Porous Particle-Reinforced Bioactive Gelatin Scaffold for Large

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Porous Particle Reinforced Bioactive Gelatin Scaffold for Large Segmental Bone Defect Repairing Yang Cui, Tengjiao Zhu, Ailing Li, Bingchuan Liu, Zhiyong Cui, Yan Qiao, Yun Tian, and Dong Qiu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19010 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Applied Materials & Interfaces

Porous Particle Reinforced Bioactive Gelatin Scaffold for Large Segmental Bone Defect Repairing Yang Cuia,b, #, Tengjiao Zhuc, #, Ailing Lia, Bingchuan Liud, Zhiyong Cuid, Yan Qiaoa, Yun Tiand,*, Dong Qiua,b* a

Beijing National Laboratory for Molecular Sciences, State Key Laboratory of

Polymer Physics and Chemistry, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b

University of Chinese Academy of Sciences, Beijing 100190, China

c

Orthopedic Department, Peking University International Hospital, Beijing 102206,

China d

Orthopedic Department, Peking University Third Hospital, Beijing 100191, China

#

These authors contributed equally to the work.

*Correspondent authors.

ABSTRACT: Large segmental bone defect repairing remains a big challenge in clinics and synthetic bone grafts suitable for this purpose are still highly demanding. In this article, hydrophilic composite scaffolds (BHP-gel scaffold) composed of bioactive hollow nanoparticle and crosslinked gelatin have been developed. The bioactive nanoparticles have a porous structure as well as high specific surface area, thus interact strongly with gelatin, to overcome the swelling problem that hydrophilic polymer scaffold will usually face. With this combination, these BHP-gel scaffolds Page 1 of 32

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showed similar porous structure and mechanical properties to the cancellous bone. They also showed excellent bioactivity and cell growth promotion performance in vitro. The best of them, 10BHP-gel scaffold was evaluated in vivo on a rat femur model, where it was found the 5-mm segmental bone defect almost healed with new bone tissue formed in 12 weeks and the scaffold itself degraded at the same time. Thus, 10BHP-gel scaffold may become a potential bone graft for large segmental bone defect healing in the future. KEYWORDS: composite scaffold, bioactivity, gelatin, segmental bone defect, bone regeneration

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INTRODUCTION Large bone defects caused by trauma, congenital malformation or tumor resection have long been a highly challenging clinical problem.1-2 They are usually characterized by low regeneration potential thus will need more specialized surgical management. Bone loss and blood insufficiency are two major negative factors in bone regeneration process.3-5 Bone loss can be tackled by implanting bone grafts, including large segmental allografts,6 autografts (fibula, rib, ilium),7-8 or synthetic bone grafts.9-12 However, large segmental allografts are less osteoinductive than autografts and risky in disease transmission and immunogenic response; autografts are severely limited by availability, donor site morbidity and movement impairments; besides, they both fail to meet the requirements for geometry and size in clinical applications.13-14 Synthetic bone grafts, including hydroxyapatite,15-16 calcium phosphate,17-19 calcium sulfate20 and bioactive glasses21-22 etc., do not have some of the drawbacks mentioned above, therefore are becoming more and more attractive. Nevertheless, they have deficiencies in biological responses, for example, osteoinductivity. Silicate

bioactive

glasses

(BGs)

have

shown

good

biocompatibility,

osteoconductivity and osteoinductivity.23 They can form active bonding with both hard and soft tissues. Sun et al.24 found that the ions released from Bioglass® 45S5, especially Si ions, could promote human osteoblast proliferation. Furthermore, BGs were found to increase the expression of genes responsible for osteogenesis, including nuclear transcription factors and insulin-like growth factors, by 200-500% compared Page 3 of 32



with the control group.25-26 In vivo, Bi et al.21 found that the BG scaffolds had an excellent performance on promoting new bone formation, even not inferior to autografts. However, BGs are rarely used alone in the form of scaffold because of their poor mechanical properties, especially brittleness.27-29 Therefore, polymer composite scaffolds with BG particles as the functional fillers were developed,30-33 which were expected to have the right combination of mechanical properties and biomedical functions. Biodegradable polyesters, e.g. poly (lactic acid) (PLA), are one of the mostly used polymer matrices due to their good mechanical properties in biological environment and adjustable degradation rates. However, polyesters are hydrophobic, which may bring about a few drawbacks: i) the poor compatibility between polyesters and BG fillers may cause heavy aggregation of filler particles, thus deteriorate the overall mechanical properties. For example, in BG/PLA composite scaffolds, although addition of BG particles would improve the compressive strength at first, higher content of BG (e.g. 30 wt%) caused a decrease in compressive strength, because of inevitable aggregation of BG particles.34-38 ii) the encapsulation of BG particles by hydrophobic polymers prevents them from contacting physiological fluid, thus no bioactivity could be shown until polymers had partially degraded, which is not beneficial for early stage healing.39-40 iii) the hydrophobic nature of pore walls in the scaffold is not ideal for aqueous fluid penetration, therefore, nutrients and metabolizing wastes could not be transported in time through the scaffold, which becomes particularly problematic when large bone grafts are involved. Page 4 of 32


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On the contrary, hydrophilic polymer matrix does not have those problems. However, hydrophilic polymer has high affinity to water, thus the interfacial interactions between filler and polymer matrix need to be significantly enhanced to avoid the scaffold severely swollen by physiological fluid or at least to maintain the graft geometry during bone regeneration. Because hydrophilic polymer is usually compatible with inorganic filler particles, increasing the specific surface area of filler particle is one of the most effective approaches to enhance the interfacial interactions. Decreasing filler particle size can increase its specific surface area, therefore increase the interfacial interactions. For example, a previous study showed that the mechanical properties of bioactive particle/gelatin composite scaffolds increased when bioactive particle size was reduced41 and when nano-sized particles were used, both the compression strength and modulus improved significantly, ranging from 2.1-15.4 MPa and 82.4-602.3 MPa, respectively, much higher than those of BG/PLA composite scaffolds mentioned above. However, despite of significant advances, the geometry of those hydrophilic composite scaffolds still could not be well maintained in vivo due to their highly swollen nature. Therefore, further increase in the interfacial interactions is still needed. Bioactive filler particles with porous structures may help to address the above challenge. With the increased interfacial interactions between polymer and bioactive particles, severely swollen of composite scaffold might be avoided. In this study, we developed a bioactive hollow particle (BHP), which is bioactive and porous. It was used to prepare the bioactive composite scaffold with gelatin (BHP-gel). On one hand, Page 5 of 32



the BHP-gel scaffold was only slightly swollen by physiological fluid and could maintain its geometry very well for bone regeneration. On the other hand, the BHP-gel scaffold exhibited excellent bioactivity in vitro to promote osteoblast proliferation and in vivo to accelerate bone regeneration (as showed on a critical-sized 5 mm segmental defect model of the rat femur). Therefore, we believe that this BHP-gel composite scaffold can be used as a potential bone graft especially suitable for large segmental bone defect repairing.

EXPERIMENTAL PROCEDURES Materials. 3-(methacryloxy) propyltrimethoxysilane (TPM), potassium persulfate (KPS) were purchased from Alfa Aesar (China) Chemicals Co., Ltd. Isoamyl acetate (PEA), calcium hydroxide, glutaraldehyde solution 25% were purchased from Sinopharm Chemical Reagent Co., Ltd. Gelatin (Type A) from porcine skin was purchased from Sigma-Aldrich. Ethanol and ammonia were purchased from Beijing Chemical Works. All chemicals were used without further purification. Synthesis of Bioactive Nanoparticles. The hollow nanoparticles (HPs) were synthesized through the hydrolysis of TPM with PEA as the soft template.42 The preparation procedure is briefed as the following: 0.2 ml TPM and 0.6 ml PEA were added to 40 ml H2O. The overall pH was adjusted to 10 by adding ammonia. After ultrasonicated for 20 min, the reaction mixture was heated to 70℃ under N2 and 10 mg of KPS was added to initiate the polymerization of TPM. The reaction was stopped after 12 h. Then the formed HPs were collected by centrifugation and washed Page 6 of 32


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with ethanol for three times. Solid silica nanoparticles (SP) were prepared by Stöber method.43 Bioactive hollow particles (BHPs) were prepared by postmodification of HPs with Ca(OH)2, as previously reported.43 Briefly, 300 ml Ca(OH)2 solution (0.2 mg·ml-1) was added to 26.7 ml suspension of HP (solid content ~15 wt%) and the mixture was stirred for 24 h. Afterward, BHPs were collected by centrifugation and washed twice with pure water to remove excessive Ca(OH)2. Bioactive solid silica nanoparticles (BSPs) were also prepared by postmodification of solid silica particles (SPs) using the same method. 43 Fabrication of Composite Scaffolds. Gelatin was dissolved in water at 60℃. Gelatin solution (20 wt%) was mixed with BHP suspension to obtain three different mixtures at BHP/gelatin weight ratios of 0.1, 0.2 and 0.5, which were labeled as 10BHP-gel, 20BHP-gel, and 50BHP-gel, respectively. They were then poured into 96-well plate, aged for 24 h, and soaked in 1% glutaraldehyde aqueous solution for 24 h to form cross-linked hydrogels. Furthermore, these hydrogels were immersed in pure water for 24 h to wash off the unreacted glutaraldehyde. Finally, bioactive composite scaffolds with BHP (BHP-gel) were obtained by freeze-drying these hydrogels at -20°C for 2 d. Composite scaffolds with BSP (BSP-gel) were also prepared in the same way, named as 10BSP-gel, 20BSP-gel, and 50BSP-gel, respectively. Gelatin solution (20 wt%) without particles was cross-linked by 1% glutaraldehyde and freeze-dried to obtain scaffold, labeled as Gel for comparison. Morphology of Nanoparticles and Composite Scaffolds. The morphologies of Page 7 of 32



nanoparticles were investigated by field emission transmission electron microscope (JEM-2200FS, Japan, 200 kV). Zeta potential and particle size were measured by Zetasizer Nano ZS. The specific surface area of the nanoparticles was measured by surface area and porosity analyzer (ASAP 2020) through the nitrogen adsorption method. The morphology of swollen scaffolds was observed by field emission environment scanning electron microscope (FEI Quanta FEG 250). The porosities of scaffolds were obtained by comparing apparent density with physical density, which was measured by Archimedean method. Six samples were used for each scaffold to ensure the repeatability in porosity measurement. In vitro Bioactivity of Nanoparticles and Composite Scaffolds. The in vitro bioactivity of material was evaluated by checking the ability of forming hydroxyapatite (HA) in simulated body fluid (SBF) at 37℃ (SBF was prepared according to the reference.47) In brief, 75 mg of material were immersed in 50 ml SBF and placed in a thermostatic water bath at 37°C for 1, 3 and 7 d. Then, the samples were centrifuged and washed with pure water for three times. The products were characterized by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). XRD data was recorded on D/max 2500 (Rigaku) using filtered Cu Kα radiation (λ=1.54 Å) at 40 mA and 40 kV with scanning speed at 4°/min, 2θ from 10° to 60°. FTIR spectra were collected in the range 2000-400 cm-1 on an EQUINOX55 spectroscope (Bruker, Germany). In vitro Cytotoxicity of Nanoparticles and Composite Scaffolds. To evaluate in vitro cytotoxicity, the CCK-8 assay was used and the O.D. values of the samples were Page 8 of 32


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measured using multi-mode microplate analyzer provided by Perkin-Elmer Instruments. The preosteoblast MC3T3-E1 cells (ATCC, CRL-2593, Rockville, MD, USA) were cultured in the α-MEM medium including 10% FBS and 1% penicillin/streptomycin. For nanoparticles (SP, BSP, HP, and BHP), they were sterilized by UV radiation and dispersed in α-MEM medium for 72 h in a cell incubator (37℃, 5% CO2) to prepare the medium extracts at concentrations of 10 mg·ml-1 and 20 mg·ml-1. Then 100 µl MC3T3-E1 cells suspensions were seeded in a 96-well plate at a density of 5×103 cells/well. The cells cultured without intervene were set as the control group. For composite scaffolds, the MC3T3-E1 cells were directly cultured on them (~0.2 g each), which were 6 mm in diameter and 1 mm in thickness. The medium was changed every 2 d. Four samples for each group were tested to obtain the mean value. Setting the cell viability of control group as 100%, the

cell

viabilities

of

other

wells

were

calculated

as

following:

cell

viability=absorbance per well/absorbance of the control×100%. Mechanical Properties of Composite Scaffolds. Mechanical properties of scaffolds were evaluated by uniaxial compression test on INSTRON 3365 with a 5 kN load under a cross-head speed of 0.5 mm·min-1 until the specimen were broken. The specimen were cylinders with a height of 10 mm and a diameter of 6 mm. Each scaffold was repeated five times and the results were averaged. Swelling Behavior of Composite Scaffolds. Scaffolds were immersed in SBF at 37℃ (10 mg·ml-1) for swelling behavior study. The swelling profile was obtained by measuring the swelling ratio at different time intervals. Their original lengths and Page 9 of 32



diameters were used to calculate the initial volume V0 and similarly for the swollen volume at time t, Vt. The swelling ratio at time t is (Vt - V0) / V0×100%. Surgical Procedure of 10BHP-gel Scaffold In vivo. In vivo performance was evaluated in a rat femoral segmental defect model. All the experimental processes followed the instructions of the Experimental Research Center of the Faculty of Medicine and acquired the permissions of the Ethics Committee of Peking University (Permit number: LA2017070). In total, 18 adolescent male Sprague Dawley rats weighing 0.30-0.35 kg were used and all of them were anesthetized by ketamine hydrochloride (50 mg·kg-1, IM) and fentanyl (0.17 mg·kg-1, IM). A 5-mm segmental defect in the femur was produced using a steel wire44-46 and then fixed with a steel plate after irrigation with 0.9% sterile saline solution. The experimental group (10BHP-gel, n=9) was implanted with 10BHP-gel scaffolds, while the control group (n=9) was left blank. The wounds were sutured well and prophylactic antibiotic together with the analgesics was given for three days. The procedures were operated by the same surgeons for both groups. Radiographic Examination. Rats were immobilized in the prone position under anesthetic condition and subjected to X-ray radiography (Hologic Dual Energy X-ray Absorptiometry, America) of the bone defect 0, 3, 6, 9 and 12 weeks after surgery. Micro-CT Evaluation. The femurs were obtained from 3 rats in each group at the 9th week and from the rest 6 rats at the 12th week, respectively. And the harvested femurs were examined by the micro-CT (Inveon Scanners, SIEMENS, Germany) after removing the superficial soft tissue and then evaluated by its associated Page 10 of 32


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analyzing software. Histologic Analysis. The harvested femurs were washed with saline thoroughly, then fixed in 4% paraformaldehyde (10% neutral buffered formalin) for about 72 h and subsequently decalcified for total 6 weeks in 10% EDTA, pH 7.0 at 4℃. After complete decalcification and dehydration, the samples were embedded in paraffin wax and 5 mm serial slices were prepared by using a microtome. Surface staining was performed with hematoxylin and eosin (H&E) for microscopic observation. The slides were photographed using a digital camera (NanoZoomer-SQ, Hamamatsu Photonics K.K, Hamamatsu, Japan). Statistical Analysis. The one-way analysis of variance (ANOVA) was conducted to evaluate the differences between the means of collected data. Each experiment was repeated three times. All quantitative data are presented as mean ± one standard deviation (SD).

RESULTS AND DISCUSSION The Physicochemical Properties of Nanoparticles. BHPs were prepared by hydrolysis of TPM. Figure 1a illustrated the mechanism of BHPs immersion in SBF to fast form HA. When BHPs are soaked in SBF, the calcium ions released from their surface could induce phosphate ions from SBF to precipitate on silanol sites to form HA (the so-called bioactivity). It can be seen that BHP had an obvious cavity in the middle (Figure 1b). The specific surface area of BHP is much higher than solid particles with similar size and composition should be (Figure S2 and Table S2). This Page 11 of 32



indicates BHP is also porous, which is expected and desired for our terminal application. The morphologies of SP, BSP, HP were shown in Figure S1, it can be seen that BHP had almost the same morphologies as HP (Figure S1j), confirming that the bioactive modification did happen mainly on the surface. The same situation occurred between SP (Figure S1b) and BSP (Figure S1f). The zeta potential of nanoparticles was summarized in Table S1. Interestingly, this surface modification would not cause too much reduction of surface charge. For example, the zeta potentials of HP and BHP were found to be -25.8 ± 0.2 mV and -20.6 ± 0.7 mV (Table S1), respectively, with only slightly reduction by surface modification, thus good colloidal stabilities were still maintained.

Figure 1. (a) Illustration the process of BHP immersion in SBF to form HA, (b) TEM image of BHP, (c) XRD traces and (d) FTIR spectra of BHP for different soaking time in SBF, (e) Cytotoxcity of nanoparticles on MC3T3 cells culturing for 2 d.

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To evaluate their ability of forming HA, BHPs were soaked in SBF for 0, 1, 3 and 7 d. The characteristic diffraction peaks corresponding to HA (26°, 32°, 47°, 49°, 53°) were observed after BHPs soaked in SBF for merely 1 d (Figures 1c), demonstrating their good bioactivities in vitro. And the intensity of HA diffraction peaks became stronger with increasing soaking time, indicating further growth of HA. The HA formation was further confirmed by FTIR (Figures 1d); the double peaks near 600 cm-1 are the characteristic absorption peaks of crystalline phosphate (PO43-). BHPs started to show the peaks of PO43- after only 1d in SBF, consistent with XRD results. Bioactive solid particles (BSP) were used for comparison (Figure S1g, h). BHP had significant earlier appearance of HA characteristic peaks and higher peak intensities than BSP did, suggesting BHP was much more bioactive than BSP. This should be due to the higher specific surface area of BHPs (including the pore wall and inner surface of the cavity, see Table S2 and Figure S2), which increased incorporation of Ca2+ as well as the bioactivity. Therefore, BHP appeared to be a better bioactive filler in bone grafts. For bone graft materials, low cytotoxicity is essential. Extracts of four kinds of nanoparticle (SP, BSP, HP, BHP) were cultured with preosteoblast MC3T3-E1 cells for 2 d. The results were showed in Figure 1e. Because one scaffold weights about 200 mg, of which ~18 mg was bioactive particles, the cytotoxicity of nanoparticles was assessed at both low (10 mg·ml-1) and high (20 mg·ml-1) concentrations, to pin later studies on scaffolds. The cell viabilities of SPs and HPs were about 80% and 95% at 10 mg·ml-1, respectively, and no significant difference in cell viabilities was found Page 13 of 32



at a higher concentration (20 mg·ml-1). However, BSPs and BHPs had cell viabilities nearly 100% compared at 10 mg·ml-1, indicating they had no cytotoxicity at all. Interestingly, as their concentrations increased to 20 mg·ml-1, their cell viabilities were as high as 132% and 193%, respectively, suggesting they could indeed promote cell proliferation, probably due to the dissolved calcium ions. Obviously, BHP had higher cell viability than BSP, in consistence with the in vitro bioactivity.

The Physicochemical Properties of Composite Scaffolds In vitro. The Illustration of interactions between gelatin and BHPs in the BHP-gel scaffold was shown in Figure 2a. Because BHP had large specific surface area and pores, polymer chains could cross into the inner of particles and obtain more interaction sites, thus having stronger interactions. Figure 2b was the optical picture of obtained scaffolds with different ratios of BHP and gelatin. All the BHP-gel scaffolds had pores with sizes ranging from 50 to 200 µm, suitable for osteocytes ingrowth.37 The microstructure of 10BHP-gel was shown in Figure 2c. There were small pores about several microns in scaffolds, beneficial to cell adhesion. Moreover, nanoparticles were dispersed well in the gelatin matrix, as viewed in image with higher magnification (insets of Figure 2c). Interestingly, BHPs were found to be exposed on the wall of scaffolds, which enabled them to directly contact with SBF, thus ready to show their high bioactivity. The porosities of BHP-gel scaffolds ranged from 63% to 86% and reduced with nanoparticle content. Therefore, 10BHP-gel scaffolds had the highest porosity in the explored formulations and were more like the cancellous bones on morphology. Page 14 of 32


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Figure 2. (a) Illustration of interactions between gelatin and BHPs, (b) Optical picture of BHP-gel scaffold, (c) ESEM image of 10BHP-gel scaffold (bar=500 µm, bar of insert=20 µm), (d) XRD spectra of BHP-gel scaffolds with different particle percentages in the SBF for 7 d, (e) Compression stress-strain curves, (f) Swelling curves of Gel and BHP-gel scaffolds in SBF, (g) Optical images of 10BHP-gel scaffolds before and after swelling in SBF for 12 h, (h) Cell proliferation on 10BHP-gel scaffold. (Number by the curves represents the weight percentage of particle to gelatin.)

As for bioactive of BHP-gel scaffolds in vitro, the XRD spectra (Figure 2d) show that all the BHP-gel composite scaffolds had the characteristic diffraction peaks of HA (26°, 29°, 32° 47°) after soaked in SBF for 7 d. The intensities of HA diffraction peaks became stronger with increasing BHP content. This confirmed that the in vitro bioactivity was indeed brought about by the bioactive particles. Moreover, BHP-gel scaffolds had relatively stronger HA diffraction peaks than BSP-gel scaffolds at the Page 15 of 32



same particle content (Figure S3d), in agreement with the bioactivity ranking between BHPs and BSPs. Also, for ideal bone grafts which need to share load with surrounding bone tissues, scaffolds should have similar mechanical properties to cancellous bones. Adding BHP to gelatin matrix led to an obvious increase in the mechanical properties of scaffolds (Figure 2e). BHP was found to have a better reinforcement than BSP (Figure S3e), due to its higher specific surface area as well as stronger polymer-particle interactions. Both BHP-gel and BSP-gel composite scaffolds could match the mechanical properties of natural cancellous bones in terms of compression performance (Table S3). Besides, the mechanical properties and porous structure of composite scaffolds can also be adjusted by varying filler particle content, in order to meet various needs of different bone injury sites in practical applications. In the samples investigated above, 10BHP-gel scaffold can provide the best mechanical support (compressive strength and modulus were 147.5±17.0 MPa and 10.8±0.7 MPa) as well as porous structure for cell ingrowth. The geometry maintaining of scaffolds is an important property in the process of large bone defect healing. Composite scaffolds and Gel scaffolds were immersed in the SBF at 37℃ for 12h. The Gel scaffold had a swelling ratio of almost 100% at 1h, which was much higher than those of BSP-gel and BHP-gel scaffolds (Figure S3f and Figure 2f). The swelling ratio was reduced significantly by addition of BSP and BHP. BHP-gel scaffolds had lower swelling ratio than BSP-gel scaffolds, which is also an evidence of stronger interactions between BHP and gelatin than BSP. BHP-gel scaffolds also quickly form an HA deposition layer on their surface, further Page 16 of 32


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restraining swelling of BHP-gel scaffolds. Especially for 10BHP-gel, the swelling was much slower and the equilibrium swelling ratio was only less than 40%. It can be seen clearly in Figure 2g that 10BHP-gel scaffold nearly maintained its geometry after soaking in SBF for 12 h, which means that BHP can effectively restrict swollen of scaffold. Thus, 10BHP-gel can be used as a good candidate for large segmental bone defect reparing. Because 10BHP-gel had better mechanical and swelling properties as well as higher bioactivity, it was the most ideal sample, which was further evaluated in vitro for cell response. We used 10BHP-gel scaffold to culture MC3T3 cells and the cells cultured without scaffold were used as control. As seen in Figure 2h, the cell viability of 10BHP-gel scaffold was 85% for incubation 2 d, indicating their good cell compatibility. With incubation time increased, 10BHP-gel scaffold had higher cell viabilities than controls, due to the increased deposition of HA in scaffold and released ions, which can promote osteoblast proliferation. Therefore, 10BHP-gel scaffold also exhibited a good performance on cell proliferation in vitro and was ready for in vivo evaluations.

In vivo Evaluation of 10BHP-gel Scaffold. As shown above, the porosity, bioactivity, cell proliferation, mechanical and swelling property of 10BHP-gel could all meet the needs of bone repair in vitro, therefore its in vivo performance was further evaluated in a rat femoral segmental defect model (Figure S4). The defects were over the critical size and could not be self-healed. To evaluation the process of bone Page 17 of 32



regeneration in the osteotomy gap, X-ray radiographs were taken at the 3rd, 6th, 9th and 12th week (Figure 3). At the 3rd week, the new bone formation in the osteotomy gap was unremarkable in all groups (Figures 3a and e). However, callus formation was evident especially in the 10BHP-gel group at the 6th week (Figures 3b and f). And notably more callus was formed in 10BHP-gel group compared to control at the 9th week (Figures 3c and g). At the 12th week, the 10BHP-gel group seemed to have healed with bridging callus formation (Figure 3h). However, none of the animals in the control group had any bridging callus and the osteotomy gap was still obviously visible (Figure 3d).

Figure 3. Plain radiographs from different groups at different time points post-surgery. (a, b, c, d) Control group for 3rd, 6th, 9th and 12th week, respectively, (e, f, g, h)10BHP-gel group for 3rd, 6th, 9th and 12th week, respectively. (Dashed rectangles indicate bone defect sites.)

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The rats were sacrificed and their femora were removed for micro-CT analysis at the 6th and 12th week post-surgery. At the 6th week, sagittal images and 3D reconstructed images (Figures 4a, b, c and d) revealed newly trabecular formation in both groups, and there was no significant difference between two groups. However, at the 12th week, more trabecular bone formation was observed in the 10BHP-gel group (Figures 4f and h) compared with the control group (Figures 4e and g). The 10BHP-gel scaffold was also found to have partially degraded after 6 weeks, but completely degraded after 12 weeks, which matches the new bone formation rate rather well. Quantitative parameters for new bone formation were obtained and statistically analyzed from the region of interest at the osteotomy site, including bone volume (BV, mm3), bone volume density (BV/TV, %), trabecular number (Tb.N, mm-1) and trabecular spacing (Tb.Sp, mm). Their mean values with SD and p-values at the 6th and 12th week were summarized in Table 1. No significant difference between experimental and control groups was observed at the 6th week, but significantly better performances were found in experimental group compared with control group at the 12th week, in nearly two folds or more.

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Figure 4. (a, b, e, f) Sagittal images and (c, d, g, h) 3D reconstructed images by micro-CT imaging of different groups at 6th week and 12th week. (Dashed rectangles indicate bone defect sites.)

Table 1. Quantitative analysis of defect healing progress from micro-CT images. 6th week

BV

12th week

10BHP-gel

Control

p

10BHP-gel

Control

p

24.9±7.4

21.1±8.7

0.76

61.7±9.3

35.1±2.1

0.019

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BV/TV

0.11±0.03

0.09±0.03

0.55

0.29±0.04

0.14±0.01

0.003

Tb.N

0.48±0.05

0.43±0.07

0.58

1.47±0.32

0.56±0.08

0.021

Tb.Sp

1.9±0.3

2.2±0.4

0.55

0.6±0.2

1.8±0.3

0.011

To further assess the formation of new bone, H&E staining of bone defect site for both groups was performed at the 6th and 12th week post-surgery. At the 6th week, there exhibited new ossified areas at the host-defect interface in both groups, and the osteotomy gap in experimental group showed enhanced endochondral ossification (Figure S5). The blank area in the osteotomy gap turned out to be the residual scaffold by examining the gross specimen (Figure S6). At the 12th week, the defect had already been filled by abundant woven bone in the experimental group (Figure 5) and no residual scaffold was spotted. The gap in experimental group was significantly narrower compared to the control group, although the outer cortex healing was still incomplete. Moreover, the active endochondral ossification at the gap indicated the trend of further healing. Conversely, although the control group showed new bone formation at the interface, the fracture gap was almost filled with granulation tissues, resulting in the nonunion of femur. Therefore, 10BHP-gel scaffold proved to promote bone formation in vivo and had a degradable rate matched to new bone generation pretty well. This scaffold, with all the aspects evaluated, is very promising in large segmental bone defect repairing and worth further work on large animal models.

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Figure 5. Histology photomicrographs of H&E staining of bone defects for each group after 12 weeks. Scale bar represents 2.5 mm (left) and 500 µm (right). NB indicates new bone (enclosed area by dashed lines).

Currently, synthetic scaffolds have gradually been applied in repairing large bone defect. Biomimetic scaffold that imitate native bone microenvironment and complex structure would become a new direction of further research. For example, combination scaffold with growth factors or cells could help osteogenesis and vascularization in process of bone healing such as bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF). However, the disadvantages of these scaffolds include immune responses from exogenous cells or cytokines, loss of bioactivity or stability in body, and easy leakage. To overcome the shortcomings aforementioned, we utilize bioactive nanoparticles (BHP) to induce bone regeneration. Importantly, scaffolds for large bone graft need to provide adequate nutrients. As proposed in the introduction, hydrophilic scaffold would be advantageous for matter Page 22 of 32


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transportation, thus is beneficial for large bone defect regeneration, which otherwise would require strong blood vessel formation capabilities. Especially, for bioactive glass, direct contacting with physiological is essential for releasing bioactive ionic species as well as forming bone mineral like apatite, in this sense, hydrophilic polymer matrix is also highly preferred. Collagen is the natural composition of bone and thus has been widely adopted for artificial bone graft matrix. Gelatin as hydrolyzed product of collagen, does not have immune response and is soluble in water thus easy to process. However, gelatin matrix is highly swollen in physiological fluid. In this study, we proposed that by using the porous bioactive particles, the interactions between gelatin and particle would be greatly enhanced, thus the composite scaffold would have a better resistance to swelling. The results confirmed this hypothesis and one of the compositions, 10BHP-gel, only had an equilibrium swollen ratio of less than 40% in volume, or ~10% in size. This enables it to support bone regeneration in vivo. Furthermore, we proposed that hydrophilic scaffold would not delay the showing of bioactivity. The results also confirmed this hypothesis and it was shown that these gelatin based composite scaffolds formed bone mineral like apatite within 1 d in SBF, almost as quick as the bioactive particles themselves. Consequently, cells grew much better in those bioactive scaffolds compared to the control. The main achievement of this study is that we have demonstrated hydrophilic polymer could be used as bone grafts for large segmental bone defect repairing, provided suitable bioactive filler particles were chosen. Of course, evaluation on even larger animals would be immediately required in order to push this work forward. Page 23 of 32



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CONCLUSIONS In summary, we have successfully developed porous BHP-gel scaffolds by different ratios of BHP/gelatin, which showed excellent bioactivity and cell proliferation promotion. They have mechanical properties similar to cancellous bones and can better maintain geometry in physiological environment in spite of their hydrophilic nature. The best of them, 10BHP-gel was evaluated in vivo on a rat femur model, where it was found the 5-mm segmental bone defect almost healed with new bone tissue formed in 12 weeks and the scaffold itself degraded at the same time. Thus, 10BHP-gel scaffold may become a potential bone substitute material for large segmental bone defect healing in the future. This work also demonstrated that even hydrophilic polymer scaffolds could be used as large bone grafts, which can overcome the problem of severe swollen by enhanced polymer-filler interactions.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Y.T.) *E-mail: [email protected] (D.Q.)

ACKNOWLEDGEMENTS This

work

was

supported

by

the

National

Basic

Research

Program

(2017YFC1103300), National Natural Science Foundation of China (Project No. Page 24 of 32


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51773209) and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB12020300).

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Porous Particle-Reinforced Bioactive Gelatin Scaffold for Large

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