Inorganic Self-Assembled Bioactive Artificial Proto-osteocells Inducing

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Biological and Medical Applications of Materials and Interfaces

Inorganic Self-Assembled Bioactive Artificial Proto-osteocells Inducing Bone Regeneration Miusi Shi, Ruiwen Yang, Qin Li, Kangle Lv, Richard J. Miron, Jie Sun, Mei Li, and Yufeng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00385 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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ACS Applied Materials & Interfaces

Inorganic Self-Assembled Bioactive Artificial Proto-osteocells Inducing Bone Regeneration Miusi Shi,†# Ruiwen Yang,‡# Qin Li,‡ Kangle Lv,‡ Richard J Miron,§ Jie Sun,‡ Mei Li,*‡ Yufeng Zhang*†

† The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, P. R. China

‡ Key Laboratory of Catalysis and Materials Science of the State Ethnic Affairs Commission and Ministry of Education, College of Chemistry and materials sciences, College of Resources and Environmental Science, South-Central University for Nationalities, Wuhan 430074, P. R. China

KEYWORDS: artificial proto-osteocells, self-assembly, BMP2, BCP, bone regeneration

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ABSTRACT: Since the discovery of osteoinduction in the early 20th century, innovative biomaterials with osteoinductive potential have emerged as candidates for bone repair. Recently, artificial protocell models have demonstrated great potential for tissue regeneration. Herein, we developed

artificial

biphasic-phosphate

bioactive

particles

in

proto-osteocells the

form

of

by

self-assembly

aqueous-bone

of

biodegradable

morphogenetic

protein

2

(BMP2)-containing pickering emulsions in corn oil to fulfill the release of BMP2 with controlled and local efficacy. These artificial proto-osteocells have the advantage of (1) being directly injected into the target location to avert reported side effects of BMP2 minimizing surgical complications, (2) exhibiting the capability of osteoinduction as shown in both in vitro and in vivo models, and (3) demonstrating calcific deposition locally by utilizing the biodegradable calcium phosphate shell. The efficiency of BMP2 within the artificial proto-osteocells showed a 25 times greater bone-inducing potential when compared to control. This study demonstrates for the first time a new strategy towards utilizing material-based artificial proto-osteocells to tackle medical issues in bone tissue repair and regeneration.

1. INTRODUCTION The regeneration of bone defects is a major and common clinical problem associated with disease involving bone loss including tumors, infections, biochemical disorders and abnormal skeletal development as well as trauma.1 Since the discovery of osteoinduction in the early 20th century, innovative biomaterials with osteoinductive potential have emerged to overcome the shortcomings of autogenous bone which has long been considered as the gold standard for bone-grafting procedures.2 Although a variety of well characterized new therapies have so far been reported in the 2


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literature, but an ideal therapy to date has not yet been established and scientists are still seeking for new and improved osteoinductive biomaterials for bone regeneration. As mentioned in our previous review,1 an ideal osteoinductive material should be capable of recruiting

mesenchymal-type

osteoprogenitor

cells,

transforming

these

undifferentiated

mesenchymal cells into mature, bone-forming osteoblasts, and inducing in-growth ectopic bone formation when implanted into extraskeletal locations. Osteoinduction, historically was achieved in two ways: via the addition of ceramics to the microstructure of various bone biomaterials sintered with specific characteristics or via an external stimulus such as the addition of bone morphogenetic proteins (BMPs). Bone morphogenetic protein 2 (BMP2) is an FDA approved recombinant protein, which has been widely considered as the most potent osteoinductive growth factor for bone regeneration.3-5 However, the administration of BMP2 for various orthopedic applications has demonstrated certain drawbacks including their short biological half-lives, localized activity, and rapid local clearance.6 Especially, abnormal BMP signaling has been associated with various more severe secondary problems such as bone and vascular disorders,7 infection and osteolysis,8 immune responses and inflammation,9 and heterotopic bone formation.10 Therefore, the clinical application of BMP-based therapies maintain significant issues from the current results and outcomes reported in the literature.11 Since first demonstrated in 1957,12 artificial protocell models indeed share many excellent characters on minimality,13 modularity and controllability.14 However, most of the reported protocell applications have been reported in nonmedical areas. Due to their capability of mimicking certain cellular functions in vitro,15 artificial protocells are emerging as potential candidates in the

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field of biotechnology. Recently, solid particles stabilized Pickering emulsions have been reported as a protocell model for housing integrated biological or biomimetic functions that exhibit higher stability against coalescence, lower toxicity, eco-friendliness, with the potential to induce tissue regeneration.16-17 Considering these advantages, our research group recently designed and developed Pickering emulsion-based artificial osteoinductive protocells carrying minimal doses of BMP2 to induce osteoblasts, which are herein referred to as “artificial proto-osteocells” with the ability to induce bone formation. The artificial proto-osteocells can be directly injected into the target location, which act as an osteoblast recruiting agent by release of BMP2 in a controlled and local manner, where calcium phosphate degraded from artificial proto-osteocells contributes to the formation of mineral component of natural bone. Since the stability and type of the Pickering emulsions largely depend on the surface wettability of the particles,18 much investigation has been reported on the surface modifications of inorganic particles in order to obtain stable Pickering emulsions.19-21 However, we found that corn oil can act as both a continuous phase and a surface modifier for a one-step construction of water-in-(corn)oil Pickering emulsions stabilized by highly hydrophilic calcium phosphate particles without prior surface modification. We attribute this phenomenon to the fatty acid within corn oil that acts as modifier during the formation of Pickering emulsions. Then we take advantages of our newly developed one-step self-assembly strategy to construct novel artificial proto-osteocells by encapsulating BMP2 into biphasic calcium phosphates (BCP) particles stabilized water-in-oil Pickering emulsions, and subsequently utilize this artificial bioactive proto-osteocells for bone regeneration. The function of these novel artificial proto-osteocells were evaluated both in vitro and

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in vivo (Scheme 1). The liquid nature of the Pickering emulsions allowed direct injection of the artificial proto-osteocells into the targeted delivery location. Furthermore, the artificial proto-osteocells were hypothesized to have high osteoinductive potential due to their incorporation with BMP2, with the potential ability to generate significantly higher quantities of new bone. To the best of our knowledge, this is the first example of using inorganic protocells for bone regeneration (artificial proto-osteocells) in vivo.

2. RESULTS AND DISCUSSION Construction of bioactive artificial proto-osteocells. Biphasic calcium phosphates, which are composed of different concentrations of the stable phase, hydroxyapatite (HA), and the more soluble phase, β-tricalcium phosphate (β-TCP), have presented excellent

biocompatibility

and

osteoconductivity and have been extensively used as scaffold and graft materials for bone regeneration.22-23 BCP was chosen in the present study as a self-assembly building block to construct the artificial proto-osteocells due to their great biocompatible and osteoconductive properties. BCP particles were prepared by a simple wet precipitation method (see experimental section). The XRD pattern of as-prepared BCP particles showed the characteristic peaks of β-TCP 2θ = 31.02° (0210) (JCPDS No. 09-0169) and HA 2θ = 31.76° (211) (JCPDS No. 09-0432), respectively, indicating that the as-prepared particles were composed of both β-TCP and HA phases (Figure 1A). The intensity ratio of two peaks at 31.02° for TCP and 31.76° for HA was IT(0210)/IH(211) = 2.16 corresponding to the mass ratio of β-TCP/HA = 70/30. The BCP particles were irregular in shape with a wide range of size distribution around 0.5~1.5 µm (Figure 1B). The water contact angle measurement (31°, Figure 1C) showed that the as-prepared BCP particles were highly hydrophilic which may not be suitable for 5



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self-assembling into water-in-oil Pickering emulsions, as it is well known that the surface wettability of the particles at the oil-water interface determines the stability and types of the Pickering emulsions formed.24 Indeed, attempting to construct water-in-oil Pickering emulsions by using as-prepared BCP particles as stabilizer and organic solvent dodecane as continuous oil phase at the volume fraction of aqueous phase of φaqu = Vaqu/Voil = 0.05 (by adding 100 µl aqueous solution to the mixture of 15-20 mg BCP and 2 ml dodecane) resulted in oil-in-water-in-oil multi emulsions. Oil soluble fluorescent dye (Nile Red) indicated that the interior of the Pickering emulsion was oil phase (Figure S1D, Supporting Information). That is, without prior surface modification, hydrophilic particles tended to form oil-in-water emulsions in dodecane rather than water-in-oil emulsions. In order to obtain bioactive artificial proto-osteocells, water-in-oil Pickering emulsions were needed to encapsulate water soluble proteins in the interior. When corn oil was used instead of dodecane as an oil phase, water-in-oil Pickering emulsions stabilized by as-prepared BCP particles with a contact angle of 31° without surface modification were surprisingly obtained by using the exact same condition described above (adding 100 µl aqueous solution to the mixture of 15-20 mg BCP and 2 ml corn oil) (Figure 1E). Water soluble fluorescent dye calcein indicated that the interior was water phase (Figure 1F, G). Optical microscope images showed the presence of discrete spherical microcapsules with polydispersed size distribution between 10 and 120 µm (Figure S1g, Supporting Information). It could be observed that the BCP particles firmly placed at the interface between corn oil and water (Figure 1E). To understand why hydrophilic BCP particles could stabilize water-in-oil emulsions in corn oil but not in dodecane, several control experiments were conducted. It is well known that corn oil contains a

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large quantity of saturated and unsaturated fatty acids that were thought to play an important role in the formation of BCP stabilized Pickering emulsions. To confirm this, BCP particles were dispersed in corn oil for 6 hours and then washed thoroughly with acetone to remove any free oil residuals. The contact angle of corn oil treated and dried BCP particles showed a significant increase to 106° (Figure 1D). This implied that the particles might be modified by the fatty acids in corn oil. FTIR spectrum of treated BCP particles revealed an increase in intensity of the bands at 2850–2950 cm-1 corresponding to C-H stretching vibrations (Figure 1H), indicating the increase of organic components on the surface of corn oil treated BCP particles in comparison with as-prepared BCP particles. As expected, TGA measurements of corn oil treated BCP particles showed a small increase of weight loss ca. 0.2% than that of as-prepared BCP particles between 250-500°C, which was attributed to the thermal decomposition of organic moieties (Figure S2A, Supporting Information). Moreover, the zeta potential of corn oil treated BCP particles became more negative than that of untreated BCP particles (Figure S2B, Supporting Information), which may be due to the introduction of fatty acids or other components from corn oil on the surface of the particles. Taken together, the results indicated that, after the treatment with corn oil, the surface of the BCP particles was modified by fatty acids or other organic components from corn oil. To further demonstrate the surface modification, oleic acid was used instead of corn oil to treat BCP particles and the contact angle after treatment was increased from 31° to 120° (Figure S2C, D, Supporting Information). After modifications either with corn oil or oleic acid, the BCP particles remained the same crystal structure and the morphology as unmodified BCP particles (Figure S2E, Supporting Information). By utilizing the advantages of corn oil, we have developed a new strategy for one-step

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construction of water-in-oil Pickering emulsions stabilized by highly hydrophilic particles without prior surface modification. Corn oil could act as a continuous oil phase and simultaneously as a modifier for modification of the particles during the formation of the Pickering emulsions. However, the fatty acids within corn oil could not stabilized w/o microdroplets on its own. The controlled bioactivity and the balance between resorption/solubilisation of BCP particles make them ideal building blocks for construction of artificial bioactive proto-osteocells in the form of Pickering emulsions, which have shown the capabilities of encapsulating guest molecules, increasing pro-tissue regenerative gene expression in vitro, selective permeability and good stability.25-27 The BCP shell of these artificial proto-osteocells act as a scaffold, simultaneously provide Ca-P sources which may stimulate bone regeneration. The injectable nature of these bioactive artificial proto-osteocells gives them further advantages as they can be applied in a minimally invasive way when compared to solid scaffolds.28 Release kinetics of protein from artificial proto-osteocells. To investigate the capability of BCP particles stabilized by Pickering emulsions as protein carriers, BSA was used as a model protein to determine protein release from proto-osteocells to the surrounding environment in simulated body fluid (SBF, pH=7.45). Figure 2A showed that the mixture of BSA and BCP particles in SBF exhibited a constant concentration of BSA in the solution through the duration of 4 weeks. In contrast, the concentration of BSA released from the artificial proto-osteocells was slowly increased within 4 weeks and there was still protein kept inside the proto-osteocells at the end of 4 weeks. The slow release of the protein from BCP-containing artificial proto-osteocells was probably due to partially decomposition of BCP particles within simulated body fluid that increased the pore size of the

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microcapsules. The same release kinetics of BMP2 was also performed and the results indicated the same trend with BSA (Figure S3, Supporting Information). Osteogenic capability in vitro. In order to demonstrate the osteogenic capability of artificial proto-osteocells in vitro, we first confirmed that proto-osteocells were not toxic to pre-osteoblast cell line MC3T3-E1 by incubating the cells together with BCP particles or proto-osteocells (Figure S4A, Supporting Information). Phalloidine staining of seeded cells showed that cells grow well and spread around the proto-osteocells (Figure 2B). We then examined the effects of proto-osteocells on pre-osteoblasts differentiation. After culturing the cells for 7 days in the presence of artificial proto-osteocells, the expression of alkaline phosphatase (ALP) was analyzed in MC3T3-E1 cells. The ALP levels of MC3T3-E1 cells increased when exposed to an increasing concentration of BMP2 in the range of 5 ng to 50 ng when using BMP2 alone (Figure 2C), which is consistent with previously published research.29 When 5 ng of BMP2 was simply mixed with BCP particles, the alkaline phosphatase activity did not change much when compared to that of the 5 ng BMP2 group alone. In contrast, when 5 ng of BMP2 was encapsulated within proto-osteocells, a significant enhancement of ALP activity was observed with more than a 75% of increase in comparison to that of 5 ng BMP2 group used alone (Figure 2C, D). As ALP activity is a key event occurring during the early time points of osteogenesis,30 the results therefore indicated that proto-osteocells efficiently and effectively enhanced osteogenic activity with the same dose of BMP2. In other words, by employing proto-osteocells, only 1/4 of the dose of BMP2 was needed to achieve similar osteogenic activity compared to BMP2 alone. On the other hand, alizarin red S staining is a general method to quantitatively determine the presence of calcific deposition by cells of an osteogenic lineage as an

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early stage marker of matrix mineralization associated with bone formation. After being cultured for 14 days in the presence of BMP2-containing (5 ng) proto-osteocells, the number of mineralized nodules stained by Alizarin Red S appeared to be the largest among all the control groups (Figure 2D, Figure S4B). In contrast, by using 5 ng of BMP2 alone or simply mixing 5 ng of BMP2 with BCP particles, the alizarin red S staining result did not show obvious calcific depositions compared to the control group with 0 ng of BMP2. This may imply that the confinement and slow release of BMP2, as well as locally utilization of Ca and P source provided by biodegradable BCP particles, were crucial during the early stage of mineralization. To further elicit osteoblast differentiation in response to proto-osteocells, the gene expression of alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) was determined by qRT-PCR at day 3 and day 7 after culture (Figure S5, Supporting Information). It was once again observed that MC3T3-E1 cells cultured with proto-osteocells produced significantly higher osteogenic-related gene expression than BCP particles or the mixture of BMP2 with BCP particles. Since macrophages are central to tissue-host interactions as they play major roles in innate immunity and in the regulation of the adaptive immune response,31 we examined the effects of proto-osteocells on macrophages phenotype/polarization by fluorescent staining and real-time qPCR (Figure S6, Supporting Information). It was found that proto-osteocells promoted macrophage polarization towards an M2 wound-healing phenotype and further contributed to creating a micro-environment favoring biomaterial-induced osteogenesis. Ectopic bone formation in vivo. To test the osteoinductive ability of proto-osteocells in vivo, we

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selected the widely suggested ectopic bone formation model by directly injecting artificial proto-osteocells into the femoris of Kunming mice (KM mice, see method section and Scheme).1 Biomaterials have biocompatible, bioactive, osteoinductive and osteoconductive properties but also exert a profound impact on host immune responses, which in turn can result in significant effects on the healing and repair process. 10 days after injection, inflammatory responses of proto-osteocells was measured by Hematoxylin and eosin (H&E) staining (Figure S7). The injection of BCP particles or BMP2 solutions, respectively, caused a significant inflammatory cells infiltration, plenty of monocytes/macrophages and foreign body giant cells were observed around the BCP particles. In contrast, the water containing BCP-capsules and BMP2 containing proto-osteocells showed a minimum accumulation of inflammatory cells. We attributed this to the negative surface charge of the artificial proto-osteocells and the hydrophobicity of corn oil residues. In general, nanoparticles with negative surface charges suppress antigen presentation and immune responses as compared to those with positive or neutral surface charges.32-34 As demonstrated above, during one-step formation of the proto-osteocells, corn oil could act as continuous oil phase and, at the same time, as the modifier for the surface modification of the particles that were lower the surface charge, also gave the hydrophobic nature of the proto-osteocells. Compared to hydrophilic surfaces, hydrophobic surfaces of proto-osteocells exhibit more M2-like phenotype macrophages with the production of anti-inflammatory and osteogenic cytokines, thus contributing to the osteogenesis and ectopic bone formation.35 The images of micro computed tomography (µ-CT) analysis of bone formation were taken after injection for 3-weeks and performed by 3D reconstruction. Figure 3 showed that BMP2 have the

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ability of ectopic osteogenesis on its own and the quantity of new bone increased with the dosage of BMP2 used (Figure 3B). The high density shadows in control groups of BCP particles (15 mg) or water-containing BCP (15 mg)-microcapsules may be attributed to the images of BCP particles. Significant difference in the quantity of new bone formation was observed by injection of proto-osteocells with the quantity ratio of BCP/BMP2 = 15 mg/4 µg compared with that of the mixture of BCP and BMP2 (Figure 3A). Analysis of bone mineral density (BMD) from the µ-CT measurements demonstrated that new bone density generated by loading 4 µg of BMP2 within proto-osteocells was similar to that of 100 µg of BMP2 used alone (Figure 3C). This remarkable result indicated that we could reduce by 25 times the use of BMP2 by employing proto-osteocells in vivo. The same trend was found for new bone volume per tissue volume (BV/TV) as well as trabecular number (Tb. N) (Figure 3D, E). In contrast, the injection of the mixture of BCP particles and BMP2 at the same quantity ratio of 15 mg/4 µg resulted in less BV/TV value, which further confirmed that the proto-osteocells can stably trap and control release BMP2, as well as efficiently utilize Ca and P sources provided by the decomposition of BCP particles in vivo. The histological analysis further revealed that proto-osteocells showed a remarkable effect on ectopic bone formation. Both H&E and Masson staining results indicated that the groups treated by BCP particles or water-containing BCP-microcapsules showed no bone formation (Figure 4A) which further confirmed that the shadows displayed in µ-CT images in the groups receiving BCP particles or water-containing BCP-microcapsules treatment were due to the BCP materials added. In other words, BCP bioceramics alone hardly have the ability to generate new bone matrix at early time points. As expected, in the group treated by the mixture of BCP and BMP2 (15 mg/4 µg), BCP

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materials and newly formed bone matrix were found separately. In contrast, the group receiving proto-osteocells (BCP/BMP2= 15 mg/4 µg) treatment demonstrated significant higher levels of new bone formation in muscles in the same location as the BCP materials, indicating that proto-osteocells are capable to localize the new bone formation by confining BMP2, to locally utilize BCP materials as Ca and P source and to provide scaffold for the new bone matrix. To evaluate whether artificial proto-osteocells have the function to form natural bone, type I collagen (COL-1) which is the major organic component of the mineralized bone matrix was detected by immunohistochemical staining (Figure 5A). After 3 weeks, the control groups that were treated with BCP or BCP-microcapsules without BMP2 showed no COL-1 expression. In comparison, both groups receiving either BCP/BMP2 mixture or proto-osteocells exhibited high levels of COL-1 expression. In addition, the area ratio of collagen to BCP residues of proto-osteocells group was 3 times more than that of BCP/BMP2 mixture treated group (Figure 5C), indicating that the osteogenic protein BMP2 was successfully confined by the BCP-microcapsules which minimized diffusion of BMP2 and led to its maximum efficiency within the local micro-environment. Consistent with H&E staining and Masson staining, the COL-1 positive area was more separated from BCP particles in the BCP/BMP2 group while the proto-osteocells were well trapped in the collagen matrix. Osteopontin (OPN) is a major organic component of bone which has previously been utilized as an indicator for osteogenic cells.36 The number of OPN-positive cells was also significantly higher in the proto-osteocells group than that of the BCP/BMP2 mixture group, which represented that the proto-osteocells can help recruit MSCs and promote their osteogenic differentiation. To further investigate the bone remodeling process, tartrate-resistant acid phosphatase (TRAP)

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staining was conducted to describe the osteoclastic resorption among the four treatment groups (Figure 5B). It was observed that the number of osteoclasts showed no significant differences between four groups (Figure 5E), which indicated that the osteogenic potential of proto-osteocells did not affect the normal bone resorption function and formed new bone under a normal physiological state. Natural bone formation is consists of two processes: (1) synthesis of organic matter (mostly collagen type I); and (2) mineralization of inorganic salts (calcium deposition).37 Treatment with our artificial proto-osteocells not only remarkably increased the collagen formation but also provided calcium for mineral deposition. During the degradation of the BCP ceramics (Figure S8, supplementary information), the pore size of the proto-osteocell membranes increase so that bioactive proteins could slowly escape, similar to the secretion process of real cells. It was previously reported that constant supplementation of BMP2 could effectively recruit MSCs and direct cell differentiation towards osteoblasts.29,

38-39

This higher ability for osteogenic differentiation might be the result of this

sustained release of BMP2 from the proto-osteocells to serve as the constant bone-inducing supplementation. MSCs in muscles were then recruited to the scaffold surface by release of BMP2 and the utilization of Ca and P ions degraded from BCP particles,40-41 mineral deposition was eventually generated.

3. CONCLUSION In summary, we designed and developed artificial bioactive proto-osteocells by self-assembly of inorganic biphasic-phosphate particles in the form of water-in-oil Pickering emulsions to trap BMP2 in a liquid state of food grade corn oil, which avoided the necessary use of traditional 14


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polymers avoiding potential biotoxicity of these organic solvents. This novel biomaterial in liquid state allows the proto-osteocells to be directly injected into the target location. During the degradation of calcium phosphate shells of the proto-osteocells, the artificial proto-osteocell further act as an osteoblast recruiting agent by release of BMP2 in a controlled and local manner, which may continuously and stably induce the MSCs recruitment and osteogenic differentiation. In addition, the calcium phosphate degraded from proto-osteocells also contributed to the formation of mineral component of natural bone. The system developed here represents a step towards the design and utilizing material based artificial proto-osteocells to tackle medical issues, which could have potential applications in hard tissue repair and regeneration.

4. EXPERIMENTAL SECTION Preparation and characterization of biphasic calcium phosphate (BCP) particles. All of the chemical reagents of analytical grade were purchased from Sinopharm Chemical Reagents (China). BCP particles were synthesized by a wet chemical precipitation method.42-44 Typically, 300 ml of 0.24 mol di-ammonium hydrogen phosphate ((NH4)2HPO4, AR) solution was added dropwise into 300 ml of 0.36 mol calcium nitrate tetra-hydrate (Ca(NO3)2·4H2O, AR) solution with vigorous stirring, and the temperature was increased to 55℃. Through the mixing process, the pH of the system was maintained at ~7.5 by addition of ammonia solution (NH3·H2O, 25~28%，AR) and subsequently the suspension was stirred for another 2 h. After precipitation, the white suspension was aged at room temperature for 36 h. The resultant white product was centrifuged, washed with deionized water and then dried at 80℃ in air for 12 h. Finally, the obtained powder was calcined at 1100℃ for 3 h. The phase composition of BCP particles was identified by X-ray diffraction (XRD; X’Pert Pro, 15



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Philips, the Netherlands). The morphology was observed by field emission scanning electron microscopy (FE-SEM, Hitachi S4800; Hitachi Co, Japan). The water contact angle was measured with a 2-µl droplet of distilled water on the surface of compressed tablet of particles at ambient temperature with a JC2000D1 optical contact angle meter (Shanghai zhongchen Technology Co., China). The average contact angle value was obtained by measuring the sample at four different positions, and the images were captured with a digital camera. The hydrophobicity of the particles measured by water droplet showed a contact angle of 31°. Preparation of corn oil treated particles. 0.5 g of as-prepared BCP particles were added to 35 ml corn oil, sonicating the dispersion in an ultrasound bath for 30 min, and then rotated at room temperature for 6 h. Subsequently, the dispersion was centrifuged at 10000 rpm and the top clear oil discarded. 35 ml of acetone was added to the precipitation, shaked for 5 min and the dispersion was re-centrifuged. Finally, the precipitation was dried at 80℃ for 12 h. The product (corn oil treated BCP particles, BCP-oil) was then used for TG, FT-IR and contact angle measurements. The zeta potential was measured via electrophoretic light scattering using a zeta potential analyser (Malvern, Zetasizer Nano, ZS90). 0.5 mg ml-1 of as-prepared BCP particles (BCP) and corn oil treated BCP particles (BCP-oil) were prepared in the solutions with pH varied from 1 to 9 adjusted by 0.01 M solutions of HCl and NaOH. Preparation of Pickering emulsion. BCP (containing 70% of β-tricalcium phosphate, β-TCP-70%) particle-stabilized water-in-oil Pickering emulsions were prepared as follows: 15~20 mg of BCP particles were mixed with 2 ml of corn oil (Arawana Brand, China) and sonicated in an ultrasound bath for 20 seconds to disperse any large aggregates. 100 µl of aqueous phase containing

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either BMP2 (provided by Jiuyuan Biotech Company, Hangzhou, China, 50 ng ml-1 ~ 40 µg ml-1, pH=4.2) or bovine serum albumin (BSA, Biosharp, 500 µg ml-1) was added into the mixture. After shaking vigorously for 5 minutes, water-in-oil Pickering emulsions were formed. For fluorescent imaging, the aqueous phase was replaced by 100 µl of calcein (Aladdin, China) solutions (100 µmol l-1 in double distilled water). For control experiments, pure water was used as aqueous phase to form Pickering emulsion that was termed as 'BCP-microcapsules' in the text. For comparison, BCP particle-stabilized emulsions using organic solvent dodecane (Aladdin, China) as continuous oil phase were prepared using the same procedure as described above. Oil-in-water-in-oil multi emulsions were formed. For fluorescent imaging, the oil phase was replaced by 100 µl of Nile Red (BR, China) solutions (100 µmol l-1 in dodecane). In vitro release studies. 20 mg BCP particles (β-TCP-70%) were mixed with 2 ml corn oil and sonicated for 20 seconds, 100 µl of BSA aqueous solution (5 µg ml-1 in double distilled water) was added as aqueous phase to construct water-in-oil Pickering emulsions. BSA was used as a model protein to determine the protein release properties. After settling, the top oil layer was removed and gently replaced by 1 ml simulated body fluid (SBF, prepared according to a reported protocol developed by Kokubo et al., pH = 7.45

45

) and kept at room temperature. 10 µl supernatant was

removed at the time of day-0.5, day-1, day-3, day-7, day-10, day-14, day-21 and day-28, respectively. Control experiments were carried out by adding 1 ml simulated body fluid to the mixture of 20 mg BCP particles and 100 µl of BSA aqueous solution. The protein concentration was measured by BCA Protein Assay Kit (Thremo Fisher Scientific Inc.).

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Cell culture. Osteoblast-like cells derived from mouse calvaria (MC3T3-E1) were cultured in α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin antibiotics (HyClone, Thermo Fisher Scientific Inc). The murine-derived macrophage cell line RAW 264.7 were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics. All cultures were maintained at 37℃ in 5% CO2 and 95% air in a humidified incubator. In vitro cellular biocompatibility. For cell cytotoxicity assays, the MC3T3-E1 cells were seeded at a density of 2000 cells per well into a 96-well plastic culture plate. The BCP particles (10 mg) and water-containing BCP-microcapsules (20 µl) were directly added into the 96-well plates. After incubation for 1 day, 3 days, 5 days, a CCK-8 (Biosharp, China) assay was applied to test the overall metabolic activity. To investigate the morphology of pre-osteoblasts in the presence of protocells, the cells were incubated with water-containing BCP-microcapsules for 3 days. After fixation with 4% formaldehyde, the cytoskeleton and nuclei were stained with phallpoidine-FITC (Sigma-Aldrich) and 4’,6-diamidino-2-phenylindole (DAPI). Confocal images were obtained by a confocal laser scanning microscope (Olympus FV1000). In vitro osteoinductive ability on osteoblast differentiation. For mineralization assessment, water-in-oil Pickering emulsions were prepared by 20 mg BCP particles (β-TCP-70%) in 2 ml corn oil and 100 µl BMP2 (50 ng ml-1). The Pickering emulsions were then cross-linked by 20 µl tetramethylorthosilicate (TMOS, Aladdin Chemical Reagent Corp.) and transferred into water as follows. After settling, the top oil phase was carefully removed and replaced with 1mL of

18


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acetone/water (7:3) mixed solution, after centrifugation at 2000 rpm for 1 min, the top solution was replaced by 1 mL of distilled water and re-centrifuged. And finally transferred into incubation buffer to give water-in-water microcompartments, and then incubated together with MC3T3-E1 cells. As control experiments, pure BCP particles (10 mg), 100 µl of BMP2 solutions (at different concentrations including 50 ng ml-1, 100 ng ml-1, 200 ng ml-1, and 500 ng ml-1), the mixture of BCP particles (10 mg) and 100 µl of BMP solution (50 ng ml-1) were also cultured respectively. The culture medium was changed every 3 days, and all groups did not get further replenishment of BMP2. After being cultured for 7 days, cells were assayed for alkaline phosphatase activity and staining. Cells were washed three times with PBS and solubilized in 0.1 % Triton X-100 (buffered in 0.1 M PBS, pH 7.3) at 4 °C for 1 h. After sonication and centrifugation, ALP activity in the supernatant was determined colorimetrically using readings of OD405/OD562. ALP and total protein was quantified by p-Nitrophenyl Phosphate (pNPP) and a BCA Protein Assay Kit. For ALP staining, the assay mixture contained

1mg

Nephthol

AS-MX

Phosphate

(Sigma

N-4875),

0,05

ml

N,

N-Dimethylformamide (DMF, Biosharp), 5 ml double distilled water (ddH2O), 5 ml 0.2M Tris-HCl and 6mg Fast blue BB salt (FBB, Sigma F-0250). After being fixed with 95% alcohol for 10 min, cells were incubation at room temperature for 2 h in the dark. To identify mineralization nodules, Alizarin Red S staining was assayed on day 14 after MC3T3-E1 cells had been cultured. The medium was removed and the cells were fixed with 95% alcohol for 10 min at room temperature. After being gently rinsed with ddH2O, the cells were stained in a solution of 0.1% Alizarin Red S (Sigma-Aldrich, pH 4.6) at 37°C for 30 min and then washed. The samples were air-dried, and microscopic images were obtained using the Nikon inverted microscope. Thereafter, alizarin red quantification was

19



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dissolved using 200 µl of 1 % cetylpyridinium chloride (Sigma-Aldrich) at room temperature for 4 h. Then 100 µl solution was transferred to 96-well plate to text OD 562nm. In vitro inflammatory effects on macrophages. An immunofluorescence assay was used to reveal the expression of M1 and M2 cell surface markers iNOs and CD206 respectively. Briefly, RAW cells were seeded into slides with proto-osteocells or control groups. After 24 hours, cells were fixed with 4% formaldehyde and washed with PBS. Rabbit anti-iNOs or CD206 (Santa Cruz, 1:100) were diluted into 2% BSA at 37°C for 1 hour. Then the cells were incubated with secondary antibodies conjugated with fluorescence (Alexa Fluor®488, Alexa Fluor®594, Abbkine, 1:200) for 1 hour. Thereafter, DAPI was added for visualizing the cell nuclei. After a final wash, confocal images were obtained by a confocal laser scanning microscope. RAW 264.7 cells were seeded with BCP particles, the mixture of BCP and BMP2, and proto-osteocells for 24 hours. Total RNA of all groups was extracted by the AxyPrepTM Multisource Total RNA Miniprep Kit (AXYGEN, Union City, California, USA) according to the manufacturer’s instruction. 1.0µg total RNA was used as a template for the synthesis of cDNA with OligodT and AMV reverse transcriptase (TaKaRa, Japan). RT-PCR primers including M1 (CD86, NOS2) and M2 (CD163, CD206) macrophages markers and inflammatory related cytokines such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and interleukins (IL). The specific primer sets are outlined in Table S1. RT-PCR was performed using 25 µl final reaction volume of QuantiFast SYBR Green PCR Kit (QIAGEN, Venlo, Holland) and the target gene expression were assayed on a CFX Connect Real-Time PCR Detection System. The delta-delta Ct method was used to calculate gene expression levels relative to house-keeping genes GAPDH and normalized to control cells.

20


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The in vivo osteogenic properties of proto-osteocells. Ectopic bone formation experiments were performed in accordance with the guidelines of the animal handling committee at the ARRIVE Guidelines. For in vivo studies, BMP2-containing proto-osteocells were prepared by dispersing 15 mg BCP particles in 2 ml corn oil and adding 100 µl BMP2 solutions (dissolved in ddH2O at 40 µg ml-1) as aqueous phases, shaken vigorously to facilitate the formation of the emulsions and then allowed to settle at the bottom of the sample vial. After removing as much as possible from the top oil layer, the as-prepared Pickering emulsions were directly injected into the femur muscles of KM mice (male, 8-week-old). As control experiments, 15 mg BCP particles, ddH2O-containing Pickering emulsions, the mixture of BCP particles (15 mg) and 100 µl BMP2 solutions (40 µg ml-1), and 100 µl BMP2 solutions (at 200 µg ml-1, 500 µg ml-1 and 1 mg ml-1) were injected, respectively. Animals were euthanized at specified time points. The legs with femur muscles were completely harvested and fixed in 4% formaldehyde solution for 24 h. After fixation, legs with femur muscles were examined using a micro-CT scanner (µCT 50, Scanco Medical AG, Bassersdorf, Switzerland). Images were quantified for bone mineral density (BMD), bone volume over tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp) as previously described.22, 46 Histological analysis. After fixation, the tissues were decalcified in 10% EDTA, dehydrated, embedded in paraffin, and sectioned to 4-µm thickness. Hematoxylin and eosin (H&E, MXB, China) staining, Masson staining (MXB, China) and tartrate-resistant acid phosphatase (TRAP, Sigma N-387A) staining were used according to the manufacturer’s protocol for general histological studies. According to previous reports,47 the number of osteoclasts was counted under a light microscope

21



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(Olympus DP71, Japan). The TRAP-positive cells with more than three nuclei were defined and counted as osteoclasts. At the same time, the expression of type I collagen (COL-1) and osteopontin (OPN) were evaluated by immunohistochemical assessment. Sections were deparaffinised in xylene two times for 10 minutes, followed by soaking in a series of graded alcohol from 100% to 70% and washed with PBS for 5 minutes. Sections were incubated with hydrogen peroxide for 15 minutes at room temperature, washed with PBS and incubated with BSA for 15 minutes at 37℃, and then the sections were incubated with the primary antibody for COL-1 (1:200, Boster SA2005, Boster Co., China) and OPN (1:200, Boster SA2005, Boster Co., China) for 1h at 37℃. After washing, sections were incubated with their secondary antibody for 20 minutes and made visible after the addition of Diaminobenzidine (DAB, MXB, China). Sections were counterstained with hematoxylin and then dehydrated. Semi-qualification of the ectopic bone formation was done according to COL-1 staining by using Photoshop CS6 software (Adobe System, USA). The area of newly formed bone (stained in brown) and BCP particles (no stain) were delineated manually and then calculated as the ratio of new bone formation area/biomaterials area. Furthermore, the ability of the samples to induce new ectopic bone formation was semi-qualitatively evaluated by two independent observers blinded to the treatment and rated according to a previously published scheme.48-49 As previously described, a score of 1 indicated the presence of particles without any bone; 2 indicated the production of new bone in one site within the section and covering less than 40% of the surface area examined; 3 indicated the production of new bone in more than one site, covering more than 40% but less than 70%, of the

22


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surface area examined; and 4 indicated the production of new bone in more than one site, covering more than 70% of the surface area examined. The overall grade for each bone graft was obtained by averaging the scores from all specimens in the group. For immune response measurements, mice were euthanized after 10 days. Sections were stain in H&E and mounted for histopathologic observation. Six fields (100 µm × 75 µm) from each section were randomly selected from histopathologic analysis according to a previous reference.50 The infiltrating cells were identified as macrophages, lymphocytes and neutrophils, based on their shape and size as previously described.51-52 Fibroblasts in the visual field were not counted.

ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxxxxx. Additional properties of proto-osteocells, cell experiment, and microscopy images images (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Present Addresses § Department of Periodontology, University of Bern, Bern, Switzerland

Author Contributions 23



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# M. Shi and R. Yang contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the funds of the National Natural Science Foundation of China (81271108 and 81570954 to YFZ, 21571192 to ML, 21477165 to J.S.), and the Fundamental Research Funds for the Central Universities (410500041).

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Scheme 1. Diagram illustrating the experimental procedure.

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Figure 1. (A) XRD spectrum of as-prepared BCP particles and standard spectra of JCPDS No. 09-0169 for β-TCP and 09-0432 for HA. (B) SEM image of as-prepared BCP particles. Scale bar = 5 µm. (C) Photograph of contact angle measurement of as-prepared BCP particles and (D) corn oil treated BCP particles. (E) Optical microscope image of BCP particles stabilized water-in-(corn)oil Pickering emulsions. Scale bar = 100 µm. (F) Fluorescence microscopy image of water soluble fluorescent dye calcein containing Pickering emulsions (scale bar = 100 µm) and (G) corresponding 3D image. (H) FT-IR spectra for as-prepared BCP particles (black) and corn oil treated BCP particles (red).

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Figure 2. The in vitro functions of artificial proto-osteocells. (A) The release profile of BSA from BCP-microcapsules over a 4-week period (red) and from the mixture of BSA+BCP particles (blue) in SBF. (B) Confocal images of proto-osteocells (DIC) and MC3T3-E1by immunofluorescence. Scare bar = 50 µm. (C) ALP activity at 7 days after osteogenic induction (*P < 0.05, **p

Inorganic Self-Assembled Bioactive Artificial Proto-osteocells Inducing

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