and the substance was dried using a hot air oven at 75\u0026deg;C for 5 h. The dried mHAP was grained into small particles by a porcelain pestle and was calcined at 450\u0026deg;C in a muffle furnace for 4\u0026ndash;5 h to eliminate unwanted impurities. Finally, the substituted ceramic was collected and stored in vials. A similar method was followed to prepare HAP without the Sr mineral.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Preparation of the mHAP/PSSS composite\u003c/h2\u003e\n \u003cp\u003eInitially, 2.0% PSSS polymer in 10 mL of an aqueous solution was refluxed for 30 min. After 30 min, a mixture of 0.5 g mHAP in 25 mL water was added to the PSSS solution. The PSSS and mHAP mixture was maintained at 80\u0026deg;C for 24 h under constant stirring. After 24 h, the reaction component was centrifuged using centrifuge tubes. The pellet was collected in a watch glass and dried in a hot air oven at 45\u0026deg;C. The obtained mHAP/PSSS composite was grained as small particles by using a porcelain pestle, collected, and stored in vials (Yazdimamaghani et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Preparation of the mHAP/PVP composite\u003c/h2\u003e\n \u003cp\u003eInitially, a 2.0% solution of PVP polymer was prepared in 10 mL of water and stirred for 30 min under a magnetic stirrer. After 30 min, 0.5 g of mHAP dispersed in 25 mL of the aqueous solution was added to the PVP solution. The reaction was maintained for 24 h at RT under constant stirring. After 24 h, the reaction mixture was centrifuged using centrifuge tubes. The pellet was collected in a watch glass and dried in a hot air oven at 45\u0026deg;C. After the composite was grained, the mHAP/PVP composite was collected and stored in vials (Lu et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Preparation of the mHAP/PSSS-PVP composite\u003c/h2\u003e\n \u003cp\u003eInitially, a mixture of 2.0% PSSS polymer and 10 mL of aqueous solution was refluxed for 30 min. After 30 min, approximately 1 g of mHAP dispersed in 50 mL of water was mixed into the PSSS solution. The reaction was maintained at 80\u0026deg;C for 24 h under constant stirring. After that, 2.0% PVP was added and stirred for 30 min. After 24 h of reaction time at 80\u0026deg;C, the reaction mixture was centrifuged using centrifuge tubes. The pellet was collected in a watch glass and dried in a hot air oven. After the composite was grained, the mHAP/PSSS-PVP composite was collected and stored in vials.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Preparation of the LNZ drug-loaded mHAP/PSSS-PVP composite\u003c/h2\u003e\n \u003cp\u003eThe final composite of mHAP/PSSS-PVP/LNZ was synthesized by adding 0.25 g of LNZ drug dispersed in 25 mL of ethanol to the above solution before centrifuging it. After adding the drug, the reaction was maintained at 95\u0026deg;C for 24 h. Then, the solution was collected in a watch glass and dried in a hot air oven. After the composite was grained, the mHAP/PSSS-PVP/LNZ composite was collected and stored on vials. Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e describes the possible interactions between the mHAP/PSSS-PVP/LNZ composite components.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Preparation of the SBF solution\u003c/h2\u003e\n \u003cp\u003eA 300 mL aliquot of DD water was placed in a 500 mL plastic beaker and stirred with a magnetic stirrer. NaCl (2.4 g) was weighed, added to the beaker, and stirred well to dissolve it completely. Then, 0.11 g of NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.07 g of KCl, 0.07 g of KHPO\u003csub\u003e4\u003c/sub\u003e, 0.09 g of MgCl\u003csub\u003e2\u003c/sub\u003e, 0.09 g of CaCl\u003csub\u003e2\u003c/sub\u003e, 0.022 g of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1.8 g of Tris buffer, and 12 mL of 1M HCl were separately added to the water solution after each salt was completely dissolved. The pH of the reaction was maintained at neutral pH (7.0) by adding either HCl or Tris buffer. The SBF solution was stored in a refrigerator (Isikli et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). (El-Ghannam and Ducheyne, \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Physicochemical characterizations\u003c/h2\u003e\n \u003cp\u003eThe synthesized composites were studied for their physical properties by Fourier transform infrared (FTIR), x-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS), HR-TEM, EDX, and Zeta potential and particle size analysis instrumentation. Details of the procedure are given below.\u003c/p\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.8.1 FTIR analysis\u003c/h2\u003e\n \u003cp\u003eThe functionality of the synthesized composites was performed to confirm all the distinct components. The FTIR characterization was performed using the KBr pellet method with IRTRACER-100 Infra-Red absorption spectroscopy (Shimadzu). The spectra were recorded within the region of 4,000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.8.2 XRD analysis\u003c/h2\u003e\n \u003cp\u003eA diffractometer system (ECO D8 ADVANCE) was used to identify the phase of all prepared composites and crystallinity with Cu K\u003csub\u003e\u0026alpha;\u003c/sub\u003e incident radiation over the 2\u0026theta; range from 10\u0026deg; to 80\u0026deg;, working at 40 kV and 30 mA and a scanning rate (2\u0026theta;) of 0.02\u0026deg;.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e2.8.3 Morphology analysis by SEM and HR-TEM techniques\u003c/h2\u003e\n \u003cp\u003eScanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) (TESCAN, VEGA3 SBH) were utilized to determine the surface morphology of the minerals present in the prepared composites. A glass plate coated with acetone-dispersed composites was used for the SEM morphological analysis.\u003c/p\u003e\n \u003cp\u003eA FEI Techni G220 S-TWIN high-resolution transmission electron microscope (HR-TEM) was employed to evaluate the microstructures of all prepared composites. Selected area electron diffraction (SAED) was used to classify the constituent phases. HR-TEM was performed on a copper grid coated with acetone-dispersed composites.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e2.8.4 Zeta potential and particle size analysis\u003c/h2\u003e\n \u003cp\u003eThe composite particle size and surface charge were assessed using dynamic light scattering (DLS) using the Delsa analyzer instrument (Beckman colter) at 25\u0026deg;C with water as a dispersant.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.8.5 Bioactivity analysis in SBF solution\u003c/h2\u003e\n \u003cp\u003eAn SBF solution with an ionic concentration comparable to blood plasma was prepared using the procedure mentioned above (Section \u003cspan class=\"InternalRef\"\u003e2.6\u003c/span\u003e). Three samples of the final composite were placed in a sealed bottle with the SBF solution and maintained for 1 day, 3 days, and 7 days at room temperature. The solution was refreshed every day. The bioactivity and hydroxyapatite-formation ability of the composites were analyzed using SEM and XRD techniques.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Biological studies\u003c/h2\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e2.9.1 \u003cem\u003eIn-vitro\u003c/em\u003e cell culture\u003c/h2\u003e\n \u003cp\u003eThe American type culture collection (ATCC PCS-500\u0026ndash;012) was the source of the human bone marrow mesenchymal stem cells (hBMSCs), which were then maintained in 24-well tissue culture plates with the addition of 10% fetal bovine serum (FBS), Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, GIBCO), and minimal essential media (Hi-Media Laboratories). To prevent bacterial infection, 100 UmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of streptomycin and 100 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of penicillin were administered for 48 h. Next, the culture environment was adjusted to a 37\u0026deg;C humidified atmosphere with 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e (Szymon et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e2.9.2 hBMSC attachment and proliferation\u003c/h2\u003e\n \u003cp\u003eAll testing samples were added to DMEM/F12 (50:50 ratio) containing 10% FBS and the bare minimum of necessary media for 150 min prior to cell seeding. Prior to this immersion, all composites were sterilized with 75% alcohol and washed three times with PBS solution. Untreated cells were included as a control group, and the cells were detached at 24 h, 36 h, and 48 h. To assess cell attachment and proliferation, they were seeded into new 96-well cell plates at a density of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e and 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well, respectively, and left for 24 h. Next, 2 mL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each sample in serum-free medium, and the samples were developed for 4 h at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. A DMSO solution (10%) was added to dissolve the formazan crystals. The composites\u0026rsquo; capacity for cell proliferation was assessed by their optical density (OD) values at 570 nm on the spectrophotometric microplate (Neo et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) using the following equation. This experiment was repeated in triplicate.\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1731326498.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e2.9.3 Osteogenic differentiation analysis\u003c/h2\u003e\n \u003cp\u003eReal-time polymerase chain reaction (RT-PCR) was used to measure the mRNA levels of the osteogenic marker genes such as osteocalcin (OCN), runt-related transcription factor (RUNx), and vascular endothelial growth factor (VEGF), in order to conduct the osteogenic differentiation study. After 24 h, 36 h, and 48 h of culture, the culture plates were washed with PBS and suspended in 1 mL of cold TRIzol Reagent (Life Technologies Co.). Each sample\u0026rsquo;s total RNA was extracted using the standard TRIzol protocol, and the extract was then resuspended in 50 \u0026micro;L of RNase-free water. Next, cDNA was produced using the transcriptase reaction mix method (SuperScript III First-Strand Synthesis System, Life Technologies), and the produced cDNA was kept frozen at \u0026minus;\u0026thinsp;20\u0026deg;C. Using a power SYBR green RT-PCR kit (Life Technologies), quantitative PCR analysis was carried out (n\u0026thinsp;=\u0026thinsp;3). The transcripts of the other relative gene were defined using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous housekeeping gene (Liao et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe results were examined statistically using one-way ANOVA in Origin Pro 8.5 software. A significance threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was established.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 FTIR characterizations\u003c/h2\u003e \u003cp\u003eThe prepared HAP, mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composite formation and the interactions between the components in the composites were determined by FTIR spectroscopy. Initially, the HAP formation was confirmed by the FTIR spectrum presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The phosphate ions (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e) are the main constituents of HAP and appear as absorbance peaks between 1,200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The HAP ceramic featured peaks at 431 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 542 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 726 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 936 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1028 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1,121 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (ν\u003csub\u003e3\u003c/sub\u003e to ν\u003csub\u003e6\u003c/sub\u003e), corresponding to PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e stretching vibrations, and a peak at 599 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e deformation vibrations. As with different key peaks, -OH ions were found to correspond to the wide-ranging band from approximately 3,700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2,500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The intensity of the peak was about 3,472 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (ν\u003csub\u003e1\u003c/sub\u003e), a typical peak of the stretching frequency of -OH ions (Wang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Characteristic vibrations of PSSS, PVP polymers, mHAP ceramics, and LNZ were noted in the FTIR spectra for mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites and shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;F. The absorption peaks at 431 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 542 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 735 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 932 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1,060 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1,130 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated the presence of the PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e ions of mHAP within the mHAP/PSSS-PVP composite. The characteristic peaks of PVP were identified as a large -OH absorption stretching band at 3,097 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which represented the polymeric affiliation of the unfastened hydroxyl organizations and bonded -OH stretching vibration (Hao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The characteristic peaks of the LNZ-loaded mHAP/PSSS-PVP composite were obtained, indicating the interactions between the mHAP, PVP, PSSS, and LNZ components. The FTIR spectrum of the mHAP/PSSS-PVP/LNZ composite showed the functional peaks of LNZ in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eF. These were 1769 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for -the C\u0026thinsp;=\u0026thinsp;O stretching, 1,226 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1,350 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the -C-O stretching, 2,722 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the asymmetric vibration, 1,072 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the -C-N stretching of the amine group, 3,298 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the -N\u0026ndash;H stretching in the presence of the 2\u0026deg; amine, and 1,075 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the -C-F stretching frequency (Bigi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The bands located at 425 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 737 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 936 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1059 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1,115 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were attributed to the PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e ions in the matrix. The mHAP/PSSS-PVP/LNZ composite revealed a specific broad absorbance at 3,090 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for -OH stretching and another at 2,721 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for alkyl C\u0026ndash;H stretching. The C\u0026ndash;O and C\u0026ndash;C stretching appeared as a sharp peak at 1,225 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The absorption peak at 1,447 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represented the symmetric bending mode of the -CH\u003csub\u003e2\u003c/sub\u003e group of the LNZ and PSSS-PVP groups. The FTIR peaks that appeared in the expected regions indicated the formation of the desired LNZ-loaded mHAP/PSSS-PVP composite. Shattering peaks in the range of 1,000\u0026ndash;1,100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e suggested phosphate group of the HAP and it may have produced a weaker crystallization after substituting Sr and the PSSS and PVP polymers (Atul and Shitalkumar, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 XRD investigations\u003c/h2\u003e \u003cp\u003eXRD evaluation was utilized to analyze the phase and crystallinity characteristics of the pure HAP, mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites. The spectra are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The diffraction peak in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eA corresponds to pure HAP ceramic. The peaks at 2θ values of 25.8\u0026deg;, 29.6\u0026deg;, 31.1\u0026deg;, 32.3\u0026deg;, 39.8\u0026deg;, 47.2\u0026deg;, and 48.6\u0026deg; correspond to the 201, 217, 211, 300, 310, 222, and 213 planes, respectively. The perceived diffraction peaks are recognized by the preferred JCPDS (File no.09-0432) report and suggest a crystalline shape of HAP (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The XRD spectrum of the mHAP matrix is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and represents the Sr substitution in HAP, showing the crystalline pattern. After substituting Sr in HAP, the crystalline behavior was found to be less intense and broader than pure HAP. This confirms the reduced crystallinity and size of the HAP crystallites due to the partial substitution of the Ca ion by a Sr ion (Pushpalatha et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The new peak of the mHAP at 2θ value 27.3\u0026deg;, corresponding to the 002 plane, appeared due to the presence of mineral Sr in the HAP lattice (Guo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The XRD spectra of the mHAP/PSSS, mHAP/PVP, and mHAP/PSSS-PVP matrix are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;F. The Sr-substituted HAP ceramics lose their crystalline behavior after the addition of polymers such as PSSS and PVP due to the dilution of the ceramics by the organic compounds. The mHAP crystalline peaks are retained in a reduced form, and these observations confirm the interactions of the polymers with the mHAP ceramics (Guo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, mHAP/PSSS-PVP/LNZ peaks at 2θ values of 27.3\u0026deg; and 32.3\u0026deg; are observed. These diffraction peaks confirm the formation of an mHAP/PSSS-PVP/LNZ composite with a reduced crystalline structure. The mHAP/PSSS-PVP/LNZ composite result showed shifted and wider distinctive peaks of HAP, including the low-intensity phases of the PSSS and PVP polymers. It confirmed the development of the composites with the interaction of mHAP, PSSS, PVP, and LNZ (Lei et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Surface morphology and elemental mapping analysis\u003c/h2\u003e \u003cp\u003eA microstructural evaluation of the composites HAP, mHAP, mHAP/PSSS, mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ was performed, and the outcomes are given in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;F. The HAP crystal exhibited a flaky particle-like morphology under the calcination process due to the influence of the sintering temperature. In the case of Sr-substituted HAP, a highly aggregated particle-like morphology can be observed for the synthesized mHAP particles in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. The mHAP/PVP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites also exhibited particle-like morphology and are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;F. The double polymer and LNZ loading played a crucial role in the morphological changes in these composites. The aggregated particles jointly formed a connected network of particles in the mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ composites (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003eThe EDX spectra are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eG. The EDX examination established that the substituted mineral (Sr) was present with the Ca, P, and C. The mHAP/PSSS-PVP/LNZ composite was formed with a Ca-to-P atomic ratio of 1.81, as evidenced by EDX and elemental mapping studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In medical applications, a small amount of porosity is required for the development of bone cells to create a successful implant attachment (Rajesh and Ravichandran, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In other words, because of its increased capacity to bond to bio-metallic devices, porosity is beneficial as it improves osseointegration. However, a substantial porosity volume may accumulate body fluid and be dangerous. Increasing the amount of porous structure also harms the mechanical properties, particularly the binding strength, which encourages fracturing (Hammood et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, it is suggested that the prepared material\u0026rsquo;s porous ratio be well controlled to ensure optimal physicochemical and biochemical efficiency. The obtained mHAP and composites are aggregated particles that jointly form a connected network of particles with a porous nature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Morphological analysis by HR-TEM\u003c/h2\u003e \u003cp\u003eThe microstructures of the mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ composites were further estimated by HR-TEM analysis and the results were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e. This showed that the two polymers and the LNZ drug particles were connected in some places by a rod-like morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This is additional evidence of the interaction between these two polymers, the drug, and the ceramic composite. The morphological changes of the aggregated particles are the positive result of the LNZ drugs adjusting the crystal morphology of the mHAP/PSSS-PVP matrix. The SAED pattern of the mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ composites is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eC \u0026amp; D. The SAED outcomes support the concept of the influence of the LNZ drug on the crystallinity of the mHAP/PSSS-PVP matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Zeta potential and particle size determination\u003c/h2\u003e \u003cp\u003eThe modulation of physicochemical properties by the LNZ drug in the mHAP/PSSS-PVP/LNZ composite will similarly modify the biological activity of the composite to favor more rapid bone healing than the mHAP/PSSS-PVP composite. The best indicator of bioactivity is the formation of apatite crystals on the composite surface. A negatively charged surface is more conducive to the development of apatite crystals than a positively charged surface (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The measurement of the composite\u0026rsquo;s surface charge will support the determination of the bioactivity of the implants. A lower zeta potential value was observed for the mHAP/PSSS-PVP/LNZ composite than for the mHAP/PSSS-PVP composite. This lower zeta potential value may have been caused by the incorporation of the LNZ drug into the mHAP/PSSS-PVP/LNZ composite because linezolid has functional groups such as nitrogen, oxygen, and fluorine that carry a negative charge. The surface charges of mHAP/PSSS-PVP and mHAP/PSSS-PVP/LNZ are found to be \u0026minus;\u0026thinsp;14.8 mV and\u0026ndash; 40.3 mV, respectively. The zeta potential results proved that the mHAP/PSSS-PVP/LNZ composite would function as a superior bioactive material in terms of the interactions of the biological molecules. The mobility distribution curve is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A and B). A particle size distribution analysis was also carried out. The particle size increased from ~\u0026thinsp;156.7 nm to ~\u0026thinsp;207.8 nm after loading the LNZ composite onto the mHAP/PSSS-PVP composite. This extra organic moiety has influenced the size of the composite, as evidenced by this analysis. The particle size distribution curve is displayed in Figs.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Bioactivity of the composite in SBF solution\u003c/h2\u003e \u003cp\u003eThe bioactive character of the composite will be demonstrated by its capacity to form bone chemical bonds. This is a major factor in determining the success of an \u003cem\u003ein-vivo\u003c/em\u003e implantation. The generated apatite particles will facilitate the osseointegration process, aiding in the formation of new bone. After soaking the mHAP/PSSS-PVP/LNZ composite implant in SBF solution for 1 day, 3 days, and 7 days, the development of apatite crystals was determined by analyzing the bone-bonding characteristics of the implant. Figures\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;C show the morphological study by SEM analysis that verified the apatite production. The apatite crystals on the mHAP/PSSS-PVP/LNZ composite appeared to be taller when the period increased and white particles became visible. The apatite crystals took the form of a particle structure after 7 days of immersion. This outcome validates the bioactive behavior of the implant, confirming its safety for additional \u003cem\u003ein-vivo\u003c/em\u003e implantation.\u003c/p\u003e \u003cp\u003eThe XRD diffraction pattern obtained after immersing the mHAP/PSSS-PVP/LNZ composite in SBF solution demonstrated that the apatite crystals that were generated were weakly crystalline (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, apatite development on the mHAP/PSSS-PVP/LNZ composite caused the peak at 32\u0026deg; to rise after the first day of immersion. These results are favorable for apatite formation and confirm that the composite has bone regeneration ability.\u003c/p\u003e \u003cp\u003eThe rapid growth of biomaterials poses significant issues when designing and preparing composite materials to repair and facilitate the regeneration of damaged or injured tissues (Gu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is anticipated that a suitable biomaterial for bone tissue regeneration would not only be bioactive but also biocompatible and would ultimately be substituted by freshly formed tissue (Metcalfe and Ferguson, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). When embedded in a body, the bone-like apatite-forming potential is a significant prerequisite for a medicinal substance to possess bone-bonding characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Biological studies\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.7.1 \u003cem\u003eIn-vitro\u003c/em\u003e cell viability and live cells\u003c/h2\u003e \u003cp\u003eThe biocompatibility of the prepared mHAP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites was assessed using human bone marrow mesenchymal stem cells (hBMSCs). Throughout the entire examination process, untreated cells were used as a control. The optical microscopic pictures of hBMSCs following varied composite treatments for varying durations of time are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Compared to the control and other treatment days, the cells looked more compact and multiplied at 7 days and 14 days of incubation, especially after receiving mHAP/PSSS-PVP/LNZ combination treatment. In addition, there were comparatively fewer dead cells than in other instances. Figure\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows that the mHAP/PSSS-PVP/LNZ composite is more biocompatible and has more potential to promote osteogenesis than the mHAP and mHAP/PSSS-PVP composites. This might be because the mHAP/PSSS-PVP and LNZ components work together, and the inclusion of LNZ does not change or lessen the mHAP/PSSS-PVP composite\u0026rsquo;s capacity to be biocompatible. The current testing and analysis did not reveal any toxic effects on bone regeneration.\u003c/p\u003e \u003cp\u003eThe mHAP/PSSS-PVP/LNZ composite showed greater cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) than the other composites. The results above are further validated by a quantitative MTT assay. After 24 h of treatment, the cells treated with the mHAP/PSSS-PVP/LNZ composite had the greatest proportion of viable cells (96%) observed. Comparing this percentage to control cells and the other two composites, mHAP and mHAP/PSSS-PVP reveals a considerable increase. After 24 h of incubation, only 85% of the cells on the mHAP ceramic were still alive.\u003c/p\u003e \u003cp\u003eFollowing the preparation of the composite using PSSS-PVP polymers, the viability was significantly enhanced, reaching 91% (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). This finding implies that these polymers are not hazardous. Subsequently, the antibiotic LNZ compound exhibited no cytotoxic effect but promoted cell proliferation. Finally, osteogenic cell proliferation is not adversely affected by adding extra ingredients to the mHAP matrix, such as polymers and antibacterial drugs. The mHAP/PSSS-PVP/LNZ composite that has been created is ideal for applications involving the regeneration of bone tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.7.2 Analysis of cell differentiation by RT-PCR analysis\u003c/h2\u003e \u003cp\u003eOCN, VEGF, and RUNx2 gene expression are among the tests that are performed using real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e09\u003c/span\u003e displays the RUNx2 (core-binding factor alpha 1) activities of hBMSCs on a range of testing samples throughout varying culture times, including 24 h. In each sample, the mHAP, mHAP/PSSS-PVP, and mHAP/PSSS-PVP/LNZ composites boosted RUNx2, OCN, and VEGF activity. According to an \u003cem\u003ein-vitro\u003c/em\u003e cell viability measurement, good cell viability was obtained at 24 h after treatment with the mHAP/PSSS/PVP/LNZ composite because of the coexistence of strontium ions and LNZ compounds. This may be explained by the fact that the mHAP/PSSS-PVP/LNZ composite\u0026rsquo;s osteoconductive properties promote increased RUNx2, OCN, and VEGF synthesis. Furthermore, as the final composite for RUNx2, OCN, and VEGF production, the blot PCR analysis on RUNx2 and OCN also demonstrated the most enhanced production of osteogenesis genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The activity on the mHAP/PSSS-PVP/LNZ composite was significantly higher than on the mHAP and mHAP/PSSS-PVP composite treatments, including the control groups. These results imply that hBMSCs treated with the mHAP/PSSS-PVP/LNZ composite achieved a good amount of cell differentiation in addition to cell proliferation.\u003c/p\u003e \u003cp\u003eThe RT-PCR analysis of OCN, VEGF, and RUNx2 gene expression provided evidence that the materials are biocompatible and can promote osteogenesis. The process of creating the apatite layer on the implant\u0026rsquo;s surface is already well-known (Diomede et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Prabakaran and Rajan, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). First, a positively charged substrate ion is produced by the calcium ions in the SBF solution electrostatically bonding with the hydroxyl substrate ion in the HAP. Second, the apatite layer is formed as a result of phosphate deposition on the cationic Ca\u003csup\u003e2+\u003c/sup\u003e layer, which positively influences the formation of the solid mineral surface of the bone. The hydroxyl ion group functions as an additional nucleation site for biomineralization in the mHAP/PSSS-PVP combination. The apatite layer\u0026rsquo;s mineralization activity has been enhanced by recent research by combining PSSS and PVA polymers.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eLNZ was successfully combined with strontium (Sr)-substituted HAP as an mHAP matrix and PSSS- and PVP-reinforced composites in this work. \u003cem\u003eIn-vitro\u003c/em\u003e experiments, such as hBMSC proliferation and differentiation, revealed that the synthesized mHAP/PSSS-PVP/LNZ composite shows superior bioactive osteogenic potential. This could be described as the synergistic osteogenic potential of strontium ions and LNZ compounds in the mHAP/PSSS-PVP/LNZ composite. The bioactivity in the SBF solution and the characterization of those samples suggest that it would be a better implant composite. The highest apatite formation was observed on the mHAP/PSSS-PVP/LNZ composite due to its more electronegative nature inducing calcium ion nucleation, and the negative charge of the materials was confirmed by zeta potential analysis such as \u0026minus;\u0026thinsp;14.8 mV and \u0026minus;\u0026thinsp;40.3 mV. The composite has good viability and can enhance osteogenic gene expression. Therefore, the current work validates that the LNZ compound with an mHAP-reinforced polymeric matrix is worthy of orthopedic application. Moreover, the divalent strontium ions also encourage osteoblast proliferation and support bone regeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHL: data curation, formal analysis, investigation, methodology, visualization, writing\u0026ndash;original draft. QD: investigation, methodology, resources, validation, writing\u0026ndash;original draft. P-YG: data curation, formal analysis, investigation, resources, writing\u0026ndash;review and editing. Y-TY: data curation, formal analysis, investigation, writing\u0026ndash;original draft. SM- data curation, formal analysis, investigation, writing\u0026ndash;reviewing and editing. AB- investigation, writing\u0026ndash;reviewing, and editing. KS- data curation, formal analysis, investigation, writing\u0026ndash;reviewing and editing. XL: conceptualization, funding acquisition, investigation, methodology, project administration, writing\u0026ndash;original draft, writing\u0026ndash;review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research, authorship, and/or publication of this article from the Priority Union Foundation of Yunnan Provincial Science and Technology Department and Kunming Medical University (202401AY070001-235). The author would like to thank Deanship of Scientific Research at Majmaah University for supporting this work under project number R-2024-1110.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmarnath Praphakar, R., Sumathra, M., Sam Ebenezer, R., Vignesh, S., Shakila, H., and Rajan, M. (2019). Fabrication of bioactive rifampicin loaded \u0026kappa;-car-MA-INH/nano hydroxyapatite composite for tuberculosis osteomyelitis infected tissue regeneration. Int. J. Pharm. 565, 543\u0026ndash;556. 10.1016/j.ijpharm.2019.05.035.\u003c/li\u003e\n\u003cli\u003eAtul, M. K., and Shitalkumar, S. P. (2017). 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Material 60, 012056. 10.1088/1757-899x/60/1/012056.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biomedical, Hydroxyapatite, Linezolid, Osteomyelitis, Polyvinyl pyrrolidone, Poly(sodium 4-styrene sulfonate)","lastPublishedDoi":"10.21203/rs.3.rs-5280375/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5280375/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe primary objective of this investigation is to rectify bacterial infections in bone (osteomyelitis) and bone regeneration by utilizing an antibiotic-loaded hydroxyapatite polymer composite. In this regard, strontium (Sr)-substituted hydroxyapatite (mHAP)-reinforced polymeric composites with linezolid (LNZ) were utilized for osteomyelitis-affected bone repair. The brittle nature of the mHAP ceramic was overcome by adding with polymers such as polyvinyl pyrrolidone (PVP) and poly(sodium 4-styrene sulfonate) (PSSS). The composite formation, crystallinity, surface morphology, and zeta potential were investigated by Fourier Transform Infrared (FTIR), x-ray diffraction (XRD), scanning electron microscopy with Energy dispersive X-ray spectroscopy (SEM-EDX), high resolution - transmission electron microscopy (HR-TEM), and Zeta potential and particle size analysis techniques. The particle size and zeta potential were noted, and the zeta potential values of mHAP/PVP-PSSS and mHAP/PVP-PSSS/LNZ composites were found to be \u0026minus;\u0026thinsp;14.8 mV and \u0026minus;\u0026thinsp;40.3 mV, respectively. The bioactive results with SBF favored apatite formation and confirmed the composite\u0026rsquo;s biocompatibility with new bone formation. The cell viability of human bone marrow mesenchymal stem cells (hBMSCs) and the gene expression analysis confirmed the osteogenic potential of the prepared materials. Because the prepared composite obtained promising results, these studies confirm that the prepared composite can release the antibiotic for the treatment of osteomyelitis-affected bone repair.\u003c/p\u003e","manuscriptTitle":"Linezolid combined Strontium substituted hydroxyapatite-Bi polymeric composite for Osteomyelitis affected bone regeneration analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 12:02:59","doi":"10.21203/rs.3.rs-5280375/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5559c2aa-2147-47ff-905f-954ab5885f97","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-20T06:54:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-11 12:02:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5280375","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5280375","identity":"rs-5280375","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}