Nanocomposite Porous Microcarriers Based on Strontium-Substituted

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Biological and Medical Applications of Materials and Interfaces

Nanocomposite Porous Microcarriers Based on Strontium-Substituted HA-g-Poly(#-benzyl-L-glutamate) for Bone Tissue Engineering Shifeng Yan, Pengfei Xia, Shenghua Xu, Kunxi Zhang, Guifei Li, Lei Cui, and Jingbo Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Nanocomposite Porous Microcarriers Based on Strontium-Substituted HA-g-Poly(γ-benzyl-L-glutamate) for Bone Tissue Engineering

Shifeng Yan,† Pengfei Xia,† Shenghua Xu,† Kunxi Zhang,† Guifei Li,† Lei Cui,*,‡ Jingbo Yin*,† †

Department of Polymer Materials, Shanghai University, 99 Shangda Road, Shanghai

200444, P. R. China ‡

Department of Orthopedics, Shanghai Tongji Hospital, Tongji University School of

Medicine, 389 Xincun Road, Shanghai 200065, P. R. China

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ABSTRACT Porous microcarriers have aroused increasing attention recently, which can create a protected environment for sufficient cell seeding density, facilitate oxygen and nutrient transfer, and well support the cell attachment and growth. In this study, porous

microcarriers

fabricated

from

hydroxyapatite-graft-poly(γ-benzyl-L-glutamate)

the

strontium-substituted

(Sr10-HA-g-PBLG)

hybrid

nanocomposite were developed. The surface grating of PBLG, the micromorphology and element distribution, mechanical strength, in vitro degradation and Sr2+ ion release of the obtained Sr10-HA-g-PBLG porous microcarriers were investigated, respectively. The grafting ratio and the molecular weight of the grafted PBLG of Sr10-HA-g-PBLG could be effectively controlled by varying the initial ratio of BLG-NCA to Sr10-HA-NH2. The microcarriers exhibited highly porous and interconnected microstructure with the porosity of about 90% and overall density of 1.03-1.06g/cm3. Also, the degradation rate of Sr10-HA-PBLG microcarriers could be effectively controlled and long-term Sr2+ release was obtained. The Sr10-HA-PBLG microcarriers allowed cells adhesion, infiltration and proliferation, and promoted the osteogenic differentiation of rabbit adipose-derived stem cells (ADSCs). Successful healing of femoral bone defect was proved by injection of the ADSCs-seeded Sr10-HA-PBLG microcarriers in a rabbit model. Keywords:

Poly(γ-benzyl-L-glutamate),

Strontium-substituted

Porous microcarriers, Femur bone defect

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hydroxyapatites,

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1. INTRODUCTION Bone defects caused by trauma, disease and congenital defects often lead to severe pain and disability and remain major clinical challenges in the field of bone tissue engineering.1,2 Over the past two decades, reconstruction and regeneration of skeletal tissues via tissue engineering strategies has gained great interest. The fabrication of appropriate three-dimensional scaffolds that support cellular growth and extracellular matrix (ECM) deposition is a key issue for tissue engineering.3 Thus, a variety of bulk porous scaffolds based on nature and/or synthetic polymers have been well developed. However, the common used preformed bulk scaffolds are noninjectable and can not meet the demand for repairing irregularly and complex shaped defects. Recently, microcarriers have received much attention because of their desirable advantages such as maintaining the cell differentiated phenotype, directly injecting and delivering the cell-seeded microcarriers to the target site that needs repair, completely filling and repairing the irregular surgical defects.4 However, the solid spherical microcarriers have low surface area-to-volume ratio, minimizing the maximum cell loading. Moreover, the surface-attached cells are vulnerable to damage. The porous microcarriers with inter-connected pores and a greater surface area are desirable to create a protected environment for sufficient cell seeding density, facilitate oxygen and nutrient transfer, and well support the cell attachment and growth. Microcarriers have been prepared from both natural polymers including gelatin,5

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chitosan,6 dextran,7 cellulose8 and synthetic polyesters such as poly(lactic acid)9 and poly(lactic-co-glycolic acid)10. Recently, we first developed porous microcarriers based on the synthetic polypeptide of poly(γ-benzyl-L-glutamate) (PBLG), which showed controllable degradation properties, cytocompatibility, and good potential for tissue engineering.11 However, the lack of bioactivity of the single component of these microcarriers limits its biological applications in bone tissue engineering. The ideal microcarriers should simulate the biochemical composition of the native extracellular matrix and natural bone, thus creating a suitable microenvironment for cell attachment and proliferation. Nano-hydroxyapatite (n-HA), a naturally occuring calcium mineral, is the main component of bone, acting as a template for the mineral phase deposition and stimulating new bone formation.12 However, it was reported that the anti-resorptive and anabolic ability of HA were unsatisfactory and new strategy was still desired.13-15 Strontium (Sr) has the potential to promote bone formation and reduce bone resorption.16,17 The Sr2+ substituted hydroxyapatite was found to stimulate osteoblast activity and inhibit osteoclast proliferation.18 Traditionally, the bioactive scaffold is based on degradable polymers and ceramics.19 Therefore, the incorporation of Sr2+ substituted hydroxyapatite into a PBLG matrix would combine the osteoconductivity and osteoinductivity of bioactive ceramics with easy processing of polymer, thus creating a bioactive composite microcarriers for bone regeneration. Meanwhile, both PBLG and Sr2+ substituted hydroxyapatite resemble the bone matrix in composition. Although the composite microcarriers based on natural polypeptide (such as gelatin20, collagen21) and hydroxyapatite have

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been reported, the fabrication of porous synthetic PBLG/hydroxyapatite composite microcarriers for bone tissue enginnering remains a challege, which is ascribed to the poor interfacial compatibility between the hydrophobic PBLG and hydrophilic hydroxyapatite. In

the

present

study,

strontium-substituted

hydroxyapatite-graft-PBLG

(Sr10-HA-g-PBLG, with 10 mol% Ca2+ replaced by Sr2+) was prepared by ring opening polymerization (ROP) of γ-benzyl-Lglutamate N-carboxyanhydride (BLG-NCA). Sr10-HA-g-PBLG porous microcarriers were then fabricated via double emulsion evaporation method using gelatin as porogen (Figure 1). The surface grafting of PBLG, the micromorphology, biodegradability, mechanical strength, in vitro degradation and Sr2+ ion release of the obtained Sr10-HA-g-PBLG porous microcarriers were investigated, respectively. Adipose derived stem cells were incorprated into the microcarriers and osteogenesis both in vitro and in vivo was assessed.

2. RESULTS AND DISCUSSION 2.1. Synthesis of Sr10-HA-g-PBLG with Controllable Grafting Ratio. The bioactive strontium-containing hydroxyapatite cements with the main component of Sr10-HA have been developed for spinal and bone fracture surgery.22,23 Strontium-substituted hydroxyapatites nanocrystallines was synthesized by coprecipitation method, and then subjected to aminated surface modification (Figure 1a). The characterization of Sr10-HA nanocrystallines and their aminated surface modification are described detailedly in the Supporting Information. The compositions and crystalline structure

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of strontium-substituted hydroxyapatites was characterized by ICP (Figure S1a) and XRD (Figure S1b), respectively. The obtained strontium-substituted HA with the expected Sr/(Sr+Ca) of 10 mol% displayed the near identical test value of 9.86 mol% using ICP. And successful amino functionality of Sr10-HA nanoparticles with APTS was determined by FTIR (Figure S1c) and XPS (Figure S1d) measurements. The covalent grafting of polypeptides onto Sr10-HA nanoparticle was performed via “grafting-from” method. The aminated Sr10-HA (Sr10-HA-NH2) nanoparticles initiated ROP of BLG-NCA monomers, leading to surface-grafting of PBLG chains and formation of Sr10-HA-g-PBLG composite nanoparticles.24,25 Surface grafting of PBLG was verified by

1

H NMR spectra and XRD. The

representative 1H NMR spectra of Sr10-HA-g-PBLG and PBLG are shown in Figure 2a. The peaks at 6.72, 4.55, 4.18, 1.88, 1.38 and 1.57 ppm were characteristic of proton peaks of PBLG,26 which were also detected in the

1

H NMR spectrum of

Sr10-HA-g-PBLG, revealing successful ROP of BLG-NCA initiated by Sr10-HA-NH2 nanoparticles. The comparison of typical XRD patterns of PBLG, Sr10-HA before and after surface grating of PBLG is displayed in Figure 2b. For PBLG, a broad peak centered at about 21° was observed, corresponding to its amorphous nature. Sr10-HA possessed the hexagonal crystal structure with the corresponding characteristic crystallization peaks.27 Sr10-HA-g-PBLG nanocomposites displayed the peaks for both PBLG and Sr10-HA. This indicated that the PBLG was grafted onto Sr10-HA nanoparticles, and the original crystalline structure of Sr10-HA nanocrystals was un-changed during surface grafting.

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As a result of thermogravimetric analysis, the grafting ratio of Sr10-HA-g-PBLG increased gradually with the mass ratio of monomer to initiator ([M]/[I]) (Figure 2c). And the measured grafting ratio was near to the theoretical value [M]/([I]+[M]) (Figure 2d), indicating the nature of living polymerization initiated by primary amines on the surface of Sr10-HA nanoparticles. Therefore, the grafting ratio of Sr10-HA-g-PBLG nanoparticles could be effectively and accurately controlled by simply changing the relative amount of BLG-NCA and Sr10-HA-NH2 (Table S1). According to GPC data, the molecular weight of the grafted PBLG increased with the theoretical grafting ratio [M]/([I]+[M]) (Figure 2d). Thus, the relative molecular weight of grafted PBLG could also be controlled by regulating the ratio of [M]/[I]. Also we could conclude that the growth of grafted PBLG chains mainly contribute to the increasing grafting ratio. 2.2 Micromorphology and Colloidal Stability of Sr10-HA-g-PBLG. The surface grafting of PBLG onto Sr10-HA nanoparticles was followed by TEM and AFM observations. The TEM micrographs of Sr10-HA before and after surface grafting are displayed in Figure 3a and 3b. The Sr10-HA was found to be needle-like with the length of about 50nm and width of about 10nm. The Sr10-HA tended to agglomerate, owing to strong inter-particle van der Waals forces as well as hydrogen bonding between surface hydroxyl groups (Figure 3a).28,29 However, the Sr10-HA-g-PBLG nanocomposites were homogeneously dispersed because the PBLG shells could efficiently weaken the inter-particle interaction (Figure 3b). Figure 3c-3e and Figure 3c’-3e’ show the AFM images of Sr10-HA, PBLG and Sr10-HA-g-PBLG. A poor

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dispersion of Sr10-HA nanoparticles was obviously observed in Figure 3c and Figure 3c’, which was consistent with TEM result (Figure 3a). The pure PBLG formed a uniform nano-fiberous structure (Figure 3d and Figure 3d’). For Sr10-HA-g-PBLG, as shown in Figure 3e and Figure 3e’, the novel core-shell structure was obviously observed. The needle-like Sr10-HA nanoparticle core was homogeneously dispersed, while the surface of which was covered with fibrous grafted PBLG shell. The colloidal stability evaluation (Figure S2) shows that the surface grafting of PBLG can effectively overcome the interparticle interaction and prevent the agglomeration of Sr10-HA nanoparticles in CH2Cl2, which is very beneficial to the homogeneous dispersion of hydrophilic inorganic Sr10-HA nanoparticles in hydrophobic organic PBLG during the solution-based fabrication of organic-inorganic composite microcarriers. 2.3. Microstructure of Porous Microcarriers. The double emulsion method is commonly employed in the fabrication of biodegradable microcapsules.30 In our study, W/O/W double emulsion method was adopted to prepare porous Sr10-HA-g-PBLG microcarriers. The gelatin served as a porogen because it has a phase transfer temperature near room temperature. Thus, the gelatin solution of inner droplets rapidly gelled after being immersed into ice-water bath. And the gelatin porogen could subsequently be leached out easily by soaking in a warm water bath, leading to porous Sr10-HA-g-PBLG microcarriers (Figure 1b). Figure 4 shows the microstructure of Sr10-HA-g-PBLG microcarriers with different grating ratios. All of the Sr10-HA-g-PBLG microcarriers exhibited a spherical

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morphology (Figure 4a-4c). The Sr, Ca, C and P elements were uniformly distributed, as shown in the EDX mapping of the surface of Sr10-HA-g-PBLG microcarriers, revealing that Sr10-HA nanoparticles were evenly dispersed within the porous microcarriers matrix. A relatively narrow size range between 200 and 300μm could be obtained by sieving (Figure 4d). Furthermore, the microcarriers possessed a highly porous and interconnected microstructure. Sr10-HA-g-PBLG microcarriers with grafting ratios of 70%, 80% and 90% showed high porosity of 89.73%, 90.05% and 91.34%, respectively (Figure 4e). It was reported that large pores, adequate porosity and high interconnectivity of the porous microcarriers provide adequate nutrient and oxaemic conditions, faciliating the growth and uniform distribution.31-33 The Sr10-HA-g-PBLG porous microcarriers with the grafting ratios of 70%, 80% and 90% showed the overall density of 1.061g/cm3, 1.048g/cm3 and 1.031g/cm3, respectively (Figure 4e). Obviously, the density of porous microcarriers decreased slightly with the grafting ratio. The overall density of the porous microcarriers was close to that of the culture medium (typically between 1.02 and 1.10), making it easy for the Sr10-HA-g-PBLG porous microcarriers to be dispersed and suspended in the culture medium by gentle agitation and permitting the desired cell–microcarriers interaction.34,35 2.4. Mechanical Strength of Porous Microcarriers. Mechanical strength of the porous microcarriers is vital to hold, support, and protect the cells during in vitro culture, injection, and post-implantation into the body.36 Figure 5 displays the mechanical properties of the porous PBLG microcarriers and Sr10-HA-g-PBLG

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microcarriers with similar microstructure (particle size: about 250μm, pore size: 30~50μm, porosity: 90.0±1%). Rheological properties were studied via oscillatory rheology, which reflect the stability of the porous microcarriers.37 Compared to the pure PBLG microcarriers, the Sr10-HA-g-PBLG microcarriers with the grafting ratios of 70%, 80% and 90% showed a 4–9, 5-14, and 4-14 fold higher magnitude of G’ in the frequency range of 1–10 rad/s (Figure 5a), indicating greatly improved the ability to resist shear failure. The measurement of compressive strength reflects the ability of the microcarriers to sustain compressive loading in vivo. During the compression testing using DMA, all the microcarriers were observed to be able to recover their original shapes within seconds after mechanical deformation, which would be useful when injecting the microcarriers in vivo, since these microcarriers should be compacted into a small fraction of their original size.38,39 As shown in Figure 5b, the compressive strengh of the pure PBLG microcarriers was about 0.013Mpa. The compressive strengh of Sr10-HA-g-PBLG porous microcarriers with the grafting ratios of 70%, 80% and 90% reached 0.039±0.001Mpa, 0.030±0.013Mpa, and 0.038±0.002Mpa, respectively, showing little change with grafting ratio. When compared to pure PBLG microcarriers, the porous Sr10-HA-g-PBLG microcarriers exhibited significantly improved storage modulus (Figure 5a) and compressive strength (Figure 5b). The mechanical enhancement might be ascribed to the uniform dispersion of the Sr10-HA nanoparticles in PBLG matrix, as well as the greatly enhanced interfacial combination between Sr10-HA nanoparticles and PBLG

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matrix.40 2.5. In Vitro Degradation and Ion Release. Bone tissue engineering scaffolds are usually expected to be resorbable, giving way to new bone regeneration in vivo to allow the full restoration of native tissue structure and function.41,42 Thus tailoring the degradation rate of the matrix is very crucial for microcarriers to be utilized in bone tissue engineering. The morphological change during degradation process was visually observed by SEM (Figure 6a-6e). The Sr10-HA-g-PBLG microcarriers (80% grafting ratio) exhibited regular spherical and porous structures prior to degradation (Figure 6a). After 4 weeks of degradation, the microcarriers almost kept unchanged in terms of micromorphology (Figure 6b). When the degradation time was further increased to 8 weeks, most microcarriers began to deform and fracture, as shown in Figure 6c. After 12 weeks, the vast majority of microcarriers were decomposed into pieces of debris, only a small number of the microcarriers maintained a globular structure (Figure 6d). When the in vitro degradation time was extended to 18 weeks, as shown in Figure 6e, all microcarriers were broken into small fragments. Quantitatively, the biodegradable degree was determined by weight loss, as shown in Figure 6f. After 18 weeks of degradation in vitro, the pure PBLG microcarriers and Sr10-HA-g-PBLG microcarriers with grafting ratios of 70, 80 and 90% showed the weight losses of 41.1%, 67.1%, 52.8% and 61.7%, respectively. It was well known that the degradation rate of polymer was greatly dependent on its relative molecular weight.43 As mentioned above, the molecular weight of the grafted PBLG increased with the theoretical grafting ratio (Figure 2d, Mn

PBLG=301700).

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The decrease of

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degradation rate with the grafting ratio of Sr10-HA-g-PBLG could be ascribed to the increasing molecular weight of grafted PBLG. Thus, the degradation rate of Sr10-HA-g-PBLG microcarriers could be effectively controlled to meet the application of bone regeneration. The elemental concentrations of Ca2+ and Sr2+ released from Sr10-HA-g-PBLG microcarriers were shown in Figure 6g. The surface grafting of PBLG ensured that the Sr10-HA was stable as a core in the Sr10-HA-g-PBLG composite. With the degradation of Sr10-HA-g-PBLG porous microcarriers, the Ca2+ and Sr2+ were released gradually. The average concentration of Sr2+ released from Sr10-HA-g-PBLG microcarriers with grafting ratios of 70, 80 and 90% were 1-5 mg/L within 12 weeks. The Ca2+ and Sr2+ ions were released at a relatively stable Ca2+/Sr2+ molar ratio of 8.8~9.1. The values were found to be close to the equivalent Ca2+/Sr2+ ratio of 9 (Figure 6h). The ion release was greatly dependant on the grafting ratio. Higher grafting rate lead to thicker PBLG shell and more compact coating of Sr10-HA nanopaticles, thus delaying the Sr2+ release. A dose-dependent effect of Sr on bone formation has previously been reported by Verberckmoes et al.44 Too high or too low a dose of Sr2+ was not conducive to promoting osteogenesis. It was reported that both nodule formation and mineralization were normal for the 2 mg/L and 5 mg/L doses. So the Sr10-HA-g-PBLG porous microcarriers with appropriate Sr2+ concentrations might be the ideal injectable vehicle for bone tissue engineering. 2.6. Proliferation and Osteogenic of ADSCs within the Microcarriers. Macroporous microcarriers were developed to provide large surface area for

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high-density cell growth in the interconnected pores, as well as to give protection for the cells within the interior of the microcarriers against high fluid shear forces.45,46 The attachment and proliferation of ADSCs within microcarriers were examined at 1 day, 7 days and 14 days after seeding. The confocal microscopic examination demonstrated that the ADSCs attached on the external surface or within the internal cavities of both the PBLG and Sr10-HA-g-PBLG microcarriers after 1 day of culture. Then cells bagan to penetrate into the interior of the porous microcarriers and occupy most of the pore surfaces (Figure 7a). For Sr10-HA-g-PBLG microcarriers, labeled cells were detected at 1/8, 2/8, 3/8 and 4/8 diameter depth by confocal microscopic observation, respectively (Figure 7b), further illustrating the infiltration and proliferation of the ADSCs in the innermost regions of the microcarriers. Figure 7c shows relative DNA content in the microcarriers, which indicated the continue proliferation of the cells onto the porous microcarriers within 14d of cultivation. At 1 day and 7 days, the DNA content in Sr10-HA-g-PBLG microcarriers was much higher than that in PBLG microcarriers, indicating the incorporation of Sr10-HA nanoparicles was beneficial for cellular proliferation at the beginning of the culture stage. However, the difference in the DNA content was no longer obvious after 14 days. It was reported that Sr2+ ions displayed the ability to stimulate the alkaline phosphatase (ALP) activity.47 As expected, the expression of ALP in ADSCs loaded in Sr10-HA-g-PBLG microcarriers was much higher than that growing in PBLG microcarriers at 7 days and 14 days post-seeding (Figure 7d), which revealed the existence of Sr10-HA and the release of Sr2+ promoted the osteogenic differentiation

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of ADSCs. 2.7. Repair of Femur Defect in Rabbits. The potential of Sr10-HA-g-PBLG microcarriers to promote bone repair was assessed by a critical bone defect in a rabbit model. As shown in Figure 8a, the operation was performed by direct injection of the microcarriers into the femur defects without additional surgical fixation. Micro-CT scans were performed at 3 months and 6 months after operation. As shown in Figure 8b, compared with the bare microcarriers group, the ADSCs-seeded Sr10-HA-g-PBLG microcarriers group showed some bone-like tissue formation. Figure 8c-8f and Figure 8c’-8f’ show the representative histological staining of the implanted cell-microcarrier constructs at 3 months and 6 months postimplantation. As shown in the H&E staining histology tissue images (Figure 8c, Figure 8e, Figure 8c’, and Figure 8e’), no acute inflammation was observed for both ADSCs-seeded Sr10-HA-g-PBLG microcarriers group and the bare microcarriers group, indicating good biocompatibility of the microcarriers.48 In the bare microcarriers group, at both 3 months and 6 months, there was no obvious osteogenesis (Figure 8c and Figure 8c’). Appreciably different from the bare microcarriers group, osteoblasts uniformly distributed in the new born bone in ADSCs-seeded Sr10-HA-g-PBLG microcarriers group. H&E staining also showed the formation of bone marrow tissue, as well as neo-generated bone. The new bone formation appeared to have an obvious upward trend in the ADSCs-seeded Sr10-HA-g-PBLG microcarriers group, and the area of new bones at 6 months was much larger than that at 3 months after implantation (Figure

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8e and Figure 8e’). Masson staining histology tissue images were presented in Figure 8d, Figure 8f, Figure 8d’ and Figure 8f’. Both the ADSCs-seeded Sr10-HA-g-PBLG microcarriers group and the bare microcarriers group showed a mixture of blue and red regions, indicating the deposition and mineralization of bone collagen. Compared with the bare microcarriers group (Figure 8d and Figure 8d’), the ADSCs-seeded Sr10-HA-g-PBLG microcarriers group displayed more intensive positive red staining, thus reveling that the extent of mineralization on ADSCs-seeded Sr10-HA-g-PBLG microcarriers was more than that on bare microcarriers.49,50 The area of red staining in ADSCs-seeded Sr10-HA-g-PBLG microcarriers group at 6 months was much larger than that at 3 months after implantation, also revealing the increasing bone mineralization with time (Figure 8f and Figure 8f’).

3. CONCLUSIONS Sr10-HA-g-PBLG hybrid nanocomposites with controllable grafting ratio and the molecular weight of the grafted PBLG were synthesized. And the Sr10-HA-g-PBLG porous microcarriers were fabricated. Well controllable Sr2+ release and degradation rate were achieved by adjusting the grafting ratio of nanocomposite. The Sr10-HA-g-PBLG microcarriers allowed in-growth and distribution of cells, and promoted the osteogenic differentiation of ADSCs in vitro. In vivo studies showed that the ADSCs-seeded Sr10-HA-g-PBLG microcarriers successfully repaired the femoral defect. These results demonstrate that the porous Sr10-HA-g-PBLG

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microcarriers possess great potential as an injectable vehicle for bone tissue engineering.

4. EXPERIMENTAL SECTION 4.1. Materials. All chemicals used were analytically pure and obtained from Sinopharm

Chemical

reagent

Co.,

Ltd.

(Shanghai,

China).

Dioxane

and

dichloromethane (CH2Cl2) were predried by refluxing over Na and CaH2, respectively. BLG-NCA was synthesized in our lab.51 Gelatin, polyvinyl alcohol (PVA), γ-benzyl-L-glutamate, 3-aminopropyltriethoxysilane (APTS), ammonia water, Ca(OH)2, Sr(OH)2 and all other chemicals were used without further purification. 4.2. Preperation and Amino-Functionality of Sr10-HA Nanoparticles. Sr10-HA nanoparticles, with Sr/(Ca + Sr) =10 mol %, were synthesized by coprecipitation method at 100oC for 24 h. H3PO4 aqueous solution (150ml, 0.20 mol/L) was added dropwise into 200ml Sr(OH)2 /Ca(OH)2 aqueous solution. The Sr/Ca molar ratio was set at 1/9. And the Ca/P was in the appropriate molar ratio to obtained the target Sr10-HA nanoparticles with (Ca + Sr)/P molar ratio of 1.67. The pH value was adjusted to about 11 by adding ammonia water. The sediment was kept still for 2 days and then washed for three times by deionized water. Finally, the precipitate was freeze dried overnight. Aminated modification of Sr10-HA nanoparticles was performed by using APTS. 2.21 g APTS was dissolved in 500ml of ethanol/water (1:9, v/v) mixed solvent. Then, the Sr10-HA nanoparticles (n(Sr10-HA)/ n(APTS)=1:2) were added into the mixed

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solvent and stirred for 3h. The pH value was adjusted to 11 ± 0.5 by addition of ammonia water. The mixture was stirred for 3h. The amino-functionalized Sr10-HA nanoparticles (Sr10-HA-NH2) were obtained by centrifugation followed by repeated washing. Finally, the Sr10-HA-NH2 nanoparticles were vacuum dried overnight. 4.3. Synthesis of Sr10-HA-g-PBLG Composite Nanoparticles. Sr10-HA-NH2 was used to initiate the surface grafting of PBLG. The typical recipe for the preparation of Sr10-HA-g-PBLG composite nanoparticles was shown in Table S1. Sr10-HA-NH2, BLG-NCA and the solvent were added into a 100mL dry flask. The synthesis procedure was performed under dry nitrogen atmosphere. After being ultrasonically treated for 15 minutes and stirred for 3 days at room temperature, the mixture was precipitated in ethanol, filtrated, washed with dioxane and vacuum dried. Fianally, the Sr10-HA-PBLG composite particles were obtained. 4.4. Preparation of Sr10-HA-g-PBLG Porous Microcarriers. Gelatin aqueous solution (3.5mL, 8 wt%) was added into Sr10-HA-g-PBLG/CH2Cl2 solution (0.07g of Sr10-HA-g-PBLG in 20mL of CH2Cl2) to prepare a water-in-oil (W/O) emulsion by using a homogenizer at 20,000 rpm for 30s. Then, the W/O emulsion was added into PVA aqueous solution (150mL, 0.1 wt%), the mixed solution was stirred at 500 rpm for 30s followed by being transferred to ice-water bath. After CH2Cl2 was volatilized, the microcarriers were transferred to warm water (50oC, 3h) to remove gelatin, leading to porous microcarriers after lyophilization. 4.5. Characterization of Sr10-HA-g-PBLG Composite Nanoparticles. The composition and surface aminization of Sr10-HA nanoparticles were confirmed by

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X-ray diffractometer (XRD) (18KW D/MAX2500V+/PC, Japan), Fourier transform infrared spectra (FTIR) (AVATAR 370, Nicolet, USA), Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) (PERKINE 7300DV, USA) and X-ray Photoelectron Spectroscopy (XPS) (VG Scientific Co., UK). The 1H NMR spectra of Sr10-HA-g-PBLG were characterized using an AV 500 NMR from Bruker. CF3COOD was used as solvent for all the samples. The morphology of the Sr10-HA nanoparticles and Sr10-HA-g-PBLG was observed via Transmission Electron Microscope (TEM) (200CX, Japan) and Atomic Force Microscope (AFM) (5550 AFM/SPM, Bruker Scientific, USA). The solution or suspension of the samples was dropped onto copper grids for TEM observation. The samples on the surface of the mica sheet for AFM observation were analyzed in tapping mode. The grafting ratio of Sr10-HA-g-PBLG composite nanoparticles was measured by TGA (Q500, TA Instruments, USA) and calculated according to previously reported method.52 The molecular weight of grafted PBLG chains obtained after dissolution of inorganic

core

of

Sr10-HA-g-PBLG

was

determined

by

Gel

permeation

chromatography (GPC) (Waters GPC e2695, USA).53 The colloidal stability of the samples was carried out by dispersing Sr10-HA, Sr10-HA-NH2, Sr10-HA-g-PBLG with different grafting ratios, Sr10-HA/PBLG mixture and Sr10-HA-NH2/PBLG mixture in CH2Cl2. For all the samples, the concentration of inorganic Sr10-HA was set at 0.7mg/mL. For the samples of Sr10-HA/PBLG mixture and Sr10-HA-NH2/PBLG mixture, the concentration of organic PBLG was set at

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2.8mg/mL, which was equal to the organic matter concentration of the Sr10-HA-g-PBLG sample with the grafting ratio of 80%. 4.6.

Characterization

of

Sr10-HA-g-PBLG

Porous

Microcarriers.

The

micromorphology of the porous microcarriers was observed by a Scanning Electron Microscopy (SEM) (Apollo 300, CamScan, UK) Surface element content and distribution for Sr10-HA-g-PBLG porous microcarriers was characterized by Energy Dispersive X-ray Spectroscopy (EDS). The mean particle size and distribution of the porous microcarriers dispersed in water were characterized using Malvern Laser Particle Size Analyzer (MASTERSIZER 2000, UK). The density and porosity of the porous microcarriers were measured according to the previously reported method.54-55 The rheological properties were performed on a rheometer (AR2000, TA Instruments, USA).56 Before testing, porous microcarrier (0.05g) were dispersed in 4ml of water to form a suspension by agitation for 1min. The relationship between storage modulus G’ and frequency were plotted. The compressive strength was measured via a Dynamic thermomechanical analysis (DMA Q800, Waters, USA). The crosshead speed was set at 0.5 mm/min. The test specimens (ф12mm) were prepared by spreading porous microcarriers aqueous suspension with the concentration of 12.5mg/mL on the bottom of the plastic cylindrical molds. Degradability of the dried samples of the microcarriers in phosphate-buffered

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saline (PBS) was determined gravimetrically.56 The micromorphology of the microcarriers during degradation was also observed by SEM. The Sr2+ and Ca2+ release profiles were measured by inductively-coupled plasma atomic emission spectrometry (ICP-AES, ICAP 6300, USA). 0.1g Sr10-HA-g-PBLG porous microcarriers were immersed in 5 mL of PBS, which was replaced every day by a fresh solution. The ion release was analyzed at different time points. 4.7. Seeding, Growth and Osteogenic of ADSCs within Microcarriers. Adipose-derived stem cells (ADSCs) were isolated from adipose tissue of New Zealand rabbits.57 All the microcarriers were sterilized with

60

Co γ irradiation,

followed by being suspended in culture medium and evenly mixed with the cells.11 The cell infiltration, proliferation and osteogenic activity were evaluated. The ADSCs were labeled with DiO pior to seeding.57 At 1 day, 7 days, and 14 days post-seeding, the labeled ADSCs/microcarriers construct was observed by confocal laser microscopy (Nikon Y-FL, Japan) to examine the cellular growth and spatial distribution. The cell numbers within the microcarriers were quantified by DNA assay as previously reported.58 An alkaline phosphatase (ALP) assay reagent kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) was applied to detect the osteogenic activity of cells in Sr10-HA-g-PBLG microcarriers and PBLG microcarriers, according to the procedure reported by Gao et al.59 4.8. In vivo Transplantation of ADSCs /Microcarrier Complex and Healing of Femur Defect. New Zealand rabbits (6 months, 4.0 ± 0.5 kg) were used in this study.

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The full-thickness segmental defects of the femur were created according to the procedure previously reported.60 The microcarriers incorporating ADSCs were injected into the defects created in the femoral condyle parallel to the joint surface. The rabbits injected with microcarriers without loading ADSCs served as control. The rabbits were euthanatized at 3 months and 6 months postimplantation. The implants extracted were subjected to Micro-computed tomography (μCT) scan (North Star Imaging, U.S.) and histological analysis (H&E, Masson’s Trichrome) to analyze the bone formation in the defect.49,50 4.9. Statistical Analysis. Statistical analysis was conducted using ANOVA. All the experimental data were expressed as means ± standard deviations (SD). Statistically significant differences was set at p ≤ 0.05.

ASSOCIATED CONTENT Supporting Information Expected and measured (by using ICP-AES) elemental composition (Ca/Sr ratios) of the samples; XRD pattern of HA and Sr10-HA; FT-IR Spectra of APTS, Sr10-HA and Sr10-HA-NH2; XPS spectra of Sr10-HA before and after aminated modification; The colloidal stability of Sr10-HA and Sr10-HA-g-PBLG; Typical recipes for the preparation of Sr10-HA-g-PBLG composite nanoparticles.

AUTHOR INFORMATION Corresponding Authors

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*E-mail: [email protected]. Tel.: +86-21-66138055. Fax:+86-21-66138069; *E-mail: [email protected]. Tel.: +86-21-57689506. Fax:+86-21-57689506. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The work was supported by the Science and Technology Commission of Shanghai Municipality (Grant No. 15JC1490400) and the National Science foundation of China (Nos. 51773113, 51473090, 81471798).

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Figure 1. Schematic illustration for the fabrication of porous Sr10-HA-g-PBLG microcarriers. (a) PBLG was grafted to amino-modified Sr10-HA nanoparticles surface via the ring opening polymerization of Ncarboxyanhydride. (b) A double emulsion was prepared by homogeneously mixing the Sr10-HA-g-PBLG dichloromethane solution and aqueous gelatin, followed by addition of the emulsion to aqueous PVA solution (1 wt%). The porous microcarriers were obtained by cooling the mixed solution, removing gelatin, and freeze-drying. Finally, adipose-derived stem cells (ADSCs) was seeded in microcarriers and injected into the femur defects of the rabbits to evaluate the new bone formation in vivo. 532x389mm (96 x 96 DPI)

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Figure 2. Synthesis of Sr10-HA-g-PBLG with controllable grafting ratio. (a) 1H NMR spectra of PBLG and Sr10-HA-g-PBLG with different grafting ratios. (b) XRD of PBLG, Sr10-HA and Sr10-HA-g-PBLG with different grafting ratios. (c) TGA curve of Sr10-HA, PBLG and Sr10-HA-g-PBLG with different grafting ratios. (d) Comparation between theoretical and measured grafting ratios and number-average molecular weight of grafted PBLG. 251x190mm (120 x 120 DPI)

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Figure 3. Micromorphology of Sr10-HA and Sr10-HA-g-PBLG nanoparticles. TEM micrographs of Sr10-HA (a) before and (b) after surface grafting. AFM images of (c, c’) Sr10-HA, (d, d’) PBLG and (e, e’) Sr10-HA-gPBLG. 206x305mm (96 x 96 DPI)

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Figure 4. The microstructure of Sr10-HA-g-PBLG porous microcarriers. The microscopic morphology and EDS images of hybrid microcarriers (the colours of green, red, blue and white corresponding to surface element distribution of Sr, Ca, P and C, respectively) with different grafting ratios: (a) 70%, (b) 80% and (c) 90%. (d) Particle distribution for Sr10-HA-g-PBLG porous microcarriers with different grafting ratios. (e) Porosity and density of Sr10-HA-g-PBLG porous microcarriers with different grafting ratios. 404x411mm (120 x 120 DPI)

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Figure 5. Mechanical strength of Sr10-HA-g-PBLG porous microcarriers. (a) Storage modulus of Sr10-HA-gPBLG porous microcarriers measured by Rheometer. (b) Compressive strength of Sr10-HA-g-PBLG porous microcarriers measured by DMA. 263x105mm (120 x 120 DPI)

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Figure 6. In vitro degradation and ion release. The morphological change in degradation process of Sr10-HAg-PBLG (grafting ratio: 80%) porous microcarriers was visually observed by SEM after degradation for (a) 0, (b) 4, (c) 8, (d) 12 and (e) 18 weeks. (f) The weight change for pure PBLG porous microsphere and Sr10HA-g-PBLG porous microcarriers with different grafting ratios. Ion release from Sr10-HA-g-PBLG microcarriers with different grafting ratios: the variation of (g) Ca2+ and Sr2+concentration and (h) Ca2+/Sr2+ molar ratio with incubation time in PBS solution. 504x751mm (96 x 96 DPI)

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Figure 7. Proliferation and osteogenic of ADSCs within the microcarriers. (a) Confocal fluorescence images of ADSCs in Sr10-HA-g-PBLG porous microcarriers at 1 day, 7 days and 14 days post-seeding. (b) Confocal microscope images showed that ADSCs could infiltrate into the innermost regions of microcarriers. (c) The number of cells on microcarriers at 1 day, 7 days and 14 days post-seeding (*p ≤ 0.05, n = 3 for each group). (d) ALP activity of ADSCs within the microcarriers after culture in vitro for 1 week and 2 weeks (*p ≤ 0.05, n = 3 for each group).

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Figure 8. Repair of femur defect in rabbits by using the bare microcarriers and ADSCs-seeded Sr10-HA-gPBLG microcarriers. (a) Surgical procedure: (1) defect created on the femur bone, (2) during and (3) after injection of microcarriers into the bone defect. (b) Appearance and Micro-computed tomography for the repaired femora at 3 months and 6 months post-implantation. Representative histological sections of tissues repaired by the (c, d, c’, d’) bare microcarriers and (e, f, e’, f’) ADSCs-seeded Sr10-HA-g-PBLG microcarriers at 3 months and 6 months post-implantation. Sections were stained with (c, e, c’, e’) H&E and (d, f, d’, f’) Masson’s trichrome to visualize bone formation. NB=new bone; BM = bone marrow. 119x122mm (300 x 300 DPI)

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