Hierarchically Porous Hydroxyapatite Hybrid Scaffold Incorporated

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Hierarchically Porous Hydroxyapatite Hybrid Scaffold Incorporated with Reduced Graphene Oxide for Rapid Bone Ingrowth and Repair Kai Zhou, Peng Yu, Xiaojun Shi, Tingxian Ling, Weinan Zeng, Anjing Chen, Wei Yang, and Zongke Zhou ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b04723 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Hierarchically Porous Hydroxyapatite Hybrid Scaffold Incorporated with Reduced Graphene Oxide for Rapid Bone Ingrowth and Repair Kai Zhou,†,₴ Peng Yu,‡ Xiaojun Shi,† Tingxian Ling,† Weinan Zeng,† Anjing Chen,† Wei Yang,‡,* Zongke Zhou†,*

†Department

of Orthopaedics, West China Hospital of Sichuan University, Chengdu,

610041, Sichuan, China ‡College

of Polymer Science and Engineering, Sichuan University, State Key

Laboratory of Polymer Materials Engineering, Chengdu, 610065, Sichuan, China ₴State

Key Laboratory of Biotherapy and Cancer Center, West China Hospital,

Sichuan University and Collaborative Innovation Center, Chengdu, 610041, China

Corresponding Authors E-mails: [email protected] (W Yang) and [email protected] (Z Zhou)

Tel: 0086-028-85422570; Fax: 0086-028-85423438

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Table of Content

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ABSTRACT: Hydroxyapatite (HA), the traditional bone tissue replacement material was widely used in the clinical treatment of bone defects because of its excellent biocompatibility. However, the processing difficulty and poor osteoinductive ability greatly limit the application of HA. Although many strategies have been reported to improve the machinability and osteointegration ability, the performance including mechanical strength, porosity, cell adhesion, etc. of material still can not meet the requirements. In this work, a soft template method was developed and a porous scaffold with hierarchical pore structure, nano surface morphology, suitable porosity and pore size, and good biomechanical strength was successfully prepared. The hierarchical pore structure is beneficial for cell adhesion, fluid transfer, and cell ingrowth. Moreover, the loaded reduced graphene oxide (rGO) can improve the adhesion and promote the proliferation and spontaneous osteogenic differentiation bone marrow mesenchymal stem cells. The scaffold is then crushed, degraded and wrapped by the newly-formed bone and the newly-formed bone gradually replaces the scaffold. The degradation rate of the scaffold well matches the rate of the new bone formation. The hierarchical porous HA/rGO composite scaffolds can greatly accelerate the bone ingrowth in the scaffold and bone repair in critical bone defects, thus providing a clinical potential candidate for large segment bone tissue engineering. KEYWORDS: hierarchical porous structure, HA/rGO composite scaffold, bone defect, cell adhesion, bone ingrowth

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With the rapidly aging population, patients with bone defects are increasing due to trauma, tumors, infections, etc.1 Bone defects that exceed the critical criterion are often difficult to repair, which can easily to cause delayed or even non-healing.2 The clinical treatment of critical bone defects is one of the most challenging topics in orthopedics, and it is necessary to implant materials to repair the defects.3 As the “gold standard” for bone transplantation, autologous bone graft should have good osteoconductivity, osteoinductive and osteogenic matrix, which can effectively improve the fracture healing. However, there are complications such as pain, bleeding, nerve injury and bone fracture in the bone donor area, and the bone mass in the donor area is limited.4 Allogeneic bone has poor osteoinductive activity, possible disease transmission, and social ethical controversy.5 Therefore, developing an ideal artificial bone graft has important clinical practical values and has always been a goal for biomaterial engineering. Inorganic

materials,

especially

calcium

phosphate

ceramics

such

as

hydroxyapatite (HA), owing to the similarity in the inorganic components of bone tissues and good biocompatibility, degradability, and osteoconductivity, have become the most popular bone substitute materials.6-8 However, the present HA artificial bone repair materials show poor cell crawling and cell adhesion, slow replacement and difficult ingrowth, which limits the clinical application of HA material.9-11 One of the reasons is that they fail to provide a microenvironment suitable for bone growth, which should be a highly interconnected three-dimensional hierarchical structures with macropores and micro-nano structures.12-14 The interconnected macropores can 4


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provide cell ingrowth channels.15,16 The micro-nano structure provides more adsorption sites to bioactive molecules and improves the nutrient and metabolic waste transportation.17,18 Moreover, this kind of interconnected three-dimensional hierarchical structure can induce early osteogenesis from the surrounding cells and tissues through cell adhesion, penetration, proliferation, and tissue ingrowth.15 So, the preparation of HA bone repair materials with hierarchical structures is a key problem to be solved in artificial bone repair materials.19 A variety of methods are currently available for the preparation of hierarchical structures, such as sintering, freeze-casting,20,21 3D printing.22,23 Among them, sintering has the advantages of simplicity, repeatability, etc.24 In addition, the reasons that artificial bone materials cannot replace the autologous bones at present are that the structure is not conducive to the growth of cells, and that it is difficult for mesenchymal stem cells (MSCs) and other related stem cells homing to the nest inside the materials.25 When a critical size bone defect occurs, even if the local growth factor exists in the material, it is difficult to form a sufficient number of osteoblasts due to the insufficient local MSC enrichment and ingrowth, resulting in the difficulty in bone defect repair. Multiple factors are important for the MSC homing, and simply tuning some factors in the bone defects cannot satisfactorily promote MSC homing.25,26 Therefore, it is particularly important to accumulate multiple factors at the bone defect site and then the enriched multiple factors provide a homing effect for MSCs. Graphene derivatives, oxidized graphene (graphene oxide, GO), which has a large 5


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amount of π-π stacking, hydrogen bonding and electrostatic binding force, can not only promote the fibronectin extracellular protein adsorption to directly mediated cell adhesion,27,28 but also enrich various factors such as growth factors and hormones in the body fluid,29,30 which constitutes a series of biological activities and promotes the initial attachment of stem cells and thus the proliferation and differentiation.31-33 However, many studies have suggested that GO may have possible cytotoxicity to a certain extent,34,35 and the toxicity has been demonstrated to be contact time, dose and/or concentration, and surface chemistry dependent.36,37 According to reports, GO can be degraded by endocytosis38 and protein corona reduces its cytotoxicity due to a significant weakening of the lipid-graphene interaction39 in vivo. But the good water dispersibility of GO limits its application. Reduced graphene oxide (rGO) is obtained by reducing GO to partially remove some partial oxygen functional groups.40 The reduction of GO into rGO lowers down the water dispersibility, allowing GO to be stable in the body and reduce the possible cytotoxicity.41,42 In general, rGO has better biosafety and stability than GO in vivo.42 Various studies have demonstrated that rGO is biocompatible, making it suitable for cell culture, biosensing, tissue engineering, and other biomedical applications.40,43 However, the duration of reported studies about rGO composite bone repair materials was generally insufficient for the examination of scaffolds degradation.44 The long term effects and associating risks, if any, of using graphene in tissue scaffolds in vivo are unclear and require a more thorough assessment prior to practical use.45

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In this study, we prepared three-dimensional porous HA/rGO scaffolds with a hierarchical structure, nano surface morphology, suitable porosity and pore size, and good biomechanical strength. The hierarchical pore structure is beneficial for the cell adhesion, fluid transfer, and cell ingrowth. The loaded rGO can improve the adhesion and promote the proliferation and spontaneous osteogenic differentiation bone marrow mesenchymal stem cells (BMSCs). The scaffold can be degraded and wrapped by the newly formed and the newly formed bone gradually replaces the location of the scaffold. The degradation rate of the scaffold well matches the rate of new bone formation. The porous HA/rGO composite scaffolds prepared by this strategy are highly potential to be clinical candidates of scaffolds in bone defect repair tissue engineering.

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RESULTS AND DISCUSSION The formation mechanism of the hierarchical pore structure is schematically given in Figure 1. Firstly, melamine foam was immersed in HA/Chitosan (CS) composite slurry until the sponge was full of the slurry (Figure 1a). Then, the melamine foam full of the slurry was transferred into the vacuum oven. During vacuum drying, with the evaporation of the solvent, water, the volume of the filled slurry reduced, so the vacancy uniformly appeared in the foam. Vacuum suction guided the gradual and uniform expansion of vacancy during the evaporation of the solvent (Figure 1b). At last, the expanding vacancy connected with each other and formed a through-hole structure in the foam. During the solvent evaporation, CS molecular chains shrunk. Because of the strong hydrogen bonding between CS molecular chains and nano-HA, the shrinking of CS molecular chains dragged the nano-HA and made it tightly stuck to the frame of the melamine foam. Thus, uniform through-hole structure was formed in the melamine foam. The schematic diagram was shown in Figure 1b. After sintering in air atmosphere, melamine foam and CS were burned out and pure porous HA ceramics were obtained as shown in Figure 1c. During sintering, nano-HA was sintered together and porous HA ceramic formed. Simultaneously, the micropores formed on the through-hole structure because of the space occupied by needle-like nano-HA. After the introduction of GO, large GO sheets attached on the surface of the through-hole structure and small GO sheets embedded on the walls of the pores and wrapped into the internal of the micropores because of Van der Waals force (Figure 1d). Then, thermal reduction was carried out at 1000 oC under nitrogen 8


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atmosphere. After heat reduction, the reduced graphene sheets closely integrated with HA because of the shape changes of GO sheets during the reduction and the electrostatic interaction between graphene sheets and HA (Figure 1e).46,47

Figure 1. Diagram of the formation mechanism of the porous HA/rGO composite scaffold.

The microstructures of the composite scaffold were characterized with SEM as shown in Figure 2. Figure 2a shows that a uniform porous structure was formed in the scaffold of HA/rGO-6/0.3. SEM images of the prepared scaffolds HA-6, HA-6-CS (Without CS) and HA-6-D (Non-vacuum drying) (Detail preparation method were shown in experiment part) after drying were shown in Figure S1a,b and c. The uniform porous structure was only formed in scaffold HA-6, so the conditions of vacuum drying and the addition of CS are necessary for the formation of the uniform porous structure. After sintering, the melamine foam was burned, and the contrast of 9


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the microstructure is more obvious (Figure S1d,e and f). To guarantee desirable cell inﬁltration and growth inside the scaffolds, the pore size should be sufficiently large depending on the types of cells. For bone tissue engineering, the minimum pore size should be approximately 100 μm due to the requirement of the cell size, migration, and transport.48-50 In the enlarged view of the porous structure (Figure 2b,d), the pore structure is regular and the pore size of these scaffolds ranged from 90 to 130 μm. More importantly, the pores are through holes and conducive for cells to grow into the interior of the material. What’s more, hierarchical pore structure was formed, and micropores were formed on the through-hole structure (the red circles in Figure 2f). This kind of miropores can provide the cell adhesion points. After the loading of graphene, the graphene attached on the surface of the hole wall (Figure 2e) successfully. The rich folds and functional groups on the surface of graphene can promote cell adhesion in the scaffolds.51-53 Some smaller graphene sheets get into the hole wall (Figure 2c), which can help the cell adhere to the cross-section after the cleavage of the scaffold in the process of degradation in vivo. In the enlarged view of the cross-sectional structure of the hole wall (Figure 2f), the graphene folds are clearly seen as shown in the yellow circles. As we can see, the graphene tightly fits on the surface of HA, and the combination between HA and graphene is strong after thermal reduction.

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Figure 2. SEM image of (a) the porous structure in HA/rGO-6/0.3 composite scaffold, (b) and (d) the pore structure, (c) cross-sectional structure of hole wall, (e) pore wall structure and (f) Enlarged view of the cross-section of hole wall. (g) Pore structure of HA/rGO-6/0.3 and EDS (h) Ca, (i) P, (j) C mapping images of Figure 2g. (k) Porosity and compression modulus of HA-3, HA-6, HA-8, HA-6-D and HA-6-CS. (l) Specific surface area of HA-6, HA/rGO-6/0.1, HA/rGO-6/0.3, HA/rGO-6/0.6 and HA/rGO-6/0.9.

The uniform distribution of graphene on the wall of the pores and in the holes can also be proved by EDS mapping images. In Figure 2g,h,i and j, EDS Ca and P 11


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mappings of HA/rGO-6/0.3 (Figure 2h,i) are bright and the EDS C mapping (Figure 2j) is relatively dark, but a uniform distribution of C can be clearly observed in the scaffold. It is worth mentioning that the through-holes can be blocked by the graphene sheet when the amount of graphene is too high, as shown in the red circles in Figure S2b of sample HA/rGO-6/0.9. As a comparison, the internal pore can be clearly seen in HA/rGO-6/0.3 in the red circle in Figure S2a. Except for the morphology, pore size and interconnectivity, the mechanical strength and porosity of the scaffold should also be considered.49,54,55 The porosity and compression modulus of samples are shown in Figure 2k. The compression modulus increases while the porosity decreases with increasing content of HA. We can see that HA-6 shows a relatively high compression modulus on the premise of high porosity. The compression modulus of sample HA-6-D, HA-6-CS prepared without vacuum drying or CS are quite low, and the reason is that the HA nanopowder cannot stick and sinter together because of the absence of hydrogen bonding and vacuum suction effect during the drying process. Actually, the compression modulus is closely related to microstructures. In Figure S3, SEM images of the surface of the pore walls of HA-6, HA-6-D, and HA-6-CS are shown. The HA nanopowder is sintered together in HA-6 while the microstructures of HA-6-D and HA-6-CS are relative loose. So, the compression modulus of HA-6-D and HA-6-CS are much lower than that of HA-6 although the same amount of HA nanopowder was introduced into the samples. The specific surface area (SSA) can be achieved by analyzing N2 12


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adsorption/desorption at 25 ºC (Figure 2l).56,57 The specific surface area of the scaffolds increases with increasing amount of graphene in the samples, and we believe that the increased specific surface area provides more sites for cell adhesion and promotes the proliferation and differentiation of the cells. Considering that the through-holes are blocked by graphene when the amount of graphene is too high, sample HA/rGO-6/0.3 was selected for the subsequent biological analysis.

Figure 3. (a) TG loss rate of scaffolds HA-6, HA/rGO-6/0.1, HA/rGO-6/0.3, HA/rGO-6/0.6 and HA/rGO-6/0.9. (b) FT-IR spectra of HA, GO, rGO and HA/rGO-6/0.3. (c) XRD spectra of GO and rGO. (d) Raman spectra of GO and rGO and XPS spectra of (e) GO and (f) rGO. The inset image of Figure 3f is the digital photo of HA/GO-6/0.3 and HA/rGO-6/0.3 immersed in physiological saline for 3days under shake at 37 ℃.

In order to figure out the content of graphene in the scaffolds, thermogravimetric (TG) analysis was performed and the TG loss rate was calculated according to the TG 13


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curve of sample HA-6, HA/rGO-6/0.1, HA/rGO-6/0.3, HA/rGO-6/0.6, and HA/rGO-6/0.9. The results are shown in Figure 3a. The TG loss rate of HA-6 was very low, meaning that CS and melamine foam were burned out during the first sintering process, which can also be proved by the XRD patterns in Figure 3c. The characteristic peaks of CS and melamine disappear in the curve of HA-6, and only the characteristic absorption peaks of HA are left, showing that sample HA-6 which only contains HA can be fully biodegraded in vivo and the TG loss ratio is approximately identical to the content of graphene in different samples. It is noticed that the TG loss ratio increases with increasing content of GO in alcohol during the preparation. However, the TG loss ratio of HA/rGO-6/0.9 which has the highest graphene content is only around 2 %, so the composite scaffolds can be mostly biodegraded in vivo after implantation. FTIR spectra of GO, rGO, HA, and HA/rGO-6/0.3 are shown in Figure 3b. The absorption peaks at 1727 cm−1 (C=O stretching vibrations), 1224 cm−1 (epoxy groups) and 1051 cm−1 (C-O stretching vibrations) in the curve of GO disappear in the curve of rGO, indicating that GO has been reduced. The characteristic absorption peaks of HA at 1037 cm−1 and 962 cm−1 (O-P stretching vibrations)58-60 appear in the curve of HA/rGO-6/0.3, indicating that the chemical structure of HA does not change in the process of sample preparation. The presence of HA in the scaffolds is also evident in XRD characterization in Figure 3c, in which the characteristic diffraction peaks of HA appear60-62 in the pattern of HA-6. It is worth mentioning that the intensity of the characteristic peak of HA-6 is higher than that of HA nanopowder, because the 14


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nanopowder has been sintered together to form HA ceramics and HA recrystallizes into a more perfect structure. The XRD pattern of GO shows typical peaks at 10.5° and 42.2° while in the pattern of rGO, the two typical peaks of GO disappear, confirming the reduction of GO during sample preparation. Raman spectra of GO and rGO (Figure 3d) show typical D and G peaks at around 1588 cm-1 and 1345 cm-1 which can be attributed to D band (symmetry A1g mode) and G band (E2g mode of sp2 carbon atoms), respectively. The increased D/G intensity ratio of rGO compared to that of GO demonstrates the removal of oxygen groups and restoration of the sp2 network during reduction. A more precise evaluation of the reduction achieved during heating treatment can be obtained by XPS (Figure 3e and f). Oxygen-containing groups were found to connected on graphene sheets, and the XPS spectra of GO (Figure 3e) can be fitted into four peaks corresponding to sp2 carbon (C=C, 284.5 eV), epoxy/hydroxyls (C-O, 286.5 eV), carbonyl (C=O, 287.4 eV), and carboxylates (O=C-O, 288.4 eV).46,63,64 In Figure 3f, the intensity of the C-C/C=C peak increases significantly, indicating that more aromatic rings are present after the transformation of GO to rGO. At the same time, the peaks associated with oxygen functionalities decreases, demonstrating that most oxygen functional groups are removed by heat reduction.65,66 As a result, the composite scaffolds are mostly biodegradable and GO has been successfully reduced. Sample HA/GO-6/0.3 and HA/rGO-6/0.3 were immersed in physiological saline for 3 days under shake at 37℃ to evaluate the stability in physiological solution environment. The supernatant of HA/rGO-6/0.3 is much clearer (inset image of Figure 3f) and the dispersibility of GO 15


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in aqueous solution greatly decreases after reduction into rGO. So sample HA/rGO-6/0.3 is stable in aqueous solution without the leakage of graphene.

Figure 4. SEM image of the morphology and adhesion of BMSCs on (a), (b) HA/rGO-6/0.3 and (c), (d) HA-6 surface. Fluorescent staining of BMSCs grown on (e) HA/rGO-6/0.3, (f) HA-6 and (g) control surface. (h) Detection of cell proliferation on HA/rGO-6/0.3, HA-6 and control by the cell counting kit-8 assay (up to 7 d). Calcification of cell matrix after incubation on (i) HA/rGO-6/0.3, (j) HA-6 and (k) control for 21d. (l) Quantitative analysis of three groups of positive staining for calcium nodules. Osteogenesis related genes expression of BMSCs including type I collagen (COL-I), runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP-2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) after (m) 7d, (n) 14d and (o) 21d incubation at HA/rGO-6/0.3, HA-6 and control by qRT-PCR. 16


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The in vitro study results are shown in Figure 4. The details of the BMSCs adhesion onto the scaffolds were examined by SEM after culturing for 7 days (Figure 4a-d). More cells cover the surface of the scaffolds and grow into the pores in scaffold HA/rGO-6/0.3 (Figure 4a) than in scaffold HA-6 (Figure 4c). The cells on both groups protrude pseudopod and adhere to the scaffold through the micropore (Figure 4b,d). It demonstrates that the scaffold promotes cell adhesion and rGO can accelerate cell proliferation. The cells are flattened with a widely spread cytoskeleton on both HA/rGO-6/0.3 (Figure 4e) and HA-6 (Figure 4f) scaffolds, whereas the cell number and state on the control group are inferior and the cell shrinkage is obvious (Figure 4g). The SEM image and cytofluorescence staining show that in the HA/rGO-6/0.3 scaffold higher cell density and larger spreading area are achieved. The time-related proliferation of BMSCs was evaluated by the cell counting kit-8 (CCK-8) assay and the results are presented in Figure 4h. The BMSC proliferation in each group is similar within 3 days. When the time is extended to day 5 and day 7, the proliferation of BMSCs on the scaffold HA/rGO-6/0.3 is faster (p < 0.001, respectively). These results indicate that the presence of rGO alters the initial cellular adhesion of BMSCs and HA/rGO-6/0.3 scaffold was more advantageous to BMSC proliferation and induces no apparent toxicity to the BMSCs. It can be attributed to the high-temperature reduction of GO to rGO, which reduces the potential cytotoxicity. The CCK-8 experiment did not find that the HA/rGO-6/0.3 composite scaffold shows obvious cytotoxicity or value-inhibiting effect, and HA/rGO-6/0.3 scaffold promotes 17


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the cell proliferation. So, we chose HA/rGO-6/0.3 composite scaffold for the following experiments. Extracellular matrix (ECM) mineralization of BMSCs determined by alizarin red staining is shown in Figure 4i-k. The BMSCs exhibit slightly up-regulated ECM mineralization on HA/rGO-6/0.3 (Figure 4i) compared to HA-6 (p < 0.001, Figure 4j) and control (p < 0.001, Figure 4k) group, as shown by the quantitative analysis (Figure 4l). The osteogenesis-related gene expressions were quantitatively measured to assess the osteogenic differentiation of BMSCs after culturing for 7 days (Figure 4m), 14 days (Figure 4n) and 21 days (Figure 4o). In general, BMSCs on scaffold HA/rGO-6/0.3 exhibit a stronger osteogenic differentiation tendency on genetic levels. Compared with scaffold HA, the BMP-2 expression is up-regulated nearly 1.5-folds after 7 days and 14 days cultivation on scaffold HA/rGO-6/0.3 and is nearly doubled after 21 days. The alkaline phosphatase (ALP) expression is nearly doubled after 7 days and nearly 1.5-folds after 14 days, and then maintains to day 21. The osteopontin (OPN) expression is up nearly 1.5-folds after 14 days and then maintains to day 21. The OPN expression is up nearly 1.3-folds after 14 days and then maintains to day 21. To enhance the bone regeneration performance, some studies tried to combine the scaffold with an osteoinductive protein, such as bone morphogenetic protein 2 (BMP-2).67,68 However, the actual application effect is not satisfactory, which may be attributed to the rapid degradation of BMP-2. It is difficult to maintain a locally suitable concentration of BMP-2 so the osteogenesis could not stably occur.67 By simply increasing the BMP concentration, it probably leads to unwanted 18


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local side effects, such as ectopic bone formation, osteoclast activation and soft-tissue swelling.69 The present HA/rGO scaffold is designed to avoid these problems. The HA/rGO scaffold-loaded rGO can continuously and effectively absorb BMP-2 in surrounding body fluid, and maintain its precise role in local repair sites. This can simultaneously avoid the side effects caused by exogenous addition of BMP-2 and the poor bone formation effect in the scaffold material caused by BMP-2 insufficience. The enhanced osteogenic differentiation of BMSCs in the HA/rGO-6/0.3 scaffold may be due to the ability of rGO to increase the adhesion of matrix proteins on the scaffold surface and local aggregation of osteogenic inducers.31,32 rGO contain several oxygen-containing functional groups such as carboxyl, carboxylic acid, carbonyl, and hydroxyl groups. It can non-covalently bond to biologically active substances such as extracellular growth factors through surface hydrogen bonding, electrostatic binding, and π-π stacking. It adsorbs extracellular proteins such as fibronectin and dexamethasone to directly mediate cell adhesion and constitute a basis for a series of biological activities that promotes stem cell initial attachment and thus value-added differentiation.28,31-33

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Figure 5. Specimen gross pictures and X-ray images of (a) (d) HA/rGO-6/0.3, (b) (e) HA-6 scaffolds and (c) (f) control group showing the healing of bone defects. (g), (h) Rendered 3D images and representative tomographic images of micro-CT of the implanted (g) HA/rGO-6/0.3 and (h) HA-6 scaffolds after 8 weeks, with the newly formed bone marked by green highlight. (i) Quantification of the newly formed bone in the implant. BV/TV refers to bone volume/total volume, Tb.Th refers to trabecular thickness and Tb.Sp refers to trabecular separation. Histological images of the newly formed bone in scaffold (j) HA/rGO-6/0.3, (k) HA-6 and (l) control group after 8 weeks. The red staining represents the new bone and the black area represents the scaffolds. (m) Quantification of areal new bone to material ratio in the histological samples and the comparison of bone mineral apposition rate in the HA/rGO-6/0.3 and HA-6 sample. Fluorescent stained image of newly formed bone in the (n) HA/rGO-6/0.3, (o) HA-6 and (p) control group at week 8. (q) Quantification of fluorescence expression in the fluorescent 20


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stained image, IL.WI refers to interlabeled width, MAR refers to mineral apposition rate.

The results of in vivo studies are shown in Figure 5. According to the gross specimens images and X-ray images, the HA/rGO-6/0.3 scaffold is firmly bonded to the host bone (Figure 5a,d), and the fusion is good and no obvious gap is observed. But for the HA-6 scaffold, there is a clear outline and a clear boundary with the host bone (Figure 5b,e). The defect area in the control group is filled with fibrous tissue (Figure 5c,f). To evaluate the new bone formation within the scaffold, we performed micro-CT and histological analysis. Due to the interconnected porous structure, the ingrowth of bone is deep into the scaffold. In the HA/rGO-6/0.3 group, the new bones was widely distributed inside the material (green highlight), and the porous structure are gradually decomposed (Figure 5g). In the HA-6 scaffold, scattered distribution of new bone can be found inside the scaffold (green highlight), and the porous structure of the material remains relatively intact (Figure 5h). In accordance with the X-ray results, the HA/rGO-6/0.3 scaffold shows a better integration with the host bone tissue at the end of week 8. Quantification of the static parameters of bone formation by micro-CT analysis reveals that the BV/TV and the Tb.Th is significantly higher, and the Tb.Sp is significant lower in the scaffold HA/rGO-6/0.3 group compared to those of the scaffold HA group (p < 0.05, p < 0.05, p < 0.01, respectively) and blank control group (p < 0.05, p < 0.01, p < 0.01, respectively. Figure 5i). Histological analysis was performed with magenta-methylene blue staining. The material and the newly formed bone completely reach the bony bond and fuse well 21


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with the host bone in the scaffold HA/rGO-6/0.3 group (Figure 5j). A large number of newly formed bones are filled inside the scaffold. The porous structure of the scaffold is degraded and surrounded by the bony callus, which bridges each other in most areas. But for scaffold HA-6, the newly formed bones are closely attached to the wall of the inner pores and they do not reach bony bond. Moreover, the three-dimensional porous structure is not cracked (Figure 5k). For the control group, the defect area is filled with a large number of fibrous tissue, and no new bone formation can be observed in the gap (Figure 5f). The histomorphometric analysis of new bone formation shows significantly higher positive staining proportion in the scaffold HA/rGO-6/0.3 group (33.7 ± 7.66 %) than in the scaffold HA-6 group (14.7 ± 5.2 %, p < 0.01) and the control group (3.5 ± 0.82 %, p < 0.01) after implanting for 8 weeks (Figure 5m). The results of the sequential fluorescent labeling are presented in Figure 5n and p. The fluorescent strip and strip spacing are wide in the scaffold HA/rGO-6/0.3 group (Figure 5n), but the fluorescent strip is thin and the spacing is narrow in the scaffold HA-6 group (Figure 5o). No obvious fluorescent strip is formed in the control group (Figure 5p). Quantification of the dynamic parameters of bone formation by fluorescent labeling reveals that the IL.WI and the MAR are significantly higher in the scaffold HA/rGO-6/0.3 group than in the scaffold HA group (p < 0.001, p < 0.001, respectively. Figure 5q). The histological observation confirms the existence of the new bone via micro-CT thresholding. All of these results demonstrate that the HA/rGO-6/0.3 groups show better host bone integration and osteoconductive ability. 22


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It is widely accepted that the internal architecture of scaffolds has an important role to play in tissue engineering applications, along with the mechanical and biological properties.13,15,18,19 In this study, we successfully fabricate a bone repair scaffold with three-dimensional hierarchical structures, which can not only allow the nutrients to infiltrate and provide pathways for tissue ingrowth, but also have appropriate strength to withstand in vivo stresses at the site of application until newly formed bones occupy the biodegradable scaffold matrix via new bone regeneration. Additionally, the π-π stacking, hydrogen bonding and electrostatic bonding of the rGO loaded on a material can adsorb macromolecules such as proteins by non-covalent binding.29 As a result, various kinds of growth factors are enriched at the bone defect site to promote the MSCs homing, thereby enabling MSC to exert osteogenic differentiation and bone repair.32-34

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Figure 6. SEM images of the newly formed bone growth into the (a), (b), (c) scaffold HA/rGO-6/0.3 and (d), (e), (f) scaffold HA-6 (M means material, NB means new bone). Rendered 3D images and representative tomographic images of micro-CT of the implanted scaffold (g) HA/rGO-6/0.3 and (h) HA-6 after 8 weeks. (i) Quantification of newly formed bone in the implant. BV/TV refers to bone volume/total volume, Tb.Th refers to the trabecular thickness and Tb.Sp refers to the trabecular separation. (j) Diagram of the the HA/rGO composite scaffold substitution and new bone formation mechanism. (k) Mechanical test performance of the surgical femur integrated with scaffold HA/rGO-6/0.3 at after 6 months.

After 6 months, SEM results show that the morphology of HA/rGO-6/0.3 scaffold 24


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becomes incomplete (Figure 6a), and the decomposed composite is wrapped by the new bones, and the decomposed composite and the newly formed bones are tightly bonded, showing excellent osseointegration ability (Figure 6a-c). On the contrary, the morphology of HA-6 scaffold is relatively complete with partial cleavage (Figure 6d). New bone growth can be seen on the surface and inside the pores of the material, and the combination of the scaffold and the newly formed bones is not as good as that in the HA/rGO-6/0.3 group (Figure 6d-f). Micro-CT scanning confirms that more bones grow in the HA/rGO-6/0.3 scaffold group. It can be seen that the internal cracks of the material are homogeneous, and the holes are filled with new bones (Figure 6g), while only a small number of new bones are observed in the holes of the HA-6 scaffold group (Figure 6h). Quantification of bone formation by micro-CT scanning reveals that the BV/TV and the Tb.Th of the HA/rGO-6/0.3 scaffold group are almost double than those in the scaffold HA group (p < 0.001, p < 0.001, respectively) and in the control group (p < 0.001, p < 0.001, respectively), and the Tb.Sp is significant lower compared to the HA scaffold group (p < 0.05) and the control group (p < 0.001, Figure 6i). Mechanism diagram of cleavage and degradation of the composite scaffold in vivo is shown in Figure 6j. The stem cells rapidly grow into the scaffold because the suitable microenvironment provided by the multi-level pore structure and rGO can improve cell adhesion and promotes the proliferation and differentiation of stem cells. With the increasing size of the new bone, the scaffold gradually cracks into pieces because of the extrusion of new bone. With the cleavage of the scaffold, more rGO 25


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inside the scaffold is exposed and the exposed rGO further promotes the cell adhesion, the proliferation and differentiation of stem cells. As a result, the cracked scaffold is degraded and wrapped by the new bone and the new bones gradually occupy the scaffold, and the degradation rate of the scaffold well matches the rate of new bone formation. At last, the scaffold is degraded and completely wrapped. rGO is wrapped in the new bones. To confirm the long-term biosafety of the HA/rGO-6/0.3 material, HE staining of rat heart, liver, spleen, kidney, lung, and pancreas after implantation for 6 months were performed. No obvious abnormalities are found in all the observed organizations (Figure S5). The repaired femur critical bone defect with the help of scaffold HA/rGO-6/0.3 shows good mechanical strength. The repaired bone can lift the weight of 500 g vertically and horizontally (Figure 6k). The tensile stress-strain curves of the repaired bone with the help of HA/rGO-6/0.3 and HA-6 are shown in Figure 6k, and the breaking strength of the repaired bone with the help of scaffold HA/rGO-6/0.3 is satisfactory (20.0 MPa) and much higher than that of HA-6 (9.8 MPa) which demonstrates that the cooperation of hierarchical porous structure and rGO can greatly promote the bone tissue repair. In summary, rGO-incorporated hierarchical porous HA composite scaffolds show faster bone ingrowth and good repairing effect.

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CONCLUSIONS This study successfully fabricated hierarchical porous HA/rGO composite scaffolds with an appropriate degradation rate and mechanical strength. The hierarchical porous structure and loaded rGO help to promote the osteogenic differentiation of BMSCs in vitro and faster bone repair in vivo. The rGO-incorporated hierarchical porous HA composite scaffolds are good clinical potential candidates for faster bone defect repair.

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METHODS

Materials. Natural graphite flakes (NG) with a purity of over 99.9 % were purchased from Shenghua Research Institute (Changsha, China) and were used without further purification. Hydroxyapatite (HA) nanopowder (purity > 97 %, particle size < 100 nm (BET)) was purchased from Aladdin (Shanghai, China). Chitosan (CS) with an acetylation degree of 90 %, a viscosity of 50 MPa·s was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd. (Nanjing, China). The through-hole melamine foam with the diameter between 100-300 μm was purchased from Beiyou building materials Co., Ltd. (Shanghai China).

Scaffold Preparation. There are three steps in the preparation of the scaffolds. First, 200 μl acetic acid was added into 10 ml distilled water to dissolve CS and 0.3 g CS powder was added into the aqueous acetic acid and the mixture was kept stirring for 1 h. Then, different amount of HA powder (3 g, 6 g, and 8 g) were added into the CS solution slowly and kept stirring for another 2 h to get a homogenous slurry. Second, melamine sponge cubes cut into hexahedral shape (3×3×10 mm3) were immersed in each slurry until the sponge is full of the slurry. Then, the melamine sponge cubes full of slurry were kept in a vacuum oven for 12 h at 60 ºC with the vacuum degree of 80 kPa to get porous nano-HA scaffolds. The porous nano-HA scaffolds were then transferred into the tube furnace and sintered at 1350 ºC for 2 h under air atmosphere to get porous hydroxyapatite ceramics. The porous 28


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hydroxyapatite ceramics were named as HA-3, HA-6, and HA-8 according to the adding amount of HA during the preparation process. Third, the alcohol solution of graphene oxide synthesized by two-step oxidation of NG flakes with concentrations of 0.1 wt%, 0.3 wt%, 0.6 wt%, and 0.9 wt% were prepared and ultrasonically treated for 2 hours. HA-3, HA-6, and HA-8 ceramics were soaked in the alcohol solution of graphene oxide for 10min and then were dried in the oven at 60 ºC. At last, the composite scaffolds were sintered at 1000 ºC for 5 min under nitrogen atmosphere to get porous HA ceramic/graphene composite scaffolds. The porous HA ceramic/graphene composite scaffolds were named as HA/rGO-X/Y (X was the amount of HA and the Y is the concentration of graphene oxide (GO) in the alcohol solution), such as HA/rGO-6/0.1, HA/rGO-6/0.3, HA/rGO-6/0.6 and HA/rGO-6/0.9 and so on. For comparison, sample HA-6 without the addition of CS was also prepared and was named as HA-6-CS. The sample HA-6 dried for 12 h at 60 ºC under atmospheric pressure was named as HA-6-D. In order to prove that GO was reduced into graphene, pure GO was used to experience the same reduction process of the third step. The reduced GO was named as rGO.

Scaffold Characterizations. Thermogravimetric analyzer (TGA, Q600, TA instrument, USA) was used to figure out the content of graphene in the composite scaffolds at a heating rate of 10 ºC /min from 50 to 1100 ºC in an air stream with a 29


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flow rate of 20 mL /min. XRD was carried out at room temperature with a DX-1000 X-ray diffractometer (Dandong Fanyuan Instrument Co. LTD, China). The samples were scanned over the range of diffraction angle 2θ = 5-80º at a scan speed of 2 º/min using CuKα radiation (λ = 0.154056 nm). Fourier transform infrared (FTIR) spectra were recorded with a Nicolet 6700 FTIR spectrometer (Thermo Nicolet, Madison, WI, USA) with a resolution of 4 cm-1 in transmission mode. Raman spectra were obtained using a LabRAM HR Raman Spectrometer (HORIBA Jobin-Yvon, France) with a laser at the excitation wavelength of 532 nm. X-ray photoelectron spectroscopy (XPS) characterization of GO and rGO was carried out using an XSAM800 instrument (Kratos Company, UK) with AlKa radiation (hv = 1486.6 eV). The morphology of the samples was examined with a JOEL JSM-5900LV field-emission scanning electronic microscope (FESEM, Japan) at an accelerating voltage of 5 kV. The porosity was measured by liquid displacement technique with ethanol. Specific surface area (SSA) of the composite scaffold were obtained by analyzing N2 adsorption/desorption

through

a

Brunner-Emmet-Teller

(BET)

measurement

(Autosorb iQ/ASiQ, Quantachrome, USA). The outgas time is 4 h, and the outgassing temperature is 80 ºC.

In Vitro Studies. The rat bone mesenchymal stem cells (BMSCs, provided by Stem Cell Bank, Chinese Academy of Science, Shanghai, China) were cultured in the -minimum essential medium (-MEM, Gibto-BRL, USA) with 10 % fetal bovine serum (FBS, Hyclone, USA), 1% antimicrobial of penicillin. The cells were seeded on 30


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the HA-6, HA/rGO-6/0.3 samples (4×4×1 mm3) or a 12-well plate at a density of 1.6×105 cells per well (four replicates). After 7 days cultivation, the cell morphology on the samples was examined by SEM as above mentioned. In addition, 200 μL FITC-labeled phalloidin (200 μM) was added in the medium for 30 min and then 200 μL DAPI (100 nM) was added to counter-stain for 30 min. The cytoskeleton and cellular morphology on the material was observed under a laser confocal microscope (A1RMP+, Nikon, Japan). After cultivation for 1, 3, 5 and 7 days, the cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was employed to quantitatively determine the cell proliferation and viability on the samples. The cell proliferation profile was determined as the percentage ratio of the optical density in the cells to that in the non-treated control at day 1. After cultivation for 21 days, the Alizarin red S (ARS) stain was used to evaluate the extracellular matrix (ECM) calcium accumulation. The culture plates were photographed under an optical microscope with a digital camera (Zeiss, German). The integrated optical density (IOD) was measured using Image Pro Plus (IPP) image analysis software (Version 6.0, Media Cybernetics). Then the average optical density value = IOD/Area was calculated. After cultivation for 7, 14 and 21 days, the expressions of six typical osteogenesis-related genes including type I collagen (COL-I), runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP-2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) were quantified by real-time PCR (LightCycler 480 Real-Time PCR System, Roche, USA) to investigate cell differentiation. The primers for different genes are listed in Table S1. 31


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In Vivo Studies. All procedures in the experiment were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan University. Forty-eight 6-8 weeks old male Sprague Dawley (SD) rats (306.7 ± 27.4 g) were randomly assigned to 3 groups corresponding to HA/rGO-6/0.3, HA-6, or blank control (n = 16 per group). The 6 mm critical-size segmental diaphyseal bone defect model was used and the surgical procedures were conducted under sterile conditions as previously described.70 The specific surgical operations were shown in the supplement information. At 2, 4, and 6 weeks post-operation, different fluorochromes were administered intraperitoneally (n = 4) at a sequence of 30 mg/kg alizarin red S (Sigma-Aldrich, USA), 25 mg/kg tetracycline hydrochloride (Sigma-Aldrich, USA), and 20 mg/kg calcein (Sigma-Aldrich, USA) to characterize the new bone formation and mineralization, as previously described.71 The rats were sacrificed and the femurs were harvested at 8 weeks and 6 months post-operation. The X-rays images were obtained by X-ray apparatus (Phillips, Netherland) with the parameters as 26 kV, 18 mAS, and micro-CT scan (Quantum GX, PerkinElmer, USA) was performed as pre-described.70 The bone volume fraction (bone volume/total volume, BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of the newly formed bone were analyzed as previously described (n = 4).71,72 All the micro-CT scanning and analysis were collected by an expert (see the acknowledgment) who was blinded to the study design. The tensile curves were

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obtained using a universal material testing machine (Instron5967, USA) with a stretching rate of 10 mm/min. After micro-CT scan, the specimens were embedded in polymethylmethacrylate (PMMA) and cut along the femur longitudinal for fluorescence labeling and histological observation. The fluorescence images were captured by a fluorescence microscope (AX10 imager A2, Zeiss, German). Then, the same sections were stained with 1 % methylene blue (Sigma-Aldrich, USA) and 0.3 % fuchsine (Sigma-Aldrich, USA). Histological observation was performed using an optical microscope with a digital camera (Discovery V8, Zeiss, German) and histomorphometry of the bone formation in the stained sections was performed as predescribed.73 When the rats were sacrificed, the heart, liver, spleen, kidney, lung, and pancreas were harvested and then stained by HE to observe histological changes. Three randomized non-overlapping areas were analyzed per specimen using the IPP software. All data were collected by a single observer who was blind to the grouping of the samples. The data were averaged for each individual specimen and then for each group. At last, the specimens were examined by SEM as above mentioned.

Statistical Analysis. All the data were expressed as means ± standard deviation (SD). One-way ANOVA followed by SNK post hocor paired t-tests were performed to determine significant difference between groups. Statistical analysis was performed using SPSS 13.0 software. p < 0.05 was considered to be statistically significant.

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AUTHOR INFORMATION Corresponding Authors [email protected] (Z Zhou) and [email protected] (W Yang). Tel: 0086-028-85422570; Fax: 0086-028-85423438 ORCID Zongke Zhou: 0000-0002-9037-4756 Wei Yang: 0000-0003-0198-1632 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Kai Zhou and Peng Yu contributed equally to this work.

ACKNOWLEDGMENTS The authors would like to thank Dr. Li Chen from Analytical & Testing Center Sichuan University for her help with micro-CT scanning and analysis. The authors would like to thank for the funding support from 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University, and National Natural Science Foundation of China (81672135, 81873987, 81871780, 51873126 and 51422305).

ASSOCIATED CONTENT SUPPORTING INFORMATION AVAILABLE: SEM images of samples; Surgical procedures; HE staining results of viscera; Primer pairs used in real-time PCR 34


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analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES (1) Hayrettin, K.; Egemen, A.; Unlu, M. C.; Ali, S.; Saffet, K. Predictors of Mortality in Elderly Patients with an Intertrochanteric or a Femoral Neck Fracture. J. Trauma Acute Care 2010, 68, 153-158. (2) Borrelli, J.; Prickett, W. D.; Ricci, W. M. Treatment of Nonunions and Osseous Defects with Bone Graft and Calcium Sulfate. Clin. Orthop. Relat. Res. 2003, 411, 245-254. (3) Jing, W.; Smith, A. A.; Liu, B.; Li, J.; Hunter, D. J.; Dhamdhere, G.; Salmon, B.; Jiang, J.; Cheng, D.; Johnson, C. A.; Chen, S.; Lee, K.; Singh, G.; Helms, J. A. Reengineering Autologous Bone Grafts with the Stem Cell Activator WNT3A. Biomaterials 2015, 47, 29-40. (4) Long, W. G. D.; Einhorn, T. A.; Kenneth, K.; Michael, M. K.; Wade, S.; Roy, S.; Tracy, W. Bone Grafts and Bone Graft Substitutes in Orthopaedic Trauma Surgery. A Critical Analysis. J. Bone Joint Surg. Am. 2007, 89, 649-658. (5)

Radcliff, K.; Hwang, R.; Hilibrand, A.; Smith, H. E.; Gruskay, J.; Lurie, J. D.; Zhao, W.; Albert,

T.; Weinstein, J. The Effect of Iliac Crest Autograft on the Outcome of Fusion in the Setting of Degenerative Spondylolisthesis: a Subgroup Analysis of the Spine Patient Outcomes Research Trial (SPORT). J. Bone Joint Surg. Am. 2012, 94, 1685-1692. (6) Shao, N.; Guo, J.; Guan, Y.; Zhang, H.; Li, X.; Chen, X.; Zhou, D.; Huang, Y. Development of Organic/Inorganic Compatible and Sustainably Bioactive Composites for Effective Bone Regeneration. Biomacromolecules 2018, 19, 3637-3648. (7) Das, A.; Pamu, D. A Comprehensive Review on Electrical Properties of Hydroxyapatite Based Ceramic Composites. Mater. Sci. Eng. C Mater. Biol. Appl. 2019, 101, 539-563. (8) Degli Esposti, M.; Chiellini, F.; Bondioli, F.; Morselli, D.; Fabbri, P. Highly Porous PHB-Based Bioactive Scaffolds for Bone tissue Engineering by In Situ Synthesis of Hydroxyapatite. Mater. Sci. Eng. C Mater. Biol. Appl. 2019, 100, 286-296. (9) Oliveira, H. L.; Da Rosa, W. L. O.; Cuevas-Suarez, C. E.; Carreno, N. L. V.; da Silva, A. F.; Guim, T. N.; Dellagostin, O. A.; Piva, E. Histological Evaluation of Bone Repair with Hydroxyapatite: A Systematic Review. Calcif. Tissue Int. 2017, 101, 341-354. (10) Hao, Y.; Yan, H.; Wang, X.; Zhu, B.; Ning, C.; Ge, S. Evaluation of Osteoinduction and Proliferation on Nano-Sr-HAP: A Novel Orthopedic Biomaterial for Bone Tissue Regeneration. J. Nanosci. Nanotechnol. 2012, 12, 207-212. (11) Lixia, M.; Jiaqiang, L.; Jinglei, Z.; Jiang, C.; Lunguo, X.; Lingyong, J.; Xiuhui, W.; Kaili, L.; Bing, F. Effect of Micro-Nano-Hybrid Structured Hydroxyapatite Bioceramics on Osteogenic and Cementogenic Differentiation of Human Periodontal Ligament Stem Cell via Wnt Signaling Pathway. Int. J. Nanomed. 2015, 10, 7031-7044. (12) Lin, Z. Y.; Duan, Z. X.; Guo, X. D.; Li, J. F.; Lu, H. W.; Zheng, Q. X.; Quan, D. P.; Yang, S. H. Bone Induction by Biomimetic PLGA-(PEG-ASP)n Copolymer Loaded with a Novel Synthetic BMP-2-Related Peptide In Vitro and In Vivo. J. Control. Release 2010, 144, 190-195. (13) Raimondi, M. T.; Eaton, S. M.; Lagana, M.; Aprile, V.; Nava, M. M.; Cerullo, G.; Osellame, R. Three-Dimensional Structural Niches Engineered via Two-Photon Laser Polymerization Promote Stem Cell Homing. Acta Biomater. 2013, 9, 4579-4584. (14) Li, J.; Zheng, Q.; Guo, X.; Zou, Z.; Liu, Y.; Lan, S.; Chen, L.; Deng, Y. Bone Induction by Surface-Double-Modified True Bone Ceramics In Vitro and In Vivo. Biomed. Mater. 2013, 8, 035005. (15) Ramay, H. R. R.; Zhang, M. Biphasic Calcium Phosphate Nanocomposite Porous Scaffolds for 36


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of

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through

a

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Two-Stage

Method:

A

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