Inorganic Strengthened Hydrogel Membrane as Regenerative

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX

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Inorganic Strengthened Hydrogel Membrane as Regenerative Periosteum Tianwen Xin,†,‡ Yong Gu,†,‡ Ruoyu Cheng,†,‡ Jincheng Tang,‡ Zhiyong Sun,‡ Wenguo Cui,*,‡,§ and Liang Chen*,‡ ‡

Department of Orthopedics, The First Affiliated Hospital of Soochow University, Orthopedic Institute, Soochow University, Suzhou, Jiangsu 215007, P. R. China § Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin second Road, Shanghai 200025, P. R. China S Supporting Information *

ABSTRACT: Periosteum plays the pivotal role in neomineralization, vascularization and protection during bone tissue regeneration. However, many artificial periosteum focused only on protection and lacked of the osteogenesis and angiogenesis functional capacity. In this study, we developed a novelty inorganic strengthened gelatin hydrogel membrane via inorganic and organic co-cross-linked double network as artificial periosteum for enhancing the durable angiogenesis and osteogenesis in bone reconstruction. Mesoporous bioactive glass nanoparticles (MBGNs) chemically modified with photo-cross-linkable gelatin derivative (GelMA) were further incorporated into GelMA to fabricate an organic/inorganic co-cross-linked hydrogel membrane (GelMA-G-MBGNs). The GelMA-G-MBGNs hydrogel membrane displayed better mechanical property, durable degradation time, pH stable, biomineralization and long-term ion release. In vitro study demonstrated that, when compared with GelMA or GelMA/MBGNs, the GelMA-G-MBGN membrane significantly promoted osteogenic differentiation while maintaining stable local pH, which is conducive to cell adhesion and proliferation. Finally, the GelMA-G-MBGN membrane shows a superior artificial periosteum with superior capacity in angiogenesis and osteogenesis for accelerating new and mature lamellar bone formation in rat calvarial critical size defect. This co-cross-linked hydrogel membrane implied a promising strategy for the development of advanced periosteum biomaterials with excellent handle and bone repairing properties. KEYWORDS: periosteum, vascularization, hydrogel, bone tissue regeneration

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and chondroblasts4 after a fracture. More importantly, periosteum provides nourishment for osteogenesis through its vast capillary network. Therefore, regeneration of microvascular network is of great importance during periosteum engineering. Recently, several attempts have been made to build an artificial periosteum to promote bone regeneration. Although most of them simply mimic the structure of fibrous layer,5,6 few efforts have been focused on the angiogenesis capacity of these membranes, and there still remains a great challenge to find a new kind of biomaterial to act with both osteogenic and

INTRODUCTION Bone defect repairing is a critical problem in clinical practice, which occurred in the cases of splintered fracture, tumor resection, or cleaning osteomyelitis lesions.1,2 Bone defects caused by high-energy trauma usually accompanied by coloboma of periosteum which as we know performs the key role in regeneration of bone defect. Hence absence of periosteum may compromise the progress of bone regeneration, resulting in prolonged healing time and even nonunion. Periosteum is composed of two layers, outer “fibrous layer” and inner “osteogenic layer”. The fibrous layer contains fibroblasts, along with nerve and capillary network,3 whereas the osteogenic layer attaches on the surface of the bone and contains progenitor cells that could develop into osteoblasts © XXXX American Chemical Society

Received: August 31, 2017 Accepted: November 7, 2017

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DOI: 10.1021/acsami.7b13167 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Inorganic Strengthened Hydrogel Membrane for Regenerative Periosteum

Figure 1. Progress of (A) fabricating methacrylic acid modified gelatin, (B) fabricating amino modified MBGNs and GelMA-MBGNs(G-MBGNs), (C) preparing GelMA/MBGNs and GelMA-G-MBGNs.

especially in neovascularization. However, usage of many ECMderived biomaterials, including gelatin, collagen, and hyaluronic acid, are confined by lack of osteogenic factor or other ions which could enhance mineralization, and so GelMA hydrogel.11−14 Osteoconductivity of pure GelMA hydrogel is also limited due to its lack of any inorganic component. In addition, the flexibility of photo-cross-linking GelMA hydrogel is unsatisfactory and the construction of GelMA membrane is brittle, and difficult to handle, so it is not conducive to be applied as artificial periosteum. Taken the aforementioned into consideration, we expected to fabricate a kind of organic− inorganic combined bioinduce ability hydrogel to promote early stage bone regeneration via enhancing neovascularization and mineralization, meanwhile, this hydrogel membrane owns the flexibility similar to periosteum which could be easily handled and convient for clinical application.

angiogenic bioactivity and promote early stage recovery of defected periosteum. Nowadays, recombinant protein, angiogenic growth factors gene transfer or endothelial cells have been used in neovascularization.7 However, the progress of neovascularization needs extracellular matrix (ECM) as a scaffold for vascular ingrowth. Among the various biomaterials, hydrogel has been an excellent choice for vascular network regeneration thanks to its intrisinic similarity to natural excellular matrix which provided three-dimensional supports for cellular growth and tissue formation.8 Since its first synthesis reported in 2000,9 methacrylate gelatin (GelMA) hydrogel, a kind of photocross-linking hydrogel, have been thoroughly studied in terms of physical and biochemical properties for many different applications ranging from tissue engineering to drug and gene delivery.10 As a kind of derivative of gelatin, GelMA hydrogel is an ideal biomaterial for periosteum and bone regeneration, B


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ACS Applied Materials & Interfaces

Figure 2. (A) (a, b) SEM images of MBGNs and G-MBGNs and (c) TEM images of MBGNs and G-MBGNs. (B) Particle size analysis of MBGNs and G-MBGNs. (C) FTIR spectra of MBGNs, amination-modified MBGNs, and G-MBGNs. (D) Small-angle X-ray scattering of MBGNs and GMBGNs.

In this study, bioactive glasses (BGs, SiO2-CaO-P2O5) have been applied as the inorganic component in fabricating of the organic−inorganic composite hydrogel. BGs have gained much attention since first being reported by Hench et al. in 197115 because of their biocompatibility, bioactivity, and osteoconductivity. The BGs will degrade in body fluid and release calcium (Ca) ions, which is reported to induce the migration, maturation, and organization of endothelial progenitors.16 Furthermore, mesoporous bioactive glasses nanoparticles (MBGNs) synthesized via templating with a block copolymer17 is reported to produce particals with much higher specific surface area and pore volume to accelerate the ions release. However, fast degradation of pure MBGNs in body fluid may cause significant fluctuation of [Ca2+] concentration in microenvironment which is reported to be ∼10 mM where optimal cell response is obtained. In addition, the directly addition of bioactive glass into the polymer system, will cause the mechanical properties of polymer system decreased due to the existence of two-phase interface incompatibility. In this regard, it has been proved that chemical conjugation of BG with the polymer could tackle these issues and produce structures with tunable physicochemical properties.18,19 Therefore, a chemical interaction between the organic and inorganic substances is required to get better control over the material degradation rate, as well as the mechanical properties of the whole matrix. Hence, we combined MBGNs and GelMA hydrogel through a co-cross-linking method to modify MBGNs surface using GelMA macromolecular and further fabricate a kind of cocrosslinking hydrogel using GelMA and MBGNsGelMA (MBGNs-G), which could be applied to neovascularization and bone regeneration through activating the neo-

vascularization and mineralization through the ions released from the MBGNs. In this work, we made the MBGNs surface modified with light cross-linking macromolecule and then co-cross-linked with GelMA to fabricate a kind of inorganic strengthened hydrogel membrane for regenerative periosteum (Scheme 1). This artificial biomimic organic/inorganic composite periosteum may accelerate the periosteum regeneration through neovascularization and then further promote the bone regeneration by releasing the irons like Ca and Si. We evaluated the mechanical properties of this hydrogel with different concentration of MBGNs to find the best composite ratio. The cell proliferation and differentiation activity on the co-cross-linked hydrogel was assessed by in vitro assays. Lastly, a rat calvarial critical size defect model was prepared to evaluate the osteogenesis and angiogenesis potential of the membrane.

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RESULTS AND DISCUSSION Fabrication of MBGNs, G-MBGNs, and Hydrogel. The GelMA was successfully fabricated using Gelatin (Type B, 250 bloom from porcine skin) and methacrylicanhydride (MA, Sigma, USA) (Figure 1A). MBGNs were successfully fabricated with calcium nitrate tetrahydrate (CN, Guanghua Chemical, China), triethylphosphate (TEP, Aladdin, Shanghai, China), and tetraethyl orthosilicate (TEOS, Thermo Fisher Scientific, USA) in Tris-HCl buffer solution (pH 8.0) as a catalyst and CTAB (Cetyltrimethylammonium Bromide, Sigma-Aldrich, USA) as template agent. MBGNs were amino-alkylated with APTES so that they had the ability to further possess other groups, and then the GelMA single strand was grafted onto the MBGNs surface via carboxy group by NHS/EDC reaction (EDC,1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hyC


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ACS Applied Materials & Interfaces drochloride; NHS, N-Hydroxysuccinimide), giving them the ability to participate in GelMA photo-cross-linking (Figure 1B). The GelMa/MBGNs hydrogel were successfully synthesized with MBGNs directly mixed with GelMA and photo-crosslinked under UV light (Figure 1C). We co-cross-linked GMBGNs with GelMA under the UV light to fabricate the enhanced mechanical property and the osteogenic hydrogel membrane called GelMA-G-MBGN membrane, which is easy to prepare and expected to accelerate the neovascularization of periosteum and then expedite bone regeneration (Figure 1C). The whole process is very simple and handle for satisfication with clinical requirements. Characterization of Nanoparticles. MBGNs were successfully sythesized via Stöber method.20,21 The scanning electron microscopy (SEM) photograph (Figure 2A) indicates that the MBGNs and G-MBGNs are smooth and neat particles with relatively uniform partical size. However, inner structure of the particle observed via TEM (Figure 2A) shows hollow channels with a diameter of approximately spread accross the particle, thus proving the mesoporous structure of MBGNs and G-MBGNs. Laser particle size analysis (Figure 2B) shown that the average particle size of MBGNs is 460.9 nm with PDI of 0.236 and the average particle size of amination-modified MBGNs is 249.1 nm with PDI of 0.098, the particle size of MBGNs became smaller after grafting of GelMA (the differences were statistically significant), the reason for this phenomenon may be that the dispersibility of aminationmodified MBGNs is much better than MBGNs. SEM and TEM proved that MBGNs and G-MBGNs are all regular spherical particles with good dispersibility. The zeta potential result is shown in Figure S2, and the zeta potential of MBGNs is −14.4 mV; after amination modification, the zeta potential turned to +20.4 mV, which could prove successful modification of MBGNs. After grafting with GelMA, the zeta potential turned back to −1.08 mV. The FTIR (Figure 2C) shown peaks at 468, 863, and 1095 cm−1 that correspond to Si−O−Si bonds, whereas peaks at 569 and 605 cm−1 correspond to crystallized P−O bonds in MBGNs, both of which prove the existence of MBGNs. As for modified MBGNs, with the major part similar to MBGNs, new peaks were found at 1635 and 1535 cm−1 corresponding to NH2, 687 cm−1 corresponding to Si−C bend, and 2960 cm−1 corresponding to C−H bend. The peak at 1635 cm−1 in the line of G-MBGNs indicated that the protein content increased. These results proved that we modified the MBGNs and grafted GelMA on the surface of MBGNs successfully. Small-angle X-ray scattering (Figure 2D) shown a peak in 2θ = 1.5 both in MBGNs and G-MBGNs, which was usually attributed to the six-square hole structure,22,23 corresponding with the TEM photograph. Results of SEM, transmission electron microscopy (TEM) photograph, and small-angle X-ray scattering indicated that there is no significant difference between the MBGNs and GMBGNs in morphology. On the basis of the above discussion, we grafted the GelMA on the surface of MBGNs successfully and kept the structure of mesoporous, and the modification and grafting method has no significant interference on the particle size and dispersing performance of MBGNs. Characterization of Hydrogel. In this study, MBGNs were added into the GelMA solution directly and photo-crosslinked to get GelMA/MBGNs hydrogel composite membrane, and G-MBGNs were also added into GelMA solution and photo-cross-linked to get GelMA-G-MBGNs hydrogel cocrors-

slinked membrane in Figure 3A. The SEM of hydrogel in Figure 3B shown that the hydrogel have sponge-like structure

Figure 3. (A) Photos of hydrogel before and after cross-link. (B) SEM images of hydrogel in low magnification(scale bar = 400 μm) and high magnification (scale bar = 20 μm) with ratio of MBGNs 3 wt %. (C) FTIR spectra of GelMA, GelMA/MBGNs, and GelMA-G-MBGNs.

with the pore size of 150 ± 21 μm. Similar sponge structure with pore size of 150 ± 10 μm was observed for GelMA/ MBGNs and GelMA-G-MBGNs hydrogel, but rougher pore wall and MBGNs could be seen in MBGNs containing samples. Previous studies proved that the pore size between 100 to 300 μm is suitable for cells’ proliferation and differentiation.24,25 As MBGNs scale up (from upper to lower in the figure respectively 1, 3, and 5 wt %) the pore size did not change significantly, and the MBGNs could evenly embedded on the surface of the pores (Figure S3). These results indicated that good dispersion of MBGNS is obtained in GelMA/MBGNs and GelMA-G-MBGNs, and the sponge sample structure of GelMA is not affected at the same time. The Fourier transform infrared spectroscopy (FTIR) of GelMA, GelMA/MBGNs and GelMA-G-MBGNs was shown in Figure 3C. With peak at 468 cm−1 corresponding to Si−O−S bonds and peak at 1069 cm−1 corresponding to P−O bond, the FTIR spectra of GelMA-G-MBGNs is similar to GelMA/ MBGNs generally, and only a new peak at 2350 cm−1 of D


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Figure 4. Mechanical test, swelling test, and weight loss analysis of (A) GelMA/MBGNs and (B)GelMA-G-MBGNs, * p < 0.05 and **p < 0.01 compared with control.

Figure 5. (A) SEM images of GelMA/MBGNs and GelMA-G-MBGNs after soak in SBF in low magnification (scale bar = 400 μm) and high magnification (scale bar = 100 μm). (B) XRD images of GelMA and GelMA-G-MBGNs after soak in SBF. (C) Concentration variation of ions in SBF. (* p < 0.05 compared with GelMA-G-MBGNs, **p < 0.01 compared with GelMA-G-MBGNs, #p < 0.05 compared with GelMA/MBGNs).

GelMA-G-MBGNs corresponding to C−N bond could prove the successful co-cross-link of GelMA-G-MBGNs. To explore whether addition of MBGNs and co-cross-linking method enhanced the mechanical property of hydrogel, we tested the mechanics of the hydrogel and the results are shown in Figure 4. At the same content of MBGNs, the compression modulus of GelMA-G-MBGNs is higher than that of GelMA/ MBGNs. However, only when the content of MBGNs is 3 wt %, the compression modulus of GelMA/MBGNs (31.4 ± 2.13 kPa) is significantly better than GelMA/MBGNs (*p < 0.05, **p < 0.01). As we could find in the figure, the compression modulus is gradually improved with higher content of MBGNs. Yet when the proportion of MBGNs is greater than 3 wt %, the mechanical properties began to decline. The cause of this phenomenon may be when the ratio of MBGNs is very low, the MBGNs brought little impact on the reticular structure of GelMA hydrogel, but when the amount of MBGNs is greater than 5%, significant aggregation of MBGNs could inversely impact the structure stability of hydrogel, thus leading to weaker mechanical property. In GelMA-G-MBGNs, although

there are bonds between MBGNs and GelMA, when the ratio of MBGNs is greater, the fragility of GelMA-G-MBGNs is greater too, and this factor will also affect the mechanical property. The swelling experiments are tested for evaluating the structural stability of the hydrogel, and the results are shown in Figure 4. After immersion for 12 h, a more significant drop of swelling ratio in GelMA-G-MBGNs group was observed when compared to that of GelMA/MBGNs group. The cocrosslinking group of GelMA-G-MBGNs shows even lower swelling ratio relative to GelMA. The results indicated that the swelling property of the blends is significantly higher than that of the cocross-linked groups, which also reflects the structural stability of the chemical co-cross-linked group of GelMA hydrogel when same content of MBGNs is incorporated. The weight loss performance could also reflect the stability of hydrogel, and the results are shown in Figure 4. After the hydrogel was soaked in phosphate buffered solution (PBS) for 28 days, the weight loss ratio of all GelMA/MBGN hydrogels with different MBGN ratios is significantly higher than that of E


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Figure 6. Cell adhesion and proliferation assays of hydrogel. (A) SEM images of cells on the hydrogel after seeding for 1, 3, and 7 days. (B) Cell proliferation of MC3T3-E1 cultured on different hydrogel using a CCK-8 kit. Statistically significant differences are indicated with * p < 0.05 and **p < 0.01 compared with control. (C) Live−dead staining of cells cultured on the hydrogel after 7 and 21 days.

the GelMA-G-MBGN hydrogel. For the GelMA/MBGN group, the weight loss speed increased with increasing ratio of MBGNs, However, the weight loss speed of GelMA-GMBGN hydrogel is lower when compared to GelMA/MBGNs. On day 28, the weight loss ratio of GelMA/MBGNs with 1, 3, and 5 wt % is 88% ± 1%, 85% ± 1.6% and 80.5% ± 1.5%, respectively, and in GelMA-G-MBGNs is 76.4% ± 1.4%, 74.5% ± 1.3%, and 67.5% ± 2.5% respectively. We could infer from these results that the inorganic/organic co-cross-linking method could increase the structural stability of hydrogel when compared with the simple physical mixing. The results above indicated that when compared with pure GelMA hydrogel and GelMA/MBGN hydrogel, the GelMA-GMBGN hydrogel has better structural stability. Additionally, as indicated by the weight loss test, it is true that the degradation rate of the co-cross-linking hydrogel can be tuned to fit the regenerating speed of different tissues apart from periosteum and bone by regulating the proportion of the inorganic phase. In Vitro Mineralization and Ion Release. For in vitro mineralization assessment of hydrogel, simulating body fluid (SBF) was applied to simulate the physiological environment of human body. The photo-cross-linked hydrogel was transparent before mineralization, and became white and opaque because of uptake of mineral after mineralization. SEM was applied for analyzing GelMA/MBGNs and GelMA-G-MBGNs after being soaked in SBF for 7 days (Figure 5A). The surface of the hydrogel was fully covered with uniformly distributed, needlelike agglomerates of minerals, while retaining the 3D microstructure of the GelMA framework; as the ratio of MBGNs increased, the number of needlelike agglomerates rose. When the ratio MBGNs reached 5 wt %, the agglomerates of minerals aggregate to each other. To characterize the structure

of the deposited agglomerates within the GelMA hydrogel, we took the diffraction patterns in which the agglomerates grown within hydrogel exhibited are shaped patterns along the (002) diffraction plane (Figure 5B), indicating that the amino acid backbone in the gelatin-based organics mediates the directional growth of apatite crystals aligned with their c axes parallel to the surface of GelMA hydrogel, a phenomenon consisitent with that found in the natural growth of biominerals. A thick and apparently continuous diffraction band was found containing the 211,112, and 300 triple diffraction peaks, which are characteristic of biological apatite. Compared with the reference XRD data of hydroxyapatite (PDF#54−0022) from the International Centre for Diffraction Data (ICDD) database, X-ray diffraction analysis proved that the agglomerates to be hydroxyapatite deposited on GelMA. The iron release results in Figure 5B shown that the release of SiO32− from GelMA/ MBGNs, GelMA-G-MBGNs, and MBGNs was very similar, and the concentration reached the maximum within 24 h, and remained almost steady for the rest of the time. The concentration of PO43− reduces rapidly in the first 24 h before stabilizing. The release of Ca2+ performed very similarly in three groups, where it mainly increased after 12 h and after 3 days after immersion. Most importantly, when compared with GelMA and GelMA/MBGN, GelMA-G-MBGNs produced the minimal impact on the pH value of SBF, as it stabilize around pH 7.55. The ability to produce Hydroxyl carbonate (HCA) in SBF is an important criterion to assess the biological activity of material. Factors such as material composition, pore structure, crystallinity, particle size, surface area, and the composition of surrounding solution could significantly affect the generation of HCA. GelMA hydrogel can be used for biomimetic F


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Figure 7. Osteogenesis of MC3T3-E1 cells measured by (A) ALP staining at 7 and 14 days (B) Alizarin red staining at 14 and 21 days, and quantitative results of alizarin red staining (* p < 0.05 and **p < 0.01).

Cell Adhesion and Proliferation on Hydrogel. MC3T3E1 cells were applied to coculture with hydrogel materials to explore the effect of materials on cell proliferation and mineralization capacity. MC3T3-E1 cells and three kinds of materials coculture results were shown in Figure 6A. After 1 d, three kinds of material surface had little amount of cell adhesion. The number of cells on the surface of GelMA/ MBGN group and GelMA-G-MBGN group was more than that of pure GelMA group, also better cell extension was identified on GelMA/MBGN and GelMA-G-MBGN group. After 7 days of culture, the cell extension and contact all increased on all three hydrogels. The number of cells on the surface of GelMA/ MBGNs was further increased, and the number of cell colonies was increased. The cells on the surface of GelMA-G-MBGNs were significantly proliferated and the colonies were further expanded. Many pseudopodium were widely connected between cells and cells and materials, covering almost the entire material surface and achieving saturation. To quantify the proliferation of cells in different groups, we performed cell counting using CCK-8 as shown in Figure 6. The difference in OD value between the three groups was not statistically significant (p > 0.05) in day 1. The OD values of the GelMA-GMBGN group were higher than those of the other three groups at 3, 5, 7, and 9 days, and the differences were statistically significant (all p < 0.05); the GelMA/MBGN group was higher than the GelMA group and control group, and the difference was statistically significant (all p < 0.05). There was no significant difference between the GelMA group and the control group (p > 0.05). In addition, because of the use of a variety of chemical reagents in the production process, the residual reagent may have a certain toxic effect on the cells. To test the toxicity of the material, we showed the cells with Live− Dead staining, and the cells in the GelMA group, GelMA/ MBGN group, and GelMA-G-MBGNsgroup were all well-lived, and most of them were live cells (green fluorescence) at 7 and 21 days, with good morphology and few dead cells (red fluorescence). The adhesion of osteoblasts to the surface of bone repair material depends mainly on the two aspects, the cell itself and the material.31,32 SEM results show that the cells can adhere

mineralization in SBF with its amino and carboxyl group on the side chain electrostatically interacting with Ca2+, thus inducing the nucleation and growth of HCA crystal which could act as an nucleation site for further Ca2+ and PO43− deposition. The mineralization is embedded within the hydrogel matrix, which indicates that the hydrogel matrix absorbs SBF through the swelling effect, and starts the mineralization process simultaneosly in core and surface of the membrane.26 SEM shown that there is a strong adhesive force between the mineral and GelMA. The MBGNs and APTES-MBGNs (APTES, 3aminopropyl)triethoxysilane) formed HCA mainly through the following steps: First, Ca2+ and Na+ in solution exchanged with H+, and a large amount of silicon hydroxyl group appeared on the surface. The Si−O−Si bonds were opened in weak alkaline conditions, with soluble silicon dissolved in the form of silicate and silicate gel, and the material and solution interface appeared more silicon hydroxyl. 27−29 The siloxane is polymerized to form a negatively charged silicon-rich gel layer. Ca2+ and PO43− were deposited on the surface of the silica gel layer to form an amorphous calcium phosphate layer, which becomes HCA after crystallization.30 Because APTESMBGN still retains the original biological activity of MBGNs, the thickness and coverage of the surface of the GelMA-GMBGNs are similar to that of the GelMA/MBGNs. As the MBGNs ratio increases or the mineralization time prolongs, the thickness of the mineralization layer increases. XRD show that the characteristic diffraction peaks of GelMA-G-MBGNs and GelMA/MBGNs at specific sites indicate that HA crystals form on the surface of the complex, which proves that the addition of MBGNs enhances the mineralization of hydrogel in physiological environment. In addition, as the results of ion release indicated, for GelMA/MBGN, with the dissolution of Si4+, local body fluid environment will turn alkaline. In contrast, with the co-cross-linking method making the chemical structure of hydrogel material more stable, the release rate of SiO32− is effectively controlled, thereby reducing the fluctuation of local environment pH, to keep the local environment relatively stable, which could be a benefit for local cell proliferation and differentiation. G


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Figure 8. (A) Photo of calvarial critical-sized defect. (B) Micro-CT results of the specimen of calvarial critical-sized defects (*p < 0.05 and **p < 0.01) and (C) the bone volume faction.

shape and uneven color when compared with other groups. After 21 days, this trend became more obivous as red and brown nodules formed via strongly positive reaction could be found in GelMA-G-MBGN and GelMA/MBGN group, whereas only smaller red nodules were found in groups without MBGNs. The quantitative analysis of the mineralized nodules after dissolved by cetylpyridinium chloride is shown in Figure 7B, the mineralization activity of GelMA/MBGN group and GelMA-G-MBGN group was significantly higher than that of control group and GelMA group, and no significant difference was found between the GelMA/MBGN group and GelMA-G-MBGN group. ALP is known as the early stage marker in the process of osteogenesis differentiation, responsible for hydrolyzing organophosphate, increasing the concentration of local PO43−, promoting Ca2+ deposition, and hydrolyzing inorganic pyrophosphate with has strong inhibitory effect on mineralization, and regulating the calcification process of bone matrix.35 In this study, the degree and activity of ALP staining at different times was found to be GelMA-G-MBGN > GelMA/MBGN > GelMA group. The GelMA group was positive for calcified nodules, and the color got denser over time, indicating that GelMA hydrogel scaffolds with special surface and internal structure could promote osteogenic differentiation. The mineralized nodules of GelMA-G-MBGNs group and GelMA/MBGNs group were darker and the calcium content was higher than that of GelMA hydrogel. These results indicate that the GelMA-G-MBGNs hydrogel prepared in this experiment has a strong ability to induce mineralization. On the one hand, previous studies have shown that the presence of Si4+ has a strong effect in promoting osteogenesis, and the addition of MBGNs greatly enhances the rate of osteogenic differentiation of cells.36 On the other hand, in the GelMA-G-MBGNs hydrogel group, the degradation rate of the material is controlled by the time-dependent degradation of the material

and spread well on the surface of the three materials. The research shows that the surface of biological materials needs a certain affinity/hydrophobic balance to be beneficial for the adhesion and spread of cells.33 GelMA hydrogel membrane is suitable for hydrophilic properties, and retains the RGD sequence that promotes cell adhesion because of its high affinity for the integrin receptors on the cell membrane surface, thus facilitating cell adhesion and spreading.10 At the same time, thanks to the biological activity of MBGNs, the surface of the HCA layer provides a stable site for initial contact of the cells and material.34 In addition, as previously described, the chemical co-cross-linking method controls the degradation rate of MBGNs in the material, which makes the surrounding local pH more stable, which is more favorable for cell adhesion and proliferation. GelMA-G-MBGNs combine complex surface morphology, moderate hydrophilicity, proper charge, superior capability of inducing mineralization, and simplicity for further modification, and are conducive to cell adhesion. In Vitro Osteogenesis Evaluation. To verify the in vitro osteogenic activity of hydrogel material, we performed alkaline phosphatase (ALP) staining and Alizarin red staining and quantitative analysis experiments on osteoblasts MC3T3-E1 cocultured with materials. ALP staining shown that the staining density of groups containing MBGNs was significantly higher than that of GelMA group and control group. As time prolongs, the ALP staining of the GelMA/MBGN group and GelMA-GMBGN group got even denser and broader when compared with the control and GelMA group, as shown in Figure 7A. The Alizarin red staining was performed after the material and cells were cocultured for 14 and 21 days. As the results show in Figure 7B, at the 14th day, only a small number of mineralized nodules were observed in the control group. A small amount of red mineralized nodules was found in the GelMA group. In contrast, the GelMA-G-MBGN and GelMA/MBGN group was found to have more red mineralized nodules with irregular H


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Figure 9. (A) HE staining of hydrogel filled areas at 4 weeks and 8 weeks’ postimplantation (25×). Scale bar = 1 mm. Bonelike structures were detected in all graft groups after H&E staining. The second row represents higher-magnification images (200×) of the corresponding square boxes in the upper row. NB and HG represent new bone and hydrogel, respectively. Immumohistochemical staining of (B) Col-I and (C) CD-31; the arrows indicate the new blood vessels.

due to the high structural stability of the material. As a result, the change of pH began to stabilize after 48 h of immersion in the environment, which caused the dissolution of MBGNs to promote osteogenesis, and the stability of the local microenvironment was improved and such a microenvironment is beneficial to the stability of various metabolic processes of cells, which plays an important role in osteogenesis of cells. In Vivo Osteogenesis and Angioplasty. To provide further verification for the bioactivity of co-cross-linking hydrogel membrane, a critical bone defect model (Figure 8A) in the rat skull by surgery was adopted to implanted the material for evaluation of different groups. Micro-CT scan results are shown in Figure 8C. Apparently, the new bone coverage area in GelMA/MBGNs group and GelMA-GMBGNs group was significantly higher than that in the first two groups at 4 and 8 weeks. The newborn bone grows from the peripheral part of the defect to the central part. Figure 8B shows quantitatively that the relative volume of newborn bone

in the GelMA/MBGN and GelMA-G-MBGN group was higher than that of the GelMA and control groups. The results of HE staining and Col-I immunohistochemical staining are shown in Figure 9A, B, consistent with the quantitative analysis of microCT. The newborn bone content of GelMA/MBGNs and GelMA-G-MBGNs group and bone tissue maturation were higher than GelMA and control group. More mature bone could also be found on detailed micro-CT results of GelMA/ MBGNs and GelMA-G-MBGNs group. It will not be hard to come to the conclusion that the addition of MBGNs has a significant advantage over pure GelMA in regeneration of calvarial defect. As for the difference between GelMA-GMBGNs and GelMA/MBGNs group, co-cross-linked GelMAG-MBGNs hydrogel has the advantages of ion-controlled release and better degradation stability, by which GelMA-GMBGNs hydrogel could provide a more stable local environment than the pure GelMA group and the blended GelMA/ MBGNs group, contributing to better bone tissue regeneration. I


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stimulate gene expression, accelerate differentiation of osteoblast and promote secretion of growth factors and generation of ECM. In addition, studies have reported that Ca2+ can also promote the secretion of vascular-related cytokines, and promote adhesion and proliferation of vascular endothelial cell and formation of capillary,16 and the MBGNs could release calcium ions in the body fluid environment, so we considered that the calcium ion from MBGNs impacted the angiogenesis, and the results of in vivo experiments have supported this view. On the other hand, because of the specific organic−inorganic phase synthesis of GelMA-G-MBGNs, a structure closely mimicing natural ECM structure is created with its unique collagen-bioactive glass composite structure, making it a suitable cell stent for cell stretching to regulate cell behavior. These functions laid a good foundation for the regeneration of the periosteum to achieve the goal of accelerated bone regeneration.

It is worthwhile that many other studies based on cell tissue engineering also provide a favorable environment for cell growth, like Yufeng Dong’s works one cell sheets,37−39 and Benoit group’s works using PEG hydrogel periosteum,40−42 which is worth learning for our later studies. Platelet endothelial cell adhesion molecule (PECAM-1) of CD31 is an integrin protein that mediates intercellular adhesion of endothelial cells. Recognized as a marker of newborn endothelial cells, it is commonly used for neonatal microvessel counting to assess angiogenesis of implanted material.43 Hence immumohistochemical staining of CD31 was conducted as shown in Figure 9C. Four weeks after surgery, only a small amount of brown vascular lumen with smaller diameter was indentified within bone defects of the control group. In comparison, more CD31-positive endothelial cells surrounded by circular lumen could be found within GelMA group and a large amount of vascular lumen with larger diameter could be found in GelMA/MBGNs and GelMA-G-MBGNs. Eight weeks after operation, the bone defect of control group is filled with fibrous connective tissue with only a small amount of microvessel. Comparatively, a large number of CD31-positive endothelial cells was identified at the material - bone interface of GelMA group forming neovessels with small diameter and ring or oval shape. GelMA/MBGNs group and GelMA-GMBGNs group shown a larger number of positive-stained endothelial cells surrounded by regular annular lumen with decreased diameter. The sequence of quantified neovascularization (Figure S4) from high to low was as follows: GelMA-GMBGNs group > GelMA/MBGNs group > GelMA group > blank group at the same time after implantation (P < 0.05). With the prolongation of time, the number of neovascularization in the material group decreased except for the control group. Such a phenomenon could be attributed to the peak of angiogenesis at 2 weeks postoperative. This study selected only 4 and 8 weeks postoperative for staining when neovascularization began to shrink as time goes on, leading to the decreased diameter of blood vessels. Micro-CT results shown that the quantified regeneration of bone defect from high to low was: GelMA-G-MBGNs > GelMA/MBGNs > GelMA. The HE staining analysis also confirmed that addition of MBGNs could accelerate the formation of new bones and have highest proportion of mature lamellar bone. And the mesoporous structure of MBGNs enhances the activity of osteoblasts and macrophages, which is conducive to the degradation of materials to make way for the formation of new bone. When degradation of the material could be synchronized with the formation of the new bone, an optimized bone repair effect could be obtained. In addition, neovascularization of implanted materials and newborn tissue is of great significance because blood vessels could bring nutrition, bioactive factors, and osteogenesis-related cells for new bone formation and transport the metabolic wastes or toxic product. Therefore, abundant vascularization of the material could be as important as the ingrowth of new bone in the regeneration of critical size bone defect. The amount of neovascularization in the GelMA-G-MBGN group was the highest at both time points after surgery. The related cell cycle enzyme is activated, and the expression of cyclin increased, promoting DNA synthesis and chromosome replication, coming to the rapid synthesis and secretion of various growth factors.44 MBGNs can directly bind to the surface of bone tissue through chemical reaction, and form a certain thickness of HCA layer with gradual release of active ingredients to

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CONCLUSION In this study, we developed a new kind of strengthen GelMA co-cross-linked with GelMA modified MBGNs to promote structural stability of GelMA hydrogel membrane as artificial periosteum for enhancing the angiogenesis and osteogenesis in bone reconstruction. The surface-coupled GelMA single-chain MBGN and GelMA solution were mixed together to obtain photocatalytic activity of GelMA-G-MBGN hydrogel with double-network structure, which improved the structural stability of GelMA hydrogel, and the mechanical properties of GelMA-G-MBGN membrane is better than GelMA/MBGNs. The degradation rate of MBGNs can be controlled, and the pH of the microenvironment around the hydrogel membrane was stable. For this reason, the GelMA-G-MBGN membrane could enhance the osteogenesis differentiation better than GelMA and GelMA/MBGNs both in vitro and in vivo because the homeostasis is very important for cell proliferation and differentiation. This structural stability of the enhanced organic−inorganic GelMA-G-MBGNs hydrogel membrane can maintain localized body fluid environment stability under the premise of promoting vascular regeneration to accelerate bone tissue reconstruction, compared with the GelMA and GelMA/MBGNs, so the GelMA-G-MBGN hydrogel membrane increased bone regeneration and angiogenesis in rat cranial defects. Therefore, the application of this hydrogel membrane may comprise a powerful platform in bone tissue engineering to treat bone defects and other kinds of damage.

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MATERIALS AND METHODS

Fabrication of GelMA and MBGNs. MBGNs with a molar composition of 80% SiO2, 16% CaO, and 4% P2O5 were synthesized by a modified Stöber method as described previously,20,21 using TrisHCl buffer solution (pH 8.0) as a catalyst and CTAB (cetyltrimethylammonium bromide, Sigma-Aldrich, USA) as template agent (Figure S1). For surface modification, 5 mL of APTES was poured into 100 mL of anhydrous toluene (2 mg/mL) of MBGN samples, which were stirred under 400 rpm (24 h, 50 °C). The suspension was centrifuged and washed three times with absolute ethanol and deionized water. The APTES-MBGNs were then dried at 60 °C overnight for further use. To get GelMA-MBGNs, 0.3 g of APTES-MBGNs were dispersed in 20 mL of deionized water, and then 0.3 g of lyophilized GelMA macromer, 400 mg of EDC, and 200 mg of NHS were added, stirred at room temperature for 12 h, and then rinsed with deionized water three times and dried at 60 °C overnight. Preparation of GelMA:GelMA was synthesized using methods previously described. J


Research Article


Corning, USA), and 3 × 105 MC3T3-E1 cells were cultured in the lower chamber. Alkaline phosphatase activity (ALP), a widely exploited early biochemical marker for osteogenic activity, was measured. Mineralization was evaluated by quantifying the formation of calcium phosphate by cells using an Alizarin Red Staining (ARS) kit (Cyagen, Guangzhou, China). In Vivo Bone Formation Study. Sprague−Dawley (SD) male rats were purchased from the Experimental Animal Center of Soochow University (Suzhou, China). The animal handling and surgical procedures were conducted in accordance with protocols approved by the Ethics Committee at the First Affiliated Hospital of Soochow University. Rat Calvarial Critical Size Defect Model and Scaffold Implantation. Before transplant, all the hydrogels were irradiation sterilized and preswelled in PBS overnight to make sure they stayed within the calvarial defect. The hydrogels was implanted in defects (5 mm in diameter) in the calvarium of the SD rats, which were prepared with a dental trephine. Microcomputed tomography analysis: The three-dimensional (3D) structures of the regenerated bone tissue within the cranial defect area were evaluated with micro-CT (SkyScan 1176, SkyScan, Aartselaar, Belgium) with the following settings: 65 kV, 385 mA, and 1 mm Al filter. 3D reconstruction was performed with system software, with the density lower threshold set as high as possible (unified for 90). A cylinder ROI of 4.8 mm in diameter was used for the bone volume fraction (bone volume (BV)/tissue volume (TV), means ± standard deviations of 6 rats). Histological procedure: Five-micron-thick histological sections were cut at the center of the embedded specimens, followed by staining with hematoxylin and eosin (H&E) to assess the bone formation area. Images were obtained under a bright-field microscope (Zeiss Axiovert 200, Carl Zeiss Inc., Thornwood, NY, USA). Expression of CD31 was detected using rabbit antimouse CD31 monoclonal antibody (Abcam, Cambridge, UK). The number of CD31-positive blood vessels was calculated by Image-Pro Plus software (Media Cybernetics), randomly selected 5 fields under the microscope (Zeiss Axiovert 200, Carl Zeiss Inc., Thornwood, NY, USA) and count the microvessels with positive staining. Statistical Analysis. Experiments were performed in triplicate unless otherwise indicated. The data were expressed as the means ± standard deviations. Statistical analysis (GraphPad Software, Inc.; USA) was evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test to evaluate the differences between the groups. Differences at p < 0.01 and p < 0.05 were considered statistically significant.

Fabrication of GelMA, GelMA/MBGN, and GelMA-G-MBGN Hydrogel. To fabricate the GelMA hydrogel, we prepared GelMA prepolymer solution by mixing the lyophilized GelMA macromer (20% w/v final) and the photoinitiator (Irgacure 2959, J&K Scientific Ltd., Beijing, China) (1% w/v) in PBS at 60 °C until fully dissolved. The prepolymer solution was then exposed to UV light (7.1 mW/cm2, 360−480 nm) for 3 min at room temperature. To get GelMA/MBGN hydrogel and GelMA-G-MBGN hydrogel, we added MBGNs or amino-MBGNs in the GelMA prepolymer and photoinitiator solution described above by different ratio (1%, 3%, 5% w/v final). The prepolymer solution was then exposed to UV light (7.1 mW/cm2, 360−480 nm) for 3 min at room temperature, and finally GelMA/MBGN hydrogel and GelMA-G-MBGN hydrogel with different ratios of MBGN were obtained. Characterization of MBGNs and GelMA-MBGNs (G-MBGNs). Scanning Electron Microscopy (SEM) Analysis. To observe the morphology of the MBGNs and GelMA-MBGNs, the samples were examined using a scanning electron microscope (SEM, S-4800; Hitachi, Kotyo, Japan) at an accelerating voltage of 30 kV. Prior to characterization, the samples were added directly on top of conductive tapes mounted on the SEM sample stubs and sputter-coated with gold for 60 s using gold sputter-coating equipment (SC7620, Quorum Technologies, UK). Fourier Transform Infrared Spectroscopy. The structure of the MBGNs, Amino-MBGNs and GelMA-MBGNs were analyzed via Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo scientific, USA). For each measurement, 128 scans were obtained with a resolution of 4 cm−1, with wavelengths that ranged from 400 to 4000 cm−1. Transmission electron microscopy (TEM) analysis. To observe the internal morphology of the MBGNs and GelMA-MBGNs, the samples were examined using a transmission electron microscope (SEM, S4800; Hitachi, Kotyo, Japan) at an accelerating voltage of 30 kV. Small-Angle X-ray Scattering. Diffraction patterns were collected using a D/MAX 2200 X-ray diffractometer (Rigaku Corporation, Japan) with a Cu source, at a scan step size of 0.0066°. A generator voltage of 40 kV and a tube current of 40 mA were employed. Particle Size and Zeta Potential Analysis. MBGNs, AminoMBGNs, and GelMA-MBGNs were dispersed in ultrapure water and then the particle size and zeta potential were analyzed with a particle size analyzer (Zetasizer nano zs90, Malvern, UK). Characterization of GelMA, GelMA/MBGN, and GelMA-GMBGN Hydrogel. Scanning Electron Microscopy (SEM) Analysis. To observe the morphology of GelMA, GelMA/MBGN, and GelMAG-MBGN hydrogel, we examined the samples using a scanning electron microscope (SEM, S-4800; Hitachi, Kotyo, Japan) at an accelerating voltage of 15 kV. Fourier transform infrared spectroscopy: The structures of GelMA, GelMA/MBGNs and GelMA-G-MBGNs hydrogel were analyzed via Fourier transform infrared spectroscopy (FTIR, Nicolet 6700; Thermo scientific, USA). Mechanical testing: All samples were preconditioned in PBS at room temperature for 12 h prior to testing. Weight loss and loss testing: The hydrogel samples were made into disks 5 mm in diameter and 2 mm in thickness and the weight of the disks was recorded before and after soaking in PBS at 37 °C. Mineralization and iron releasing: Mineralization and iron releasing test of the hydrogel were proceeded in Simulated Body Fluid (SBF).45,38 The used SBF were collected to test the concentration of calcium, phosphorus, and silicon by ICP-AES (inductively coupled plasma atomic emission spectroscopy, PerkinElmer Optima 7300, Waltham, MA, USA) and test the pH value using a pH meter. Cell Adhesion and Proliferation on Hydrogel. Hydrogel disks described above were used for the cell adhesion studies. MC3T3-E1 cells were seeded onto disks in 96-well culture plate at a density of 2.0 × 103 cells/well. The morphology of the cells seeded on the cultured hydrogel surface was examined via SEM. Cell proliferation was measured using cell counting kit-8 reagent (CCK-8, Dojindo, Kumamoto, Japan). In Vitro Osteogenesis Study. The hydrogel disks were placed in the upper chamber of a 6-well Transwell plate (pore size: 8 mm,

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13167.

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Detailed experiment methods, Table S1, Figures S1 and S1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Wenguo Cui: 0000-0002-6938-9582 Author Contributions †

T.X., Y.G., and R.C. contributed equally to this work.

Notes

The authors declare no competing financial interest. K


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(17) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G. H.; Chmelka, B. F.; Stucky, G. D. Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores. Science 1998, 279 (5350), 548−552. (18) Mahony, O.; Tsigkou, O.; Ionescu, C.; Minelli, C.; Ling, L.; Hanly, R.; Smith, M. E.; Stevens, M. M.; Jones, J. R. Silica-Gelatin Hybrids with Tailorable Degradation and Mechanical Properties for Tissue Regeneration. Adv. Funct. Mater. 2010, 20 (22), 3835−3845. (19) Mahony, O.; Yue, S.; Turdean-Ionescu, C.; Hanna, J. V.; Smith, M. E.; Lee, P. D.; Jones, J. R. Silica−gelatin hybrids for tissue regeneration: inter-relationships between the process variables. J. SolGel Sci. Technol. 2014, 69 (2), 288−298. (20) Stö ber, W.; Fink, A.; Bohn, E. Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 1968, 26 (1), 62−69. (21) Manzano, M.; Aina, V.; Areán, C. O.; Balas, F.; Cauda, V.; Colilla, M.; Delgado, M. R.; Vallet-Regí, M. Studies on MCM-41 mesoporous silica for drug delivery: Effect of particle morphology and amine functionalization. Chem. Eng. J. 2008, 137 (1), 30−37. (22) Huo, Q.; Margolese, D. I.; Ciesla, U.; Feng, P.; Gier, T. E.; Sieger, P.; Leon, R.; Petroff, P. M.; Schüth, F.; Stucky, G. D. Generalized synthesis of periodic surfactant/inorganic composite materials. Nature 1994, 368 (6469), 317−321. (23) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G. H.; Chmelka, B. F.; Stucky, G. D. Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores. Science 1998, 279 (5350), 548−552. (24) Karageorgiou, V.; Kaplan, D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005, 26 (27), 5474−5491. (25) Saravanan, S.; Leena, R. S.; Selvamurugan, N. Chitosan based biocomposite scaffolds for bone tissue engineering. Int. J. Biol. Macromol. 2016, 93, 1354−1365. (26) Josset, Y.; Nasrallah, F.; Jallot, E.; Lorenzato, M.; Dufour-Mallet, O.; Balossier, G.; Laurent-Maquin, D. Influence of physicochemical reactions of bioactive glass on the behavior and activity of human osteoblasts in vitro. J. Biomed. Mater. Res. A 2003, 67 (4), 1205−1218. (27) Li, J.; Chen, Y.; Yin, Y.; Yao, F.; Yao, K. Modulation of nanohydroxyapatite size via formation on chitosan-gelatin network film in situ. Biomaterials 2007, 28 (5), 781−790. (28) Coleman, R. J.; Jack, K. S.; Perrier, S.; Grøndahl, L. Hydroxyapatite Mineralization in the Presence of Anionic Polymers. Cryst. Growth Des. 2013, 13 (10), 4252−4259. (29) Landis, W. J.; Jacquet, R. Association of calcium and phosphate ions with collagen in the mineralization of vertebrate tissues. Calcif. Tissue Int. 2013, 93 (4), 329−337. (30) Ducheyne, P.; Qiu, Q. Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function. Biomaterials 1999, 20 (23−24), 2287−2303. (31) Bajpai, I.; Kim, D. Y.; Kyongjin, J.; Song, I. H.; Kim, S. Response of human bone marrow-derived MSCs on triphasic Ca-P substrate with various HA/TCP ratio. J. Biomed. Mater. Res., Part B 2017, 105 (1), 72−80. (32) Kiroshka, V. V.; Petrova, V. A.; Chernyakov, D. D.; Bozhkova, Y. O.; Kiroshka, K. V.; Baklagina, Y. G.; Romanov, D. P.; Kremnev, R. V.; Skorik, Y. A. Influence of chitosan-chitin nanofiber composites on cytoskeleton structure and the proliferation of rat bone marrow stromal cells. J. Mater. Sci.: Mater. Med. 2017, 28 (1), 21. (33) Bacakova, L.; Filova, E.; Parizek, M.; Ruml, T.; Svorcik, V. Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants. Biotechnol. Adv. 2011, 29 (6), 739−767. (34) Redey, S. A.; Nardin, M.; Bernache-Assolant, D.; Rey, C.; Delannoy, P.; Sedel, L.; Marie, P. J. Behavior of human osteoblastic cells on stoichiometric hydroxyapatite and type A carbonate apatite: role of surface energy. J. Biomed. Mater. Res. 2000, 50 (3), 353−364. (35) Bellows, C.; Aubin, J.; Heersche, J. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner. 1991, 14 (1), 27−40.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81371930, 81601891, and 81301646), Shanghai Municipal Education CommissionGaofeng Clinical Medicine Grant Support (20171906), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (15KJB320012), Key Talented Man Project of Jiangsu Province (RC2011102), and Standardized Diagnosis and Treatment Project of Key Diseases in Jiangsu Province (BE2015641), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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REFERENCES

(1) Vallet-Regí, M.; Ruiz-Hernández, E. Bioceramics: From Bone Regeneration to Cancer Nanomedicine. Adv. Mater. 2011, 23 (44), 5177−5218. (2) Habibovic, P.; Barralet, J. E. Bioinorganics and biomaterials: Bone repair. Acta Biomater. 2011, 7 (8), 3013−3026. (3) Ellender, G.; Feik, S. A.; Carach, B. J. Periosteal structure and development in a rat caudal vertebra. J. Anat. 1988, 158, 173−187. (4) Allen, M. R.; Hock, J. M.; Burr, D. B. Periosteum: biology, regulation, and response to osteoporosis therapies. Bone 2004, 35 (5), 1003−1012. (5) Shi, X.; Fujie, T.; Saito, A.; Takeoka, S.; Hou, Y.; Shu, Y.; Chen, M.; Wu, H.; Khademhosseini, A. Periosteum-mimetic structures made from freestanding microgrooved nanosheets. Adv. Mater. 2014, 26 (20), 3290−3296. (6) Frohbergh, M. E.; Katsman, A.; Botta, G. P.; Lazarovici, P.; Schauer, C. L.; Wegst, U. G.; Lelkes, P. I. Electrospun hydroxyapatitecontaining chitosan nanofibers crosslinked with genipin for bone tissue engineering. Biomaterials 2012, 33 (36), 9167−9178. (7) Amini, A. R.; Laurencin, C. T.; Nukavarapu, S. P. Bone tissue engineering: recent advances and challenges. Crit Rev. Biomed Eng. 2012, 40 (5), 363−408. (8) Alge, D. L.; Anseth, K. S. Bioactive hydrogels: Lighting the way. Nat. Mater. 2013, 12 (11), 950−952. (9) Van Den Bulcke, An I.; Bogdanov, B.; De Rooze, N.; Schacht, E. H.; Cornelissen, M.; Berghmans, H. Structural and Rheological Properties of Methacrylamide Modified Gelatin Hydrogels. Biomacromolecules 2000, 1 (1), 31−38. (10) Yue, K.; Trujillo-de Santiago, G.; Alvarez, M. M.; Tamayol, A.; Annabi, N.; Khademhosseini, A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials 2015, 73, 254−271. (11) Xing, Q.; Yates, K.; Vogt, C.; Qian, Z.; Frost, M. C.; Zhao, F. Increasing Mechanical Strength of Gelatin Hydrogels by Divalent Metal Ion Removal. Sci. Rep. 2015, 4, 1−10. (12) Helary, C.; Bataille, I.; Abed, A.; Illoul, C.; Anglo, A.; Louedec, L.; Letourneur, D.; Meddahi-Pelle, A.; Giraud-Guille, M. M. Concentrated collagen hydrogels as dermal substitutes. Biomaterials 2010, 31 (3), 481−490. (13) Malda, J.; Visser, J.; Melchels, F. P.; Jungst, T.; Hennink, W. E.; Dhert, W. J.; Groll, J.; Hutmacher, D. W. 25th anniversary article: Engineering hydrogels for biofabrication. Adv. Mater. 2013, 25 (36), 5011−5028. (14) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2012, 64, 18−23. (15) Hench, L. L.; Splinter, R. J.; Allen, W. C.; Greenlee, T. K. Bonding Mechanisms at the Interface of Ceramic Prosthetic Materials. J. Biomed. Mater. Res. 1971, 5 (6), 117−141. (16) Aguirre, A.; González, A.; Planell, J. A.; Engel, E. Extracellular calcium modulates in vitro bone marrow-derived Flk-1+ CD34+ progenitor cell chemotaxis and differentiation through a calciumsensing receptor. Biochem. Biophys. Res. Commun. 2010, 393 (1), 156− 161. L


Research Article

ACS Applied Materials & Interfaces (36) Hench, L. L.; Jones, J. R. Bioactive Glasses: Frontiers and Challenges. Front. Bioeng. Biotechnol. 2015, 3 (194), 1−12. (37) Long, T.; Zhu, Z.; Awad, H. A.; Schwarz, E. M.; Hilton, M. J.; Dong, Y. The effect of mesenchymal stem cell sheets on structural allograft healing of critical sized femoral defects in mice. Biomaterials 2014, 35 (9), 2752−2759. (38) Shang, X.; Shu, B.; Wang, Y.; Luo, Z.; Wang, G.; Barton, S.; Morandi, M. M.; Kevil, C.; Dong, Y. Human Mesenchymal Stromal Cell Sheet Enhances Allograft Repair in a Mouse Model. Sci. Rep. 2017, 7 (1), 7982. (39) Tian, Y.; Xu, Y.; Xue, T.; Chen, L.; Shi, B.; Shu, B.; Xie, C.; Max Morandi, M.; Jaeblon, T.; Marymont, J. V.; Dong, Y. Notch activation enhances mesenchymal stem cell sheet osteogenic potential by inhibition of cellular senescence. Cell Death Dis. 2017, 8 (2), e2595. (40) Hoffman, M. D.; Benoit, D. S. Emulating native periosteum cell population and subsequent paracrine factor production to promote tissue engineered periosteum-mediated allograft healing. Biomaterials 2015, 52, 426−440. (41) Shubin, A. D.; Felong, T. J.; Schutrum, B. E.; Joe, D. S.; Ovitt, C. E.; Benoit, D. S. Encapsulation of primary salivary gland cells in enzymatically degradable poly(ethylene glycol) hydrogels promotes acinar cell characteristics. Acta Biomater. 2017, 50, 437−449. (42) Vats, K.; Marsh, G.; Harding, K.; Zampetakis, I.; Waugh, R. E.; Benoit, D. S. Nanoscale physicochemical properties of chain- and stepgrowth polymerized PEG hydrogels affect cell-material interactions. J. Biomed. Mater. Res., Part A 2017, 105 (4), 1112−1122. (43) Giatromanolaki, A.; Koukourakis, M. I.; Theodossiou, D.; Barbatis, K.; O’Byrne, K.; Harris, A. L.; Gatter, K. C. Comparative evaluation of angiogenesis assessment with anti-factor-VIII and antiCD31 immunostaining in non-small cell lung cancer. Clin. Cancer Res. 1997, 3 (12), 2485−2492. (44) Hench, L. L.; Polak, J. M. Third-generation biomedical materials. Science 2002, 295 (5557), 1014−1017. (45) Zhou, L.; Tan, G.; Tan, Y.; Wang, H.; Liao, J.; Ning, C. Biomimetic mineralization of anionic gelatin hydrogels: effect of degree of methacrylation. RSC Adv. 2014, 4 (42), 21997−22008.

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Inorganic Strengthened Hydrogel Membrane as Regenerative

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