Self-oriented hydroxyapatite nanorods in gelatin matrix via single- directional evaporation for bone defect repair

Self-oriented hydroxyapatite nanorods in gelatin matrix via single- directional evaporation for bone defect repair | 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 Research Article Self-oriented hydroxyapatite nanorods in gelatin matrix via single- directional evaporation for bone defect repair Xiaoyang Liu, Tan Chen, Yushuai Cheng, Wei He, Zhengke Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9273382/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 Bone, as a typical anisotropic material, with hydroxyapatite nanocrystals oriented in the direction of collagen fibrils. Drawing inspiration from nature, constructing bone defect repair scaffolds by mimicking the natural components and structures of bones to recreate the microenvironment at the bone defect site is an effective strategy. However, it is quite challenging to precisely control the orientation behavior of nanoscale hydroxyapatite nanorods. Here, for the first time, by adjusting the aspect ratio of HAP and the size of the hydrogel grid, the preferential orientation of HAP can be achieved through the directional evaporation of water in the hydrogel. And we introduced Fe 2+ into the hydroxyapatite lattice, while maintaining the original rod-like morphology of HAP without introducing the magnetic mineral impurity phase. Animal experiments indicated that the anisotropic gelatin/hydroxyapatite scaffolds can effectively accelerate the repair process of large-sized bone defects. hydroxyapatite nanorods gelatin self-orientation bone defect repair Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In orthopedics and cranio-maxillofacial surgery, large bone defects caused by severe trauma, tumor resection, infection, and other diseases remain widespread problems. Such severe bone defects not only cause patients pain and reduce their quality of life, but also shorten their expected lifespan, posing an urgent global medical challenge that needs to be addressed. Currently, most research on scaffolds developed for bone repair and regeneration is mainly focused on optimizing the composition of organic/inorganic materials. Commonly used natural macromolecules for bone repair scaffolds include collagen 1 , gelatin 2 , chitosan 3 , sodium alginate 4 , silk fibroin 5 , agarose 6 , hyaluronic acid 7 , etc. They exhibit excellent biocompatibility and facilitate the adhesion and growth of osteoblasts. However, their poor spatial stability and osteoinductivity limit their large-scale application. The inorganic components in bone repair scaffolds can mainly be classified into two types: phosphates and silicates. Among them, bioactive glass 8 (silica glass containing calcium and phosphate), hydroxyapatite (HAP) 9 , 10 , and tricalcium phosphate (TCP) 11 – 13 are the most common. Due to their chemical composition being similar to that of natural bone, inorganic scaffolds show excellent biocompatibility and promote the proliferation and differentiation of osteoblasts. However, calcium phosphate ceramics encounter difficulties in processing, and the lack of flexibility limits their application in load-bearing and deformed position 12 . In summary, scaffolds made of a single biomaterial have many problems. Currently, the best solution is composite scaffolds, which combine inorganic ceramics and organic polymers. At present, most bone repair composite scaffolds are prepared by physically mixing polymers and ceramic (nano) particles or adding ceramic (nano) particles to polymer solutions, followed by freeze-drying and other processes 14 – 16 . This often leads to random, aggregated, and non-structured distribution of ceramic particles in the composite materials, which is quite different from the structure of natural bones 17 , 18 . At the nanoscale, bones are composed of collagen triple helices and mineralized with highly substituted carbonate hydroxyapatite (HAP) biomineral nanocrystals 19 . The c-axes of these HAP nanocrystals are parallel to the long axes of the collagen fibrils, and their (100) crystal planes are roughly parallel to each other 17 , 20 . These mineralized collagen fibrils with a diameter of about 100 nm, non-collagenous fibrillar extracellular matrix, and minerals are assembled into layered lamellar structures of bone, forming the functional units of bone. These lamellae are arranged in various ways to form two types of cortical bone and trabecular bone 21 , 22 . That is, natural bones exhibit highly aligned, anisotropic, biphasic and ultrafine structural designs, as well as complex hierarchical assembly structures 23 . Therefore, it is increasingly important to study how microstructure affects the biological activity of polymer/ceramic composites and to utilize these theories to develop bone graft substitutes that are closer to natural bone tissue. As the main natural inorganic component of bone, hydroxyapatite is one of the most commonly used inorganic components in bone repair scaffold materials, with excellent biological activity, biocompatibility, osteoconductivity, non-toxicity, and non-inflammatory properties 24 . Studies have shown that HAP is very beneficial for bone construction, as it can stimulate growth factors (such as bone morphogenetic proteins) and promote the secretion of alkaline phosphatase in mesenchymal stem cells. Of course, there are differences between synthetic HAP and natural HAP in terms of color, size, morphology, and crystallinity, which, to some extent, weaken the osteogenic effect of synthetic HAP 25 . Interestingly, the special crystal structure of hydroxyapatite allows Ca 2+ in the lattice to be replaced by a series of metal ions of similar size, such as Mg 2+ , Zn 2+ , Sr 2+ , Fe 2+ , etc. 26–28 . Although the degree of these isomorphic substitutions is small, they greatly adjust many properties of HAP, such as biological activity, interaction between bone minerals and calcium phosphate-based implant materials, surface chemistry, charge, and morphology. The introduction of metal ions not only affects the crystal growth, the efficiency of drug delivery, the dissolution rate and solubility, but also exhibits some special properties, such as antibacterial and superparamagnetic properties 29 . This research successfully introduced Fe 2+ into the hydroxyapatite lattice (Fe-HAP) through the hydrothermal method. Unlike the previously reported irregular sheet-like or needle-like aggregates 30 – 34 , Fe-HAP is a shape-regular rod-shaped crystal without magnetite impurities. Thanks to the double-layer oleic acid ligands on the particle surface, Fe-HAP nanoparticles are uniformly dispersed in the gelatin (Gel) matrix. During the drying process, as the water in the Gel/Fe-HAP composite hydrogel is directedly evaporated, the Fe-HAP rod-shaped crystals spontaneously align along the direction of water evaporation. Animal experiments showed that the oriented arrangement of Gel/hydroxyapatite scaffolds can effectively promote the repair process of critical-sized bone defects. Results and discussion In the traditional research on Fe 2+ /Fe 3+ doped hydroxyapatite, whether by hydrothermal method, wet chemical precipitation method, or mechanochemical synthesis, the introduction of Fe is always accompanied by the appearance of magnetite or magnetite oxide 30 , 32 , 35 – 38 . Based on the coordination environment of Ca in the lattice of HAP, it can be classified into two types: Ca (I) forms coordination with six oxygen atoms belonging to four phosphate groups, while Ca (II) coordinates with seven oxygen atoms, among which six come from the four phosphate groups, and one comes from the hydroxyl group 36 . The tendency of Fe 2+ and Fe 3+ to replace Ca (I) or Ca (II) in the HAP unit cell is completely different since the differences in charge and ionic radii, which also lead to changes in the unit cell parameters 39 . According to density functional theory (DFT) calculations, Fe 3+ ions preferentially occupy the Ca(II) sites, while the preference of Fe 2+ for the Ca (I) and Ca (II) sites is comparable in terms of energy 39 , 40 . Considering that Fe 3+ would cause greater lattice distortion and the introduction of magnetite impurity phases 41 , this research selected Fe 2+ as the dopant. As shown in Fig. 1 B, the infrared calcium absorption peak of Fe-HAP was almost identical to that of the HAP nanoparticles prepared under the same conditions. Compared with HAP, the XRD peak position of Fe-HAP was slightly shifted, and the crystallinity was reduced (Fig. 1 C). Table S1 indicated that the introduction of Fe would lead to a contraction deformation of the crystal lattice. As shown in Fig. 1 D and Fig. 1 E, compared with pure HAP, the doping of Fe made the Fe-HAP nanoparticles slimmer, but still retained the rod-like morphology of traditional hydroxyapatite (length: 70–200 nm, diameter: 10–50 nm). The hydrodynamic size of Fe-HAP nanoparticles measured by DLS was found to be 78.8 nm, while the size of HAP nanoparticles prepared under the same synthesis conditions was 84.14 nm. The difference in hydrodynamic sizes between the two was the more elongated morphology of Fe-HAP. From the DLS particle size distribution graph (Fig. S1 ), the size distribution of Fe-HAP particles was wider, which was also consistent with the TEM images. Moreover, no Fe 3 O 4 impurity phase was observed in the TEM images, which was consistent with the XRD spectrum. To ensure the EDS signal, electron energy spectrum scanning (EDS) was performed at the Fe-HAP enriched position. As shown in Fig. 1 F, the EDS element mapping diagram indicated the uniform distribution of Ca, O, Fe, and P in the Fe-HAP aggregates, and no Fe 3 O 4 particle was found in the TEM image, indicating that Fe was successfully incorporated into the HAP lattice without Fe 3 O 4 impurity. In addition, through inductively coupled plasma spectroscopy, it can be confirmed that the Fe content in Fe-HAP is 22.83% (relative to the molar ratio of Ca). Traditional HAP nanoparticles exhibited diamagnetism 42 , 43 , and the introduction of Fe transformed the diamagnetism into superparamagnetism (with a saturation magnetization over 2.876 emu/g), thereby endowing Fe-HAP with potential applications in fields such as magnetic control orientation, magnetic thermal response, and magnetic imaging (Fig. S2). Compared with the pure gelatin scaffold, the appearance of the characteristic absorption peaks of PO 4 3− in the infrared spectrum (the absorption peaks at 1090 and 1040 cm − 1 correspond to the asymmetric stretching vibration of PO 4 3− ) indicated the successful introduction of Fe-HAP nanoparticles, while the enhancement of the methylene peaks at 2853 and 2926 cm − 1 was due to the oleic acid ligands on the surface of Fe-HAP nanoparticles(Fig. S3). The content of Fe-HAP nanoparticles in the Gel/Fe-HAP composite scaffold could be determined from the thermogravimetric curve (Fig. S4), which was 4.83 wt%. To investigate the distribution of HAP and Fe-HAP nanoparticles in the gelatin matrix, the frozen sections of Gel/HAP and Gel/Fe-HAP were placed under the TEM for observation. As shown in Fig. 2 A, HAP was uniformly dispersed in the gelatin matrix, and the long axis direction of the nanoparticles seemed to preferentially align horizontally. However, the orientation behavior was not obvious. In contrast, Fe-HAP exhibited a significant orientation arrangement in the gelatin matrix: rod-shaped nanoparticles were uniformly dispersed in the gelatin matrix, and the long axis direction was neatly arranged in the "upper left - lower right" direction (Fig. 2 B). To explore the influence of the hydrogel matrix on the orientation behavior of Fe-HAP, methylacrylated gel (GelMA) was selected to replace gelatin to prepare GelMA/Fe-HAP composite scaffolds, and the distribution of nanoparticles in the GelMA/Fe-HAP composite scaffolds was observed under TEM. Fe-HAP particles were uniformly dispersed in the GelMA matrix, but did not show any directional tendency of arrangement (Fig. 2 C). As shown in the schematic diagram on the left, within the same gelatin matrix network, HAP and Fe-HAP nanoparticles exhibited different orientation behaviors, and even for the same Fe-HAP nanoparticles, their orientation behaviors in the gelatin matrix and GelMA matrix were completely different. Considering that the orientation process of Fe-HAP nanoparticles in the hydrogel matrix was related to the three-dimensional network of the hydrogel. In this research, one-dimensional SAXs curves of gelatin (Gel) at room temperature, GelMA hydrogel at room temperature, and gelatin at 50°C (Gel 50°C) were obtained (Fig.S5). The one-dimensional SAXs curves were fitted using the gel model in the SasView software within the range of 0.01 Å −1 to 1 Å −1 . The fitting function was shown as the following formula. $$\:I\left(Q\right)\approx\:\frac{{I}_{L}\left(0\right)}{{\left(1+\left[\left(D+1\right)/3\right]{Q}^{2}{\xi\:}^{2}\right)}^{\frac{D}{2}}}+{I}_{G}\left(0\right)\cdot\:{e}^{-{Q}^{2}{R}_{g}^{2}/3}+B$$ Here, I L and I G corresponded to the Lorentz model and the Guinier model respectively; D represented the fractal dimension. When D = 2, the first term of the above equation could simplify to the Ornstein-Zernicke model; ξ represented the correlation length, which could be used to evaluate the average size of the cross-linked regions in the hydrogel, and the smaller ξ was, the smaller the size of the cross-linked regions in the hydrogel was 44 ; Rg indicated the mean square rotation radius; B represented the background scattering. Through fitting, the relevant lengths of GelMA, Gel, and Gel at 50°C were 4.67 nm, 6.52 nm, and 10.05 nm, respectively, indicating that the cross-linking network sizes of the three hydrogels gradually increased. At room temperature, the gelatin network was composed of the physical cross-linking of β-folding of gelatin molecules and the chemical cross-linking of glutaraldehyde, and the physical crosslinks unfolded at 50°C, thereby causing an increase in the hydrogel's relevant length. Furthermore, the orientation process of gelatin/Fe-HAP during the water evaporation process was analyzed by small-angle X-ray scattering (SAXs), and the two-dimensional scattering images of SAXs were integrated using Fit. 2d software to obtain the azimuthal angle curves after different evaporation times. As shown in Fig. 3 B, the original state of the Gel/Fe-HAP composite scaffold was an isotropic hydrogel. With the continuous evaporation of water, the two-dimensional scattering images gradually tended to be elliptical. After 4 hours, the Gel/Fe-HAP composite scaffold exhibited obvious anisotropy. The SAXS azimuthal angle curve indicated that as water evaporated, Fe-HAP gradually aligned along the 180° azimuthal angle in the gelatin network (Fig. 3 C). After 4 hours, Fe-HAP preferentially aligned along the z-axis direction of the hydrogel. Based on the formula, the orientation degree π at this time was calculated to be 0.752. As shown in Fig. 3 D, the x-axis and y-axis directions of the Gel/Fe-HAP composite hydrogel gradually solidified and dried as water evaporated. Taking a single grid at the center position as an example. The water molecules in the hydrogel grid could only escape along the z-axis direction, thereby forcing the Fe-HAP nanorod confined within the network to align along the z-axis direction. Due to the different sizes of the hydrogel grids, overly small grids would restrict the rotation of Fe-HAP nanoparticles, thereby hindering the orientation behavior of the nanorods. The glutaraldehyde vapor cross-linked gelatin network size was smaller at room temperature, but when dried at 50°C, the gelatin matrix underwent β-folding expansion, and the grid size increased, thereby allowing the Fe-HAP nanorods to rotate and orient (Fig. 3 A). However, the GelMA hydrogel had an overly small grid size, which restricted the torsional orientation of Fe-HAP nanoparticles (Fig. 2 C). External and internal (for the x-y plane) sections of the same Gel/Fe-HAP composite hydrogel were made, and the orientation of Fe-HAP nanoparticles was observed under TEM. As shown in Fig.S6, the outer layer of the composite hydrogel had inconsistent directions for water evaporation, and the arrangement of Fe-HAP particles was rather disordered, with only some particles orienting along the z-axis. In the internal part of the composite hydrogel, after the evaporation process, the water was discharged in a single direction, and Fe-HAP was arranged in an oriented manner, which further verified the theory proposed earlier that the single-directional evaporation of water vapor led to the orientation arrangement of Fe-HAP nanoparticles. To further verify the influence of the aspect ratio of rod-shaped nanoparticles on the orientation behavior, we prepared pure HAP nanoparticles with an aspect ratio similar to that of Fe-doped hydroxyapatite by adjusting the pH value. As shown in Fig.S7A, after adjusting the pH value of the hydrothermal reaction solution to 10.5, HAP exhibited a long rod-like morphology, with a length of 100–400 nm and a diameter of 10–30 nm. Using these HAP nanorods to prepare Gel/HAP composite scaffolds, we induced the HAP nanorods to align in the preset direction using the same method. The SAXs (Fig.S7B) and TEM (Fig.S7C) images showed that the HAP nanorods were oriented and arranged in the gelatin matrix. The above results indicated that the orientation behavior of HAP nanoparticles in the hydrogel matrix was affected by the hydrogel network and the aspect ratio of the nanoparticles: when the hydrogel cross-linking density was too high, the grid size was too small, which limited the rotational orientation of the nanorods; the aspect ratio of the nanoparticles determined the magnitude of the deflection force that the nanorods received during the directional evaporation of water, and nanoparticles with a too small aspect ratio received insufficient deflection force and could not achieve orientation arrangement. Given that Fe 2+ could promote bone formation by stimulating vascular growth 45 – 48 , in subsequent animal experiments, we selected the Gel/Fe-HAP scaffold for the bone defect repair. Considering that the bone defect repair scaffold needed sufficient porosity to ensure cell migration and proliferation, as well as the exchange of nutrients and metabolic waste, we introduced PEG-4000 as a pore-forming agent into the Gel/Fe-HAP composite hydrogel system (As shown in Fig.S8, the introduction of PEG-4000 did not affect the orientation behavior of Fe-HAP). After ethanol was used to remove PEG-4000, dense and uniform micron-sized pores were successfully formed on the Gel/Fe-HAP scaffold (Fig.S9). The rough surface also facilitated the adhesion and migration of osteogenic-related cells. In addition, this research uniformly drilled through holes on the scaffold using a micro-drill (500 µm) to further increase the porosity of the Gel/Fe-HAP scaffold. The three-dimensional morphology of the Gel/Fe-HAP scaffold was observed using micro-CT, and the distribution of the mineral phase in the scaffold and the overall porosity of the scaffold were analyzed (Fig.S10). The Gel/Fe-HAP scaffold presented a three-dimensional porous interconnected morphology, with a rough surface, micro-meter-sized pores, and a pore volume of 80.51% as analyzed by Imalytics software. By adjusting the CT signal range, the distribution of the mineral phase in the Gel/Fe-HAP composite scaffold could be obtained. As shown in Fig.S10B, Fe-HAP was uniformly distributed in the scaffold without obvious agglomeration. Of course, considering the micrometer resolution of micro-CT, the orientation behavior of Fe-HAP nanoparticles could not be obtained through CT scanning. As shown in Fig. 4 A, neither the pure gelatin (Gel) scaffold, the isotropic gelatin/Fe-HAP composite scaffold (I-Gel/Fe-HAP), nor the anisotropic gelatin/Fe-HAP composite scaffold (Gel/Fe-HAP) completely degraded after 6 weeks of implantation, showing the characteristic of gradually repairing from the defect edge to the defect center. Among the three groups of scaffolds, the Gel/Fe-HAP scaffold exhibited the best bone repair effect, with a BV/TV of 61.71%, significantly higher than 31.68% of the Gel scaffold and 46.48% of the I-Gel/Fe-HAP scaffold. At 12 weeks of repair, the differences in bone defect repair effects among the three groups were more obvious. The pure gelatin scaffold had a poor bone defect repair effect due to the lack of bone conductivity and bone induction: only partial new bone formation was observed at the edge of the defect area, and no hard tissue was generated in the defect center area. The BV/TV of the I-Gel/Fe-HAP scaffold at 12 weeks after surgery was 59.38 ± 5.36%, with a cavity remaining in the defect center area, and the newly generated bone tissue in the defect area was biased towards the bone marrow direction, with a thickness of only a thin layer, unable to restore the mechanical properties of the normal femur. The Gel/Fe-HAP scaffold group showed the best bone defect repair effect, with a BV/TV recovery to 74.63 ± 9.88% at 12 weeks, compared to the BV/TV of intact left leg femur of 85.37% (Fig. 4 B). The bone defect area was completely covered by newly generated bone tissue except for the small hole (d: 0.5-1mm) in the center, and the bone thickness at the defect center position had not yet recovered to the normal femur thickness. The excellent restorative effect of the scaffold could be attributed to its replication of the nanostructured extracellular matrix of natural bone cells, which was conducive to the migration, proliferation, and differentiation of osteogenic-related cells. In addition, the bone mineral density (BMD) of the Gel/Fe-HAP scaffold group at 12 weeks after implantation was 3.65 ± 0.61 g/cm³, the average bone trabecular thickness (Tb. Th) was 0.70 ± 0.26 mm, and the average bone trabecular separation (Tb. Sp) was 0.12 ± 0.05 mm, all of which were better than those of the Gel scaffold group and the I-Gel/Fe-HAP scaffold group (Fig. 4 C-E). The results of HE (Fig. 5 A) and Masson (Fig. 5 C) staining indicated that the scaffold had not completely degraded at 6 weeks after implantation. In the pure gelatin group, due to the lack of bone conductivity, new bone tissue only appeared at the edge of the defect after 12 weeks, while the center of the defect remained empty. The I-Gel/Fe-HAP scaffold contained Fe-HAP, which facilitated the migration of osteogenic-related cells towards the defect center. After 12 weeks, the newly formed bone tissue covered more than 50% of the bone defect area, but there were still obvious blank areas at the defect center. The Gel/Fe-HAP scaffold exhibited the best bone defect repair effect: the newly formed bone tissue was dense and uniform in thickness, almost completely covering the defect area (Fig. 5 B and 5 D). The immunohistochemical results of osteocalcin showed that the expression of osteocalcin in the new bone tissue of the Gel/Fe-HAP group was the highest, and the bone maturity was comparable to that of the surrounding normal bone tissue (Fig. 5 E). Furthermore, we evaluated the mechanical properties of the new bone tissue through nanoindentation tests. As shown in Fig.S11, after 12 weeks, the mechanical properties at the defect center position of the Gel/Fe-HAP group were the best, with a compressive modulus and hardness of 0.254 GPa and 5.467 GPa, respectively. Compared to the 0.293 GPa and 6.271 GPa of the normal bone tissue, the mechanical performance recovery rate was over 85%. The animal experiment results indicated that the Gel/Fe-HAP scaffold had good bone repair performance. The hydroxyapatite nanoparticles oriented along the long bone direction could effectively promote the bone defect repair process. Conclusion This research successfully prepared Fe-doped hydroxyapatite nanoparticles by taking advantage of the similarities between Fe 2+ and Ca 2+ . The Fe-HAP particles were rod-shaped and possessed superparamagnetism while avoiding the introduction of magnetite impurity phases. During the drying process of the hydrogel composite of gelatin/Fe-HAP, the directional evaporation of water caused the Fe-HAP nanoparticles to gradually align along the direction of water evaporation. An anisotropic gelatin/Fe-HAP bone repair scaffold using PEG-2000 as the pore-forming agent and supplemented with micropore drilling was prepared. In the rabbit femoral defect repair experiment, the anisotropic gelatin/Fe-HAP scaffold demonstrated excellent bone repair capabilities. The nano-hydroxyapatite with anisotropic bone orientation arrangement could effectively accelerate the bone defect repair process, and has a promising application prospect in clinical orthopedics. Declarations Acknowledgments: This work was supported by Research Project of Qizhen Innovation Concept Verification Center of Zhejiang University (No. GNYZ-2024010), National Natural Science Foundation of China (No. 51873187), Science Fund for Distinguished Young Scholars of Zhejiang Province (No.LR20E030004), National Basic Research Program of China (No. 2018YFC1004803). Author Contributions: X.L. conducted the experiments, analyzed the experimental data and wrote the paper. T.C., Y.C. and W.H. conducted the experiments. Z.W. proposed the concept, supervised this research, and revised the manuscript. Competing interests: Authors declare that they have no competing interests. Data and materials availability: All data are available in the main text or supplementary materials. References Li, X.; Li, Z. L.; Wang, P. L.; Lu, G. G.; Tong, L.; Liu, Q. Y.; Chen, Y. F.; Lin, J. L.; Luo, E.; Liang, J.; Jiang, Q.; Fan, Y. J.; Zhang, X. 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Huang, H.; Qiang, L.; Fan, M.; Liu, Y.; Yang, A.; Chang, D.; Li, J.; Sun, T.; Wang, Y.; Guo, R.; Zhuang, H.; Li, X.; Guo, T.; Wang, J.; Tan, H.; Zheng, P.; Weng, J., 3D-printed tri-element-doped hydroxyapatite/ polycaprolactone composite scaffolds with antibacterial potential for osteosarcoma therapy and bone regeneration. Bioactive Materials 2024, 31 , 18-37. Zhong, Z.; Wu, X.; Wang, Y.; Li, M.; Li, Y.; Liu, X.; Zhang, X.; Lan, Z.; Wang, J.; Du, Y.; Zhang, S., Zn/Sr dual ions-collagen co-assembly hydroxyapatite enhances bone regeneration through procedural osteo-immunomodulation and osteogenesis. Bioactive Materials 2022, 10 , 195-206. Li, X. Y.; Zou, Q.; Man, Y.; Li, W., Synergistic Effects of Novel Superparamagnetic/Upconversion HA Material and Ti/Magnet Implant on Biological Performance and Long-Term In Vivo Tracking. Small 2019, 15 (31). Tampieri, A.; D'Alessandro, T.; Sandri, M.; Sprio, S.; Landi, E.; Bertinetti, L.; Panseri, S.; Pepponi, G.; Goettlicher, J.; Bañobre-López, M.; Rivas, J., Intrinsic magnetism and hyperthermia in bioactive Fe-doped hydroxyapatite. Acta Biomaterialia 2012, 8 (2), 843-851. Zilm, M. E.; Staruch, M.; Jain, M.; Wei, M., An intrinsically magnetic biomaterial with tunable magnetic properties. Journal of Materials Chemistry B 2014, 2 (41), 7176-7185. Iannotti, V.; Adamiano, A.; Ausanio, G.; Lanotte, L.; Aquilanti, G.; Coey, J. M. D.; Lantieri, M.; Spina, G.; Fittipaldi, M.; Margaris, G.; Trohidou, K.; Sprio, S.; Montesi, M.; Panseri, S.; Sandri, M.; Iafisco, M.; Tampieri, A., Fe-Doping-Induced Magnetism in Nano-Hydroxyapatites. Inorganic Chemistry 2017, 56 (8), 4446-4458. Adamu, D. B.; Tufa, L. T.; Lee, J.; Zereffa, E.; Segne, T. A.; Razali, M. H., Facile synthesis of bismuth and iron co-doped hydroxyapatite nanomaterials for high-performance fluoride ions adsorption. Journal of Environmental Chemical Engineering 2023, 11 (6), 111196. Li, X.; Wu, J.; Li, D.; Zou, Q.; Man, Y.; Zou, L.; Li, W., Pro-osteogenesis and in vivo tracking investigation of a dental implantation system comprising novel mTi implant and HYH-Fe particles. Bioactive Materials 2021, 6 (9), 2658-2666. El-Maghrabi, H. H.; Younes, A. A.; Salem, A. R.; Rabie, K.; El-shereafy, E. S., Magnetically modified hydroxyapatite nanoparticles for the removal of uranium (VI): Preparation, characterization and adsorption optimization. Journal of Hazardous Materials 2019, 378 . Sheikh, L.; Sinha, S.; Singhababu, Y. N.; Verma, V.; Tripathy, S.; Nayar, S., Traversing the profile of biomimetically nanoengineered iron substituted hydroxyapatite: synthesis, characterization, property evaluation, and drug release modeling. Rsc Advances 2018, 8 (35), 19389-19401. Adamiano, A.; Iafisco, M.; Sandri, M.; Basini, M.; Arosio, P.; Canu, T.; Sitia, G.; Esposito, A.; Iannotti, V.; Ausanio, G.; Fragogeorgi, E.; Rouchota, M.; Loudos, G.; Lascialfari, A.; Tampieri, A., On the use of superparamagnetic hydroxyapatite nanoparticles as an agent for magnetic and nuclear in vivo imaging. Acta Biomaterialia 2018, 73 , 458-469. Isaev, D. D.; Kriventsov, V. V.; Petrov, S. A.; Bystrov, V. S.; Bulina, N. V., Substitution in the Structure of Hydroxyapatite Doped with Iron Cations Upon Mechanochemical Synthesis. Journal of Surface Investigation 2023, 17 (3), 687-693. Makshakova, O. N.; Shurtakova, D. V.; Vakhin, A. V.; Grishin, P. O.; Gafurov, M. R., Incorporation of Iron(II) and (III) in Hydroxyapatite-A Theoretical Study. Crystals 2021, 11 (10). Avakyan, L.; Paramonova, E.; Bystrov, V.; Coutinho, J.; Gomes, S.; Renaudin, G., Iron in Hydroxyapatite: Interstitial or Substitution Sites? Nanomaterials 2021, 11 (11). Pupilli, F.; Tavoni, M.; Drouet, C.; Tampieri, A.; Sprio, S., Iron-doped hydroxyapatite by hydrothermal synthesis: Factors modulating the Fe 2. Open Ceramics 2024, 18 . Nakayama, M.; Kajiyama, S.; Kumamoto, A.; Nishimura, T.; Ikuhara, Y.; Yamato, M.; Kato, T., Stimuli-responsive hydroxyapatite liquid crystal with macroscopically controllable ordering and magneto-optical functions. Nature Communications 2018, 9 . Baba, S.; Tanaka, S., Fabrication of c-axis oriented hydroxyapatite ceramics in a rotating high magnetic field using photopolymerization. Journal of the European Ceramic Society 2020, 40 (12), 4332-4339. Wisotzki, E. I.; Tempesti, P.; Fratini, E.; Mayr, S. G., Influence of high energy electron irradiation on the network structure of gelatin hydrogels as investigated by small-angle X-ray scattering (SAXS). Physical Chemistry Chemical Physics 2017, 19 (19), 12064-12074. Yang, R.; Li, G.; Zhuang, C.; Yu, P.; Ye, T.; Zhang, Y.; Shang, P.; Huang, J.; Cai, M.; Wang, L.; Cui, W.; Deng, L., Gradient bimetallic ion–based hydrogels for tissue microstructure reconstruction of tendon-to-bone insertion. 2021, 7 (26), eabg3816. Zhu, Y.; Li, Z. Y.; Zhang, Y. J.; Lan, F.; He, J.; Wu, Y., The essential role of osteoclast-derived exosomes in magnetic nanoparticle-infiltrated hydroxyapatite scaffold modulated osteoblast proliferation in an osteoporosis model. Nanoscale 2020, 12 (16), 8720-8726. Shuai, C. J.; Cheng, Y.; Yang, W. J.; Feng, P.; Yang, Y. W.; He, C. X.; Qi, F. W.; Peng, S. P., Magnetically actuated bone scaffold: Microstructure, cell response and osteogenesis. Composites Part B-Engineering 2020, 192 . Tang, Y. Q.; Wang, Q. Y.; Ke, Q. F.; Zhang, C. Q.; Guan, J. J.; Guo, Y. P., Mineralization of ytterbium-doped hydroxyapatite nanorod arrays in magnetic chitosan scaffolds improves osteogenic and angiogenic abilities for bone defect healing. Chemical Engineering Journal 2020, 387 . Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9273382","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":615267896,"identity":"a7a8e1ac-4112-4113-bf86-fe83d45869a8","order_by":0,"name":"Xiaoyang Liu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyang","middleName":"","lastName":"Liu","suffix":""},{"id":615267897,"identity":"dd11fd42-018f-4dc1-abc5-3b4567b73153","order_by":1,"name":"Tan Chen","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Tan","middleName":"","lastName":"Chen","suffix":""},{"id":615267898,"identity":"f345204b-8ad8-4b67-9cde-8ad44330e617","order_by":2,"name":"Yushuai Cheng","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yushuai","middleName":"","lastName":"Cheng","suffix":""},{"id":615267899,"identity":"05882301-1330-42d4-bcfb-a41910da19e9","order_by":3,"name":"Wei He","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"He","suffix":""},{"id":615267900,"identity":"e82b7310-ae24-4b3b-87bc-c8eb536560be","order_by":4,"name":"Zhengke Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYNCCAzYMjA1AmocELWmkazkMoYnSYnD87OGXP86ct2eekcD44G0bg7w5QS1n8tIsJG7cZmackcBsOLeNwXBnAwEtZgdyzAwMPtxmA2phk+ZtY0gwOEBIy/k3ZgYJH87xALWw/yZOy40c4wcHbhyQANnCTJQW+xtvzBgbziQbMPY8bJacc07CcAMhLZL9OcYffxyzszdsTz744U2ZjTxBW4CATQJEGjaAI1OCsHogYP4AIuWJUjsKRsEoGAUjEgAAhk5C9gofkZUAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Zhengke","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-03-31 02:39:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9273382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9273382/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105875587,"identity":"207f8a60-ed21-4bd0-934f-16b58a8ae5ea","added_by":"auto","created_at":"2026-04-01 05:41:01","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":609959,"visible":true,"origin":"","legend":"\u003cp\u003eA: The Schematic diagram of Fe-HAP, where Fe\u003csup\u003e2+\u003c/sup\u003e replaces some of the Ca\u003csup\u003e2+\u003c/sup\u003e; B: Infrared absorption spectra of HAP and Fe-HAP nanoparticles; C: Comparison of XRD spectra of HAP and Fe-HAP; TEM image of the HAP nanorods (D) and Fe-HAP nanorods (E); F: The TEM image of the enrichment area of Fe-HAP nanorods and the corresponding EDS mapping.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/1d8e27b9975fec2c5dda157c.jpeg"},{"id":105875515,"identity":"0609a48f-7da4-4ce6-bf9b-296cd70ac02e","added_by":"auto","created_at":"2026-04-01 05:40:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":688406,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram and TEM image: (A) the isotropic dispersion of HAP in the gelatin matrix network; (B) the anisotropic dispersion of Fe-HAP in the gelatin matrix network; (C) the isotropic dispersion of Fe-HAP in the methylacrylated gelatin matrix network. In the above images, i represented images at low magnification and ii for image at high magnification.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/27fb90bc37014cf2ee3620a7.jpeg"},{"id":105875528,"identity":"f252d5b3-5a75-4616-95e6-399efaf7f1a9","added_by":"auto","created_at":"2026-04-01 05:40:45","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":478484,"visible":true,"origin":"","legend":"\u003cp\u003eA: For the gelatin matrix network, during the heating process, the gelatin underwent β-helical unfolding, increasing the grid size, which provided sufficient space for the Fe-HAP orientation arrangement process; B: The SAXs images revealed the gradual orientation process of Fe-HAP nanoparticles in the Gel/Fe-HAP composite; C: The azimuthal angle curves of Gel/Fe-HAP after different volatilization times; D: A schematic diagram showing the directional arrangement of Fe-HAP nanorods in a single gelatin grid due to the directional evaporation of water molecules.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/c7dab7fef3b57ff2762a101a.jpeg"},{"id":105875518,"identity":"5fd903ad-db1a-427f-8196-27b47635660f","added_by":"auto","created_at":"2026-04-01 05:40:43","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":366451,"visible":true,"origin":"","legend":"\u003cp\u003eA: Representative axial, sagittal, and 3D micro-CT images of rabbit femur regeneration after 6 weeks and 12 weeks. The dotted orange box indicated the location of the defect. The bone volume/tissue volume (BV/TV, B), bone mineral density (BMD, C), trabecular bone separation (Tb. Sp, D), and thickness of trabecular (Tb. Th, E) were calculated by Imalytics analysis software. In Fig. B and C, the relevant bone indicators of the intact left femur at the same position were marked with purple dotted lines. Data was analysed from defects after 6 weeks and 12weeks in both groups, as indicated in the bars, and compared using an independent-samples T test. *Significant difference with P \u0026lt; 0.05; ** Significant difference with P \u0026lt; 0.01; ***Significant difference with P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/3ebfa9f9db7b27b3cb412f92.jpeg"},{"id":105875526,"identity":"ce727ad4-0d9c-436e-848f-359d6f5175b2","added_by":"auto","created_at":"2026-04-01 05:40:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1039188,"visible":true,"origin":"","legend":"\u003cp\u003eA: Representative images of H\u0026amp;E staining of each group after 6 (A) and 12 (B) weeks; Representative images of Masson staining of each group after 6 (C) and 12 (D) weeks. The black asterisk represented the new bone. E: Immuno-histological characterizations for OCN in femoral defect model. In Figure A-D, the initial defect areas were marked with two vertical dotted lines, and i, ii, and iii represented geltain scaffolds, I-Gel/Fe-HAP scaffold, and Gel/Fe-HAP scaffolds, respectively.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/8048e873232a7f555c40c5d8.jpeg"},{"id":108804592,"identity":"3e78225d-b98b-431d-944a-d575cef7943f","added_by":"auto","created_at":"2026-05-08 15:21:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3503466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/4de2562d-565a-4a04-bb68-24b7bdb0a636.pdf"},{"id":105875505,"identity":"aea2eb30-034c-4ebe-9bcf-1944ab996c56","added_by":"auto","created_at":"2026-04-01 05:40:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1825515,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9273382/v1/54acb643a6761ffa5a5fb278.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Self-oriented hydroxyapatite nanorods in gelatin matrix via single- directional evaporation for bone defect repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn orthopedics and cranio-maxillofacial surgery, large bone defects caused by severe trauma, tumor resection, infection, and other diseases remain widespread problems. Such severe bone defects not only cause patients pain and reduce their quality of life, but also shorten their expected lifespan, posing an urgent global medical challenge that needs to be addressed. Currently, most research on scaffolds developed for bone repair and regeneration is mainly focused on optimizing the composition of organic/inorganic materials. Commonly used natural macromolecules for bone repair scaffolds include collagen\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, gelatin\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, chitosan\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, sodium alginate\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, silk fibroin\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, agarose\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, hyaluronic acid\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, etc. They exhibit excellent biocompatibility and facilitate the adhesion and growth of osteoblasts. However, their poor spatial stability and osteoinductivity limit their large-scale application. The inorganic components in bone repair scaffolds can mainly be classified into two types: phosphates and silicates. Among them, bioactive glass\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e (silica glass containing calcium and phosphate), hydroxyapatite (HAP)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and tricalcium phosphate (TCP)\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e are the most common. Due to their chemical composition being similar to that of natural bone, inorganic scaffolds show excellent biocompatibility and promote the proliferation and differentiation of osteoblasts. However, calcium phosphate ceramics encounter difficulties in processing, and the lack of flexibility limits their application in load-bearing and deformed position\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In summary, scaffolds made of a single biomaterial have many problems. Currently, the best solution is composite scaffolds, which combine inorganic ceramics and organic polymers.\u003c/p\u003e \u003cp\u003eAt present, most bone repair composite scaffolds are prepared by physically mixing polymers and ceramic (nano) particles or adding ceramic (nano) particles to polymer solutions, followed by freeze-drying and other processes\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This often leads to random, aggregated, and non-structured distribution of ceramic particles in the composite materials, which is quite different from the structure of natural bones\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. At the nanoscale, bones are composed of collagen triple helices and mineralized with highly substituted carbonate hydroxyapatite (HAP) biomineral nanocrystals\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The c-axes of these HAP nanocrystals are parallel to the long axes of the collagen fibrils, and their (100) crystal planes are roughly parallel to each other\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These mineralized collagen fibrils with a diameter of about 100 nm, non-collagenous fibrillar extracellular matrix, and minerals are assembled into layered lamellar structures of bone, forming the functional units of bone. These lamellae are arranged in various ways to form two types of cortical bone and trabecular bone\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. That is, natural bones exhibit highly aligned, anisotropic, biphasic and ultrafine structural designs, as well as complex hierarchical assembly structures\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, it is increasingly important to study how microstructure affects the biological activity of polymer/ceramic composites and to utilize these theories to develop bone graft substitutes that are closer to natural bone tissue.\u003c/p\u003e \u003cp\u003eAs the main natural inorganic component of bone, hydroxyapatite is one of the most commonly used inorganic components in bone repair scaffold materials, with excellent biological activity, biocompatibility, osteoconductivity, non-toxicity, and non-inflammatory properties\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Studies have shown that HAP is very beneficial for bone construction, as it can stimulate growth factors (such as bone morphogenetic proteins) and promote the secretion of alkaline phosphatase in mesenchymal stem cells. Of course, there are differences between synthetic HAP and natural HAP in terms of color, size, morphology, and crystallinity, which, to some extent, weaken the osteogenic effect of synthetic HAP\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Interestingly, the special crystal structure of hydroxyapatite allows Ca\u003csup\u003e2+\u003c/sup\u003e in the lattice to be replaced by a series of metal ions of similar size, such as Mg\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, etc. \u003csup\u003e26\u0026ndash;28\u003c/sup\u003e. Although the degree of these isomorphic substitutions is small, they greatly adjust many properties of HAP, such as biological activity, interaction between bone minerals and calcium phosphate-based implant materials, surface chemistry, charge, and morphology. The introduction of metal ions not only affects the crystal growth, the efficiency of drug delivery, the dissolution rate and solubility, but also exhibits some special properties, such as antibacterial and superparamagnetic properties\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis research successfully introduced Fe\u003csup\u003e2+\u003c/sup\u003e into the hydroxyapatite lattice (Fe-HAP) through the hydrothermal method. Unlike the previously reported irregular sheet-like or needle-like aggregates\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, Fe-HAP is a shape-regular rod-shaped crystal without magnetite impurities. Thanks to the double-layer oleic acid ligands on the particle surface, Fe-HAP nanoparticles are uniformly dispersed in the gelatin (Gel) matrix. During the drying process, as the water in the Gel/Fe-HAP composite hydrogel is directedly evaporated, the Fe-HAP rod-shaped crystals spontaneously align along the direction of water evaporation. Animal experiments showed that the oriented arrangement of Gel/hydroxyapatite scaffolds can effectively promote the repair process of critical-sized bone defects.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eIn the traditional research on Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e doped hydroxyapatite, whether by hydrothermal method, wet chemical precipitation method, or mechanochemical synthesis, the introduction of Fe is always accompanied by the appearance of magnetite or magnetite oxide\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Based on the coordination environment of Ca in the lattice of HAP, it can be classified into two types: Ca (I) forms coordination with six oxygen atoms belonging to four phosphate groups, while Ca (II) coordinates with seven oxygen atoms, among which six come from the four phosphate groups, and one comes from the hydroxyl group\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The tendency of Fe\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e to replace Ca (I) or Ca (II) in the HAP unit cell is completely different since the differences in charge and ionic radii, which also lead to changes in the unit cell parameters\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. According to density functional theory (DFT) calculations, Fe\u003csup\u003e3+\u003c/sup\u003e ions preferentially occupy the Ca(II) sites, while the preference of Fe\u003csup\u003e2+\u003c/sup\u003e for the Ca (I) and Ca (II) sites is comparable in terms of energy\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Considering that Fe\u003csup\u003e3+\u003c/sup\u003e would cause greater lattice distortion and the introduction of magnetite impurity phases\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, this research selected Fe\u003csup\u003e2+\u003c/sup\u003e as the dopant. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the infrared calcium absorption peak of Fe-HAP was almost identical to that of the HAP nanoparticles prepared under the same conditions. Compared with HAP, the XRD peak position of Fe-HAP was slightly shifted, and the crystallinity was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e indicated that the introduction of Fe would lead to a contraction deformation of the crystal lattice.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, compared with pure HAP, the doping of Fe made the Fe-HAP nanoparticles slimmer, but still retained the rod-like morphology of traditional hydroxyapatite (length: 70\u0026ndash;200 nm, diameter: 10\u0026ndash;50 nm). The hydrodynamic size of Fe-HAP nanoparticles measured by DLS was found to be 78.8 nm, while the size of HAP nanoparticles prepared under the same synthesis conditions was 84.14 nm. The difference in hydrodynamic sizes between the two was the more elongated morphology of Fe-HAP. From the DLS particle size distribution graph (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), the size distribution of Fe-HAP particles was wider, which was also consistent with the TEM images. Moreover, no Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e impurity phase was observed in the TEM images, which was consistent with the XRD spectrum. To ensure the EDS signal, electron energy spectrum scanning (EDS) was performed at the Fe-HAP enriched position. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the EDS element mapping diagram indicated the uniform distribution of Ca, O, Fe, and P in the Fe-HAP aggregates, and no Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particle was found in the TEM image, indicating that Fe was successfully incorporated into the HAP lattice without Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e impurity. In addition, through inductively coupled plasma spectroscopy, it can be confirmed that the Fe content in Fe-HAP is 22.83% (relative to the molar ratio of Ca). Traditional HAP nanoparticles exhibited diamagnetism\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and the introduction of Fe transformed the diamagnetism into superparamagnetism (with a saturation magnetization over 2.876 emu/g), thereby endowing Fe-HAP with potential applications in fields such as magnetic control orientation, magnetic thermal response, and magnetic imaging (Fig. S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with the pure gelatin scaffold, the appearance of the characteristic absorption peaks of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e in the infrared spectrum (the absorption peaks at 1090 and 1040 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the asymmetric stretching vibration of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e) indicated the successful introduction of Fe-HAP nanoparticles, while the enhancement of the methylene peaks at 2853 and 2926 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was due to the oleic acid ligands on the surface of Fe-HAP nanoparticles(Fig. S3). The content of Fe-HAP nanoparticles in the Gel/Fe-HAP composite scaffold could be determined from the thermogravimetric curve (Fig. S4), which was 4.83 wt%.\u003c/p\u003e \u003cp\u003eTo investigate the distribution of HAP and Fe-HAP nanoparticles in the gelatin matrix, the frozen sections of Gel/HAP and Gel/Fe-HAP were placed under the TEM for observation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, HAP was uniformly dispersed in the gelatin matrix, and the long axis direction of the nanoparticles seemed to preferentially align horizontally. However, the orientation behavior was not obvious. In contrast, Fe-HAP exhibited a significant orientation arrangement in the gelatin matrix: rod-shaped nanoparticles were uniformly dispersed in the gelatin matrix, and the long axis direction was neatly arranged in the \"upper left - lower right\" direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To explore the influence of the hydrogel matrix on the orientation behavior of Fe-HAP, methylacrylated gel (GelMA) was selected to replace gelatin to prepare GelMA/Fe-HAP composite scaffolds, and the distribution of nanoparticles in the GelMA/Fe-HAP composite scaffolds was observed under TEM. Fe-HAP particles were uniformly dispersed in the GelMA matrix, but did not show any directional tendency of arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). As shown in the schematic diagram on the left, within the same gelatin matrix network, HAP and Fe-HAP nanoparticles exhibited different orientation behaviors, and even for the same Fe-HAP nanoparticles, their orientation behaviors in the gelatin matrix and GelMA matrix were completely different.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering that the orientation process of Fe-HAP nanoparticles in the hydrogel matrix was related to the three-dimensional network of the hydrogel. In this research, one-dimensional SAXs curves of gelatin (Gel) at room temperature, GelMA hydrogel at room temperature, and gelatin at 50\u0026deg;C (Gel 50\u0026deg;C) were obtained (Fig.S5). The one-dimensional SAXs curves were fitted using the gel model in the SasView software within the range of 0.01 \u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e to 1 \u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e. The fitting function was shown as the following formula.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:I\\left(Q\\right)\\approx\\:\\frac{{I}_{L}\\left(0\\right)}{{\\left(1+\\left[\\left(D+1\\right)/3\\right]{Q}^{2}{\\xi\\:}^{2}\\right)}^{\\frac{D}{2}}}+{I}_{G}\\left(0\\right)\\cdot\\:{e}^{-{Q}^{2}{R}_{g}^{2}/3}+B$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, I\u003csub\u003eL\u003c/sub\u003e and I\u003csub\u003eG\u003c/sub\u003e corresponded to the Lorentz model and the Guinier model respectively; D represented the fractal dimension. When D\u0026thinsp;=\u0026thinsp;2, the first term of the above equation could simplify to the Ornstein-Zernicke model; ξ represented the correlation length, which could be used to evaluate the average size of the cross-linked regions in the hydrogel, and the smaller ξ was, the smaller the size of the cross-linked regions in the hydrogel was\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e; Rg indicated the mean square rotation radius; B represented the background scattering. Through fitting, the relevant lengths of GelMA, Gel, and Gel at 50\u0026deg;C were 4.67 nm, 6.52 nm, and 10.05 nm, respectively, indicating that the cross-linking network sizes of the three hydrogels gradually increased. At room temperature, the gelatin network was composed of the physical cross-linking of β-folding of gelatin molecules and the chemical cross-linking of glutaraldehyde, and the physical crosslinks unfolded at 50\u0026deg;C, thereby causing an increase in the hydrogel's relevant length. Furthermore, the orientation process of gelatin/Fe-HAP during the water evaporation process was analyzed by small-angle X-ray scattering (SAXs), and the two-dimensional scattering images of SAXs were integrated using Fit. 2d software to obtain the azimuthal angle curves after different evaporation times. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the original state of the Gel/Fe-HAP composite scaffold was an isotropic hydrogel. With the continuous evaporation of water, the two-dimensional scattering images gradually tended to be elliptical. After 4 hours, the Gel/Fe-HAP composite scaffold exhibited obvious anisotropy. The SAXS azimuthal angle curve indicated that as water evaporated, Fe-HAP gradually aligned along the 180\u0026deg; azimuthal angle in the gelatin network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After 4 hours, Fe-HAP preferentially aligned along the z-axis direction of the hydrogel. Based on the formula, the orientation degree π at this time was calculated to be 0.752.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the x-axis and y-axis directions of the Gel/Fe-HAP composite hydrogel gradually solidified and dried as water evaporated. Taking a single grid at the center position as an example. The water molecules in the hydrogel grid could only escape along the z-axis direction, thereby forcing the Fe-HAP nanorod confined within the network to align along the z-axis direction. Due to the different sizes of the hydrogel grids, overly small grids would restrict the rotation of Fe-HAP nanoparticles, thereby hindering the orientation behavior of the nanorods. The glutaraldehyde vapor cross-linked gelatin network size was smaller at room temperature, but when dried at 50\u0026deg;C, the gelatin matrix underwent β-folding expansion, and the grid size increased, thereby allowing the Fe-HAP nanorods to rotate and orient (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, the GelMA hydrogel had an overly small grid size, which restricted the torsional orientation of Fe-HAP nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExternal and internal (for the x-y plane) sections of the same Gel/Fe-HAP composite hydrogel were made, and the orientation of Fe-HAP nanoparticles was observed under TEM. As shown in Fig.S6, the outer layer of the composite hydrogel had inconsistent directions for water evaporation, and the arrangement of Fe-HAP particles was rather disordered, with only some particles orienting along the z-axis. In the internal part of the composite hydrogel, after the evaporation process, the water was discharged in a single direction, and Fe-HAP was arranged in an oriented manner, which further verified the theory proposed earlier that the single-directional evaporation of water vapor led to the orientation arrangement of Fe-HAP nanoparticles.\u003c/p\u003e \u003cp\u003eTo further verify the influence of the aspect ratio of rod-shaped nanoparticles on the orientation behavior, we prepared pure HAP nanoparticles with an aspect ratio similar to that of Fe-doped hydroxyapatite by adjusting the pH value. As shown in Fig.S7A, after adjusting the pH value of the hydrothermal reaction solution to 10.5, HAP exhibited a long rod-like morphology, with a length of 100\u0026ndash;400 nm and a diameter of 10\u0026ndash;30 nm. Using these HAP nanorods to prepare Gel/HAP composite scaffolds, we induced the HAP nanorods to align in the preset direction using the same method. The SAXs (Fig.S7B) and TEM (Fig.S7C) images showed that the HAP nanorods were oriented and arranged in the gelatin matrix. The above results indicated that the orientation behavior of HAP nanoparticles in the hydrogel matrix was affected by the hydrogel network and the aspect ratio of the nanoparticles: when the hydrogel cross-linking density was too high, the grid size was too small, which limited the rotational orientation of the nanorods; the aspect ratio of the nanoparticles determined the magnitude of the deflection force that the nanorods received during the directional evaporation of water, and nanoparticles with a too small aspect ratio received insufficient deflection force and could not achieve orientation arrangement.\u003c/p\u003e \u003cp\u003eGiven that Fe\u003csup\u003e2+\u003c/sup\u003e could promote bone formation by stimulating vascular growth \u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, in subsequent animal experiments, we selected the Gel/Fe-HAP scaffold for the bone defect repair. Considering that the bone defect repair scaffold needed sufficient porosity to ensure cell migration and proliferation, as well as the exchange of nutrients and metabolic waste, we introduced PEG-4000 as a pore-forming agent into the Gel/Fe-HAP composite hydrogel system (As shown in Fig.S8, the introduction of PEG-4000 did not affect the orientation behavior of Fe-HAP). After ethanol was used to remove PEG-4000, dense and uniform micron-sized pores were successfully formed on the Gel/Fe-HAP scaffold (Fig.S9). The rough surface also facilitated the adhesion and migration of osteogenic-related cells. In addition, this research uniformly drilled through holes on the scaffold using a micro-drill (500 \u0026micro;m) to further increase the porosity of the Gel/Fe-HAP scaffold. The three-dimensional morphology of the Gel/Fe-HAP scaffold was observed using micro-CT, and the distribution of the mineral phase in the scaffold and the overall porosity of the scaffold were analyzed (Fig.S10). The Gel/Fe-HAP scaffold presented a three-dimensional porous interconnected morphology, with a rough surface, micro-meter-sized pores, and a pore volume of 80.51% as analyzed by Imalytics software. By adjusting the CT signal range, the distribution of the mineral phase in the Gel/Fe-HAP composite scaffold could be obtained. As shown in Fig.S10B, Fe-HAP was uniformly distributed in the scaffold without obvious agglomeration. Of course, considering the micrometer resolution of micro-CT, the orientation behavior of Fe-HAP nanoparticles could not be obtained through CT scanning.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, neither the pure gelatin (Gel) scaffold, the isotropic gelatin/Fe-HAP composite scaffold (I-Gel/Fe-HAP), nor the anisotropic gelatin/Fe-HAP composite scaffold (Gel/Fe-HAP) completely degraded after 6 weeks of implantation, showing the characteristic of gradually repairing from the defect edge to the defect center. Among the three groups of scaffolds, the Gel/Fe-HAP scaffold exhibited the best bone repair effect, with a BV/TV of 61.71%, significantly higher than 31.68% of the Gel scaffold and 46.48% of the I-Gel/Fe-HAP scaffold. At 12 weeks of repair, the differences in bone defect repair effects among the three groups were more obvious. The pure gelatin scaffold had a poor bone defect repair effect due to the lack of bone conductivity and bone induction: only partial new bone formation was observed at the edge of the defect area, and no hard tissue was generated in the defect center area. The BV/TV of the I-Gel/Fe-HAP scaffold at 12 weeks after surgery was 59.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36%, with a cavity remaining in the defect center area, and the newly generated bone tissue in the defect area was biased towards the bone marrow direction, with a thickness of only a thin layer, unable to restore the mechanical properties of the normal femur. The Gel/Fe-HAP scaffold group showed the best bone defect repair effect, with a BV/TV recovery to 74.63\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88% at 12 weeks, compared to the BV/TV of intact left leg femur of 85.37% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The bone defect area was completely covered by newly generated bone tissue except for the small hole (d: 0.5-1mm) in the center, and the bone thickness at the defect center position had not yet recovered to the normal femur thickness. The excellent restorative effect of the scaffold could be attributed to its replication of the nanostructured extracellular matrix of natural bone cells, which was conducive to the migration, proliferation, and differentiation of osteogenic-related cells. In addition, the bone mineral density (BMD) of the Gel/Fe-HAP scaffold group at 12 weeks after implantation was 3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 g/cm\u0026sup3;, the average bone trabecular thickness (Tb. Th) was 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 mm, and the average bone trabecular separation (Tb. Sp) was 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mm, all of which were better than those of the Gel scaffold group and the I-Gel/Fe-HAP scaffold group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of HE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and Masson (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) staining indicated that the scaffold had not completely degraded at 6 weeks after implantation. In the pure gelatin group, due to the lack of bone conductivity, new bone tissue only appeared at the edge of the defect after 12 weeks, while the center of the defect remained empty. The I-Gel/Fe-HAP scaffold contained Fe-HAP, which facilitated the migration of osteogenic-related cells towards the defect center. After 12 weeks, the newly formed bone tissue covered more than 50% of the bone defect area, but there were still obvious blank areas at the defect center. The Gel/Fe-HAP scaffold exhibited the best bone defect repair effect: the newly formed bone tissue was dense and uniform in thickness, almost completely covering the defect area (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The immunohistochemical results of osteocalcin showed that the expression of osteocalcin in the new bone tissue of the Gel/Fe-HAP group was the highest, and the bone maturity was comparable to that of the surrounding normal bone tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, we evaluated the mechanical properties of the new bone tissue through nanoindentation tests. As shown in Fig.S11, after 12 weeks, the mechanical properties at the defect center position of the Gel/Fe-HAP group were the best, with a compressive modulus and hardness of 0.254 GPa and 5.467 GPa, respectively. Compared to the 0.293 GPa and 6.271 GPa of the normal bone tissue, the mechanical performance recovery rate was over 85%. The animal experiment results indicated that the Gel/Fe-HAP scaffold had good bone repair performance. The hydroxyapatite nanoparticles oriented along the long bone direction could effectively promote the bone defect repair process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis research successfully prepared Fe-doped hydroxyapatite nanoparticles by taking advantage of the similarities between Fe\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e. The Fe-HAP particles were rod-shaped and possessed superparamagnetism while avoiding the introduction of magnetite impurity phases. During the drying process of the hydrogel composite of gelatin/Fe-HAP, the directional evaporation of water caused the Fe-HAP nanoparticles to gradually align along the direction of water evaporation. An anisotropic gelatin/Fe-HAP bone repair scaffold using PEG-2000 as the pore-forming agent and supplemented with micropore drilling was prepared. In the rabbit femoral defect repair experiment, the anisotropic gelatin/Fe-HAP scaffold demonstrated excellent bone repair capabilities. The nano-hydroxyapatite with anisotropic bone orientation arrangement could effectively accelerate the bone defect repair process, and has a promising application prospect in clinical orthopedics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Research Project of Qizhen Innovation Concept Verification Center of Zhejiang University (No. GNYZ-2024010), National Natural Science Foundation of China (No. 51873187), Science Fund for Distinguished Young Scholars of Zhejiang Province (No.LR20E030004), National Basic Research Program of China (No. 2018YFC1004803).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.L. conducted the experiments, analyzed the experimental data and wrote the paper. T.C., Y.C. \u0026nbsp;and W.H. conducted the experiments. Z.W. proposed the concept, supervised this research, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi, X.; Li, Z. L.; Wang, P. L.; Lu, G. G.; Tong, L.; Liu, Q. Y.; Chen, Y. F.; Lin, J. L.; Luo, E.; Liang, J.; Jiang, Q.; Fan, Y. J.; Zhang, X. D.; Sun, Y., Dopamine-Integrated Nanointerface between Fibrillar Matrix and Hydrophilic Nanohydroxyapatite Regulates Immune Microenvironment to Boost Endogenous Bone Regeneration. \u003cem\u003eAdvanced Functional Materials \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e33\u003c/em\u003e (16).\u003c/li\u003e\n\u003cli\u003eWang, X. C.; Tang, S. J.; Chai, S. L.; Wang, P.; Qin, J. H.; Pei, W. H.; Bian, H. Y.; Jiang, Q.; Huang, C. X., Preparing printable bacterial cellulose based gelatin gel to promote in vivo bone regeneration. \u003cem\u003eCarbohydrate Polymers \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e \u003cem\u003e270\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eTao, F. H.; Cheng, Y. X.; Shi, X. W.; Zheng, H. F.; Du, Y. M.; Xiang, W.; Deng, H. 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P., Mineralization of ytterbium-doped hydroxyapatite nanorod arrays in magnetic chitosan scaffolds improves osteogenic and angiogenic abilities for bone defect healing. \u003cem\u003eChemical Engineering Journal \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e387\u003c/em\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"hydroxyapatite nanorods, gelatin, self-orientation, bone defect repair","lastPublishedDoi":"10.21203/rs.3.rs-9273382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9273382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBone, as a typical anisotropic material, with hydroxyapatite nanocrystals oriented in the direction of collagen fibrils. Drawing inspiration from nature, constructing bone defect repair scaffolds by mimicking the natural components and structures of bones to recreate the microenvironment at the bone defect site is an effective strategy. However, it is quite challenging to precisely control the orientation behavior of nanoscale hydroxyapatite nanorods. Here, for the first time, by adjusting the aspect ratio of HAP and the size of the hydrogel grid, the preferential orientation of HAP can be achieved through the directional evaporation of water in the hydrogel. And we introduced Fe\u003csup\u003e2+\u003c/sup\u003e into the hydroxyapatite lattice, while maintaining the original rod-like morphology of HAP without introducing the magnetic mineral impurity phase. Animal experiments indicated that the anisotropic gelatin/hydroxyapatite scaffolds can effectively accelerate the repair process of large-sized bone defects.\u003c/p\u003e","manuscriptTitle":"Self-oriented hydroxyapatite nanorods in gelatin matrix via single- directional evaporation for bone defect repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 05:39:13","doi":"10.21203/rs.3.rs-9273382/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":"eb55646d-7b42-4fc2-8371-c68b129d6c52","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-04T13:12:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T22:00:12+00:00","index":24,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T13:26:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 05:39:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9273382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9273382","identity":"rs-9273382","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}