Sr-HA-graft-Poly(Î³-benzyl-l-glutamate) Nanocomposite Microcarriers

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Sr-HA-graft-poly(#-benzyl-L-glutamate) Nanocomposite Microcarriers: Controllable Sr2+ Release for Accelerating Osteogenenisis and Bony Nonunion Repair Long Gao, Zhongyue Huang, Shifeng Yan, Kunxi Zhang, Shenghua Xu, Guifei Li, Lei Cui, and Jingbo Yin Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01101 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on October 1, 2017

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Sr-HA-graft-poly(γ-benzyl-L-glutamate) Nanocomposite Microcarriers: Controllable Sr2+ Release for Accelerating Osteogenenisis and Bony Nonunion Repair Long Gao†, Zhongyue Huang‡, Shifeng Yan†, Kunxi Zhang†, Shenghua Xu†,Guifei Li†, Lei Cui,*,§, ǁ and Jingbo Yin*,† †

Department of Polymer Materials, Shanghai University, Shanghai 200444, People’s Republic

of China. ‡

§

Minhang Hospital, Fudan University, 200119, Shanghai, China. Department of Regenerative Medicine, Tong Ji University School of Medicine, Shanghai

200092, People’s Republic of China ǁ

Department of plastic surgery, Beijing shijitan hospital, capital medical university, Beijing

100038, People’s Republic of China. *E-mail: [email protected] (J. Yin), Tel +86-21-66138055, Fax +86-21-66138069 [email protected] (L. Cui),Tel +86-21-57689506, Fax +86-21-57689506 Keywords: Poly(γ-benzyl-L-glutamate), Porous microcarriers, Strontium ions, Osteoinduction, Nonunion

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Abstract The microcarrier system offers an attractive method for cellular amplification and phenotype enhancement in the field of bone tissue engineering. However, it remains a challenge to fabricate porous microcarriers with osteoinductive activity for speedy and high-quality osseointegration in regeneration of serious complication of bone fracture, like nonunion. Here, we present a facile method for the first time manufacture microcarriers with osteogenic effects and properties based on well controlled and long-term Sr2+ release. At first, Strontium substituted hydroxyapatite was prepared (Sr-HA) and a novel Sr-HA-graft-poly(γ-benzyl-L-glutamate) (Sr-HA-PBLG) nanocomposite was synthesized. Then, the microcarriers with highly interconnected macro-pores were fabricated by a double eumulsion method, which allow cells to adhere and proliferate, and secrete extracellular matrix. Besides, the microcarriers with a relatively uniform diameter of 271.5±45.0 µm are feasible for injection. The Sr-HA-PBLG microcarriers efficiently promoted osteogenic gene expression in vitro. With injection of the Sr-HA-PBLG microcarriers loading adipose derived stem cells (ADSCs) into the nonunion sites, bone regeneration was observed at 8 weeks after injection in a mice model. 1. Introduction Bone tissue engineering1-2 is a powerful approach to treat serious complications of bone fracture, like nonunion. A major problem in bone tissue engineering is the availability of a sufficient number of cells with the appropriate phenotype for damaged bone3-5. The microcarrier system is employed for cellular amplification and phenotype enhancement6-7. It has been used to fill bone defects8-10, such as the trabecular bone11 and periodontal pocket12. As a micro-scaffold, the porous microcarrier possesses a highly interconnected structure, which facilitates mass transfer of oxygen and nutrients, thus provides an ideal environment13-14. Besides, it can be applied as an


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injectable substance to fit the shape of bone defects and be easily entrapped by the bone interstices after transplantation15. At the same time, as an injectable system, the operative time and cost can be reduced16. Furthermore, it will be much more efficient for bone regeneration if the microcarriers possess the stable function to stimulate specific cellular responses and activate genes that trigger cellular differentiation. The mineral component of bone is similar to hydroxyapatite (HA) but contains other ions which are important for biological apatites17. Strontium (Sr), a trace element in human body, is considered to be a bone-seeking element that positively influences the growth of bone, and decreases the resorption of bone18-20. It is found that Sr has the capability to enhance the osteogenic differentiation for the stem cells via activating Wnt/β-catenin pathway11. It should be mentioned that an excessively high dose may induce toxicity, resulting in negative influence for the bone mineralization17, 21-22. However, low dose of Sr may be beneficial for bone formation1820, 23

. So far, many composite materials have been designed to control the release of Sr2+, such as,

incorporating Sr-containing nanoparticles (NPs) to the hydrogels by mixing23-24, in situ mineralization, and loading Sr2+ in titania nanotube arrays on titanium substrates19. However, these strategies share the limitation that there are no covalent bonds between the inorganic NPs and the polymer matrix25-27. This may imply an excessive amount of Sr2+ released from the polymer matrix to the external environment, leading to potentially toxic effects on the host tissue28. Especially for microcarriers in the 3-D suspension culture system, which require constant agitation to maintain in suspension, inorganic particles with no strong interfacial adhesion with the polymer matrix may easily fall off, and the release of Sr2+ is not stable and sustained. Therefore, it is quite necessary to form a covalent between polymer and NPs.


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In this study, we experimentally demonstrate a well-defined hybrid material based on Srsubstituted HA (Sr-HA) with a covalently attached poly(γ-benzyl-L-glutamate) (PBLG) shell. Sr-HA is surface-modified by using γ-aminopropyltriethoxysilane (APS). Sr-HA-PBLG is then obtained by ring opening polymerization (ROP) of γ-benzyl-Lglutamate N-carboxyanhydride (BLG-NCA). Sr-HA-PBLG porous microcarriers are then fabricated not only to serve as an injectable cell vehicle, but also create a bioactive microniche that could stimulate the differentiation and proliferation of osteoblasts. The effect of osteogenic gene expression of adipose derived stem cells growing in Sr-HA-PBLG microcarriers is also detected. Finally, cellseeded Sr-HA-PBLG microcarriers were injected into nonunion defects of mice femur to evaluate the new bone formation in vivo. 2. Results and discussion 2.1 Synthesis of Sr-HA-PBLG with gradient grafting rate The direct covalent conjugation of polypeptides and their derivatives to inorganic NPs are promising for the design of novel hybrid materials. With combined properties of both components , a bioactive core may stimulate the differentiation of cells by releasing ions, and the polypeptide shell may provide the system with biocompatibility, solution stability and possibility of multi-site chemical modification. PBLG becomes an interested point for its biocompatibility28, significant functionality25, and unique secondary structures29. And long-chain PBLG is also readily prepared by "graft-from” strategy27. Here, a novel nanocomposite is synthesized through covalent surface functionalization of Sr-HA by long-chain PBLG. The surface functionalization of Sr-HA is treated by APS to endow Sr-HA surface with -NH2 groups at first. The -NH2 groups at the end of APS chains are employed to


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further initiate ring opening polymerization (ROP) of BLG-NCA. The reaction between Sr-HA and APS is monitored by XPS and the result is listed in Figure2(a). The peaks at the binding energy of 532.8 and 131.9 eV should be attributed to O1s and Sr3p, respectively. The presence of Si2p and N1s signals in the XPS of amino-functionalized Sr-HA ( Sr-HA-NH2 ) confirms the existence of amino groups on the surface of Sr-HA particles. After polymerization, Sr-HA-PBLG composite NPs are obtained. 1H NMR spectra of Sr-HAPBLG and PBLG are shown in Figure2b. Compared with the 1H NMR spectra of PBLG, it is clear that the incorporated amino group successfully initiates the ROP of BLG-NCA, and NMR results confirm the coating on Sr-HA surfaces is PBLG. The grafting rate is defined as the mass ratio of grafted polymer to entire composite particle. The grafting rate of Sr-HA-PBLG particles is measured by TGA. Results show that, Sr-HA-PBLG composite particles with the improvement of grafting rate lead to greater weight loss. It could be confirmed that ROP of BLG-NCA initiated by small primary amines proceeds as a living polymerizations. The grafting rate calculated from the TGA data is close to the estimated [M]/[I] ratio ( [M] and [I] was the concentration of BLG-NCA and Sr-HA-NH2), the error is less than 5%. Therefore, the grafting rate of Sr-HA-PBLG NPs could be modulated by varying the initial ratio of BLG-NCA to Sr-HA-NH2. In order to verify which one of the longer PBLG chains and the more polymer chains contribute to the increasing grafting rate, the molecular weight of PBLG shell is measured. As showed in Figure2e, with the increase of Sr-HA-PBLG grafting rate, molecular weight is improved. There is a linear relationship between molecular weight and [M]/[I] ratio, with R2=0.982. Therefore, the increasing grafting rate is due to longer PBLG chains.


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XRD patterns of PBLG, Sr-HA, and Sr-HA-PBLG NPs are shown in Figure2(f). According to the Joint Committee on Powder Diffraction Standards (JCPDS) database (74-0565, 34-0476, 340479, 34-0482, and 33-1348), Sr-HA possesses the hexagonal crystal structure. However, the angles of the Sr-HA peaks are slightly smaller than those of HA, which should be attributed to the larger lattice constant of Sr10(PO4)6(OH)2. And the wide dispersion peak at 2θ of 22-25° should be attributed to PBLG. This dispersion peak is gradually enhanced while Sr-HA characteristic peaks are weakened due to the increase of PBLG content and reduce of Sr-HA content. Besides, results show that the polymerization induces no change in crystalline phase of Sr-HA nanocrystals, and formes no secondary phase. 2.2 Morphology and colloidal stability of Sr-HA-PBLG NPs Figure3(a) shows the TEM images of Sr-HA and Sr-HA-PBLG NPs. Sr-HA NPs are of nanorod shape and aggregate easily. PBLG brushes are observed on the surface of Sr-HA-PBLG and the nanoparticles are well dispersed. An apparent core-shell structure of Sr-HA-PBLG NPs could also be observed by AFM, as shown in Figure3(b), where the red color section is Sr-HA core and the dim coating part was PBLG shell. At a three-dimensional (3D) image, several separated elliptical NPs are scanned clearly. Moreover, a top view image and a height profile are also presented, and the mountain-shaped NPs are about 250 nm in length and 45 nm in height. At the same time, the XPS results (Figure2a) show that, after polymerization, Sr element signal is hardly detected on the surface of Sr-HA-PBLG composite NPs, indicating that the Sr-HA is completely coated by PBLG shell. NPs tend to aggregate to form microscale clusters because of inter-particle Van der Waals force and the hydrogen bonding between surface hydroxyl groups30-31. For instance, Sr-HA NPs with


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poor colloidal stability tend to precipitate from the solvents. Methods to prepare polymerinorganic composites are based on solution process. For this reason, the improvement of colloidal stability and dispersion property of Sr-HA in solution is an important issue. Monitoring of time-dependent colloidal stability of Sr-HA NPs and Sr-HA-PBLG NPs in methylene chloride (CH2Cl2) is illustrated in Figure3(c). The colloidal stability is significantly improved with the increase of grafting rate. In fact, Sr-HA was unstable and would precipitate from CH2Cl2 within several minutes. For Sr-HA-PBLG whose grafting rate is relatively low, the colloid stability increases slightly, which indicated that low grafting rate could not efficiently overcome the interparticle interaction. Especially, Sr-HA-PBLG with the highest grafting rate shows excellent colloidal stability, and keeps stable for more than half a month. 2.3 Morphology of Sr-HA-PBLG microcarriers and Sr distribution As a kind of microscaffold, porous microcarriers have large pores with a highly interconnected structure, which facilitate the effective mass transfer of oxygen and nutrients, thus provide an ideal environment6, 14, 32. As shown in Figure4, the Sr-HA-PBLG porous microcarriers with the diameter of about 200 µm present high interconnected macropores with a size range of 40–60 µm. Besides, a homogeneous distribution of Sr-HA NPs in microcarrier is a key issue to realize. The Energy Dispersive X-ray Spectroscopy (EDS) maps of the Sr-HA-PBLG hybrid microcarriers show a uniform spatial distribution of the strontium signals. These results exhibit that Sr-HA NPs are uniformly dispersed in the porous microcarriers matrix. 2.4 Structure and properties of microcarriers Several factors are crucial for the successful application of microcarriers in tissue engineering. Firstly, the diameter of microcarrier has a bearing on the number of attached cells. As reported,


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the microcarriers with diameters in the range of 100~400µm are suitable for cell adhesion6. The as-obtained microcarriers show a relatively uniform diameter of 271.5±45.0 µm (Figure5a), which are feasible for injection. Secondly, the microporous structure significantly affects cellular distribution, growth and organization especially in practical tissue engineering. Sung et al33 demonstrated that microcarriers with pore size of 40-60 µm resulted in more cells infiltrating into the interior region. The as-obtained porous microcarriers exhibited a highly interconnected porous structure. As shown in Figure5b, Sr-HA-PBLG microcarriers with grafting rate of 50, 60, 70, 80 and 90% showed the porosity of 63.3±7.6, 77.2±6.3, 89.0±6.2, 81.2±6.6, 82.4±5.0, respectively. And average pore diameters of 30.1±7.3, 37.3±7.6, 45.4±6.7, 50.3±7.5 and 52.3±7.2 µm, respectively (Figure5c). For engineered microcarriers, large pores and high interconnectivity could actively enhance the diffusion of oxygen and nutrients, contributing to in-growth and cell distribution in the constructs32, 34. Furthermore, compared with the culture medium (typical density 1.02~1.10), the microcarriers are suggested to have a higher specific overall density for the objective of retaining them in gentle-agitation suppression6. As shown in Figure5d, the overall density of SrHA-PBLG microcarriers with the grafting rate of 50, 60, 70, 80 and 90% are 1.082, 1.071, 1.057, 1.047and 1.035 g /mL, respectively. 2.5 Degradation of microcarriers and Sr2+ release Cell-seeded microcarriers used for direct bone defect site delivery should keep an ideal biodegradation rate for vivo longevity. The in vitro degradation of porous microcarriers is shown in Figure6. The Sr-HA-PBLG microcarriers with different grafting rates show different degradation rate in PBS. More quantitatively, the remaining weight (Figure6a) of Sr-HA-PBLG


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microcarriers with grafting rate of 50, 60, 70, 80 and 90% are 65.0, 69.2, 73.4, 74.2 and 81.8% respectively. Weight loss of Sr-HA-PBLG porous microcarriers is mainly due to the degradation of PBLG and the loss of Sr-HA NPs separated from PBLG shell. The degradation of PBLG is determined by molecular weight at the same conditions28. With the improvement of grafting rate, molecular weight of PBLG increases, therefore the degradation of high grafting rate Sr-HAPBLG needs longer time. Therefore, we can adjust the Sr-HA-PBLG grafting rate to enable the degradation rate of microcarriers to match the bone regeneration. Through the SEM, we can clearly observe the morphology and structure of microcarriers degrade in vitro at the indicated time point (Figure6c). Porous microspheres structure disappeared after 12 weeks. With the degradation of the microcarriers, the Sr2+ is released, which is also closely related to the Sr-HA-PBLG grafting rate. The release kinetics of Sr2+ is evaluated through immersing the SrHA-PBLG microcarriers in PBS for 8 weeks, and the result is exhibited in Figure6b. And the released Sr2+ amounts decreased with the increase of grafting rate. The average Sr2+ amounts released from Sr-HA-PBLG microcarriers with grafting rate of 50, 60, 70, 80 and 90% are 210ppm/day. No sample shows burst release at the first day. Results show that a higher of Sr-HAPBLG grafting rate can improve the effect of controlled release. Higher grafting rate results in thicker PBLG shell and slower Sr2+ release. At the same time, with higher grafting rate, the content of the Sr-HA also reduces, which is another a factor affecting Sr2+ release. Through surface PBLG grafting, Sr2+ release is well controlled, which is crucial for its application in bone tissue engineering. 2.6 Cell proliferation in vitro and osteogenesis gene expression


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A major problem in bone tissue engineering is to deliver sufficient cells to damaged tissue to participate in bone regeneration. Microcarriers provide a large surface area for cell adhesion during propagation6 Additionally, it is reported that macroporous microcarriers are effective to stimulate differentiation of cells seeded within them32, 34and enhance mineralization35. Although the exact mechanism of enhancing redifferentiation has not been explained, the approach is promising for bone engineering. As shown in Figure7a, the Sr-HA-PBLG microcarriers (with grafting rate 90%) supported DiOlabelled ADSCs attachment well at the 1st and 3rd days. With the increase of culture time at the 5th day, cells were observed to penetrate into the inner area and occupy most pore surfaces of microcarriers. Number of cells kept growing during cultivation, reached 1.78×106 /30mg microcarriers at the 14th day (Figure7b). Furthermore, labeled cells were detected at depths of 50, 75, 100 and 125 µm, respectively (Figure7c). More importantly, as shown in Figure8, RT-PCR analyses reveal that expression of ALP, Runx2, COL I, and OCN in ADSCs loaded in Sr-HA-PBLG microcarriers (with grafting rate 90%) are significantly higher than those growing in PBLG microcarriers at the 14th day after seeding. These results represent that the release of Sr2+ from microcarriers enhances the osteogenic differentiation of ADSCs seeded. 2.7 Repair of nonunion defect of femur in mice Nonunion is a severe complication of bone fracture36. Although the exact causes of delayed union and nonunion are unknown, many treatments have been attempted in repairing the bony nonunion37-38. However, most of these intervention have been reported to be associated with morbidities, finite supply, failure therapy and cost38.


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In this study, we injected ADSCs-seeded Sr-HA-PBLG microcarriers (with grafting rate 90%) into nonunion defect to evaluate its promotive effect in new bone formation, mice injected with ADSCs-seeded PBLG microcarriers were served as the control group. As shown in Figure9a-b, with our modified method, the nonunion model is successfully created in middle segment of femur in mice. After 8 weeks of injecting of ADSCs harbored Sr-HA-PBLG microcarriers, micro-CT examination revealed that regenerated bone exhibited similar callus structure as that in normal controls. As determined quantitatively by µCT scan, the ratio of bone volume (BV) /tissue volume (TV) (Figure9c) in the ADSCs/Sr-HA-PBLG group showed no significance with that in normal bone, which was much higher than that in control group. Histological examination further demonstrated that nonunion region was occupied with fibrous tissue in control group. Whereas, the gaps between fracture ends were filled with neo-generated calluses in ADSCs-seeded Sr-HA-PBLG microcarriers treated mice. 3. Conclusion Sr-HA-PBLG composite NPs with gradient grafting rate and porous microcarriers have been fabricated. Well controllable Sr2+ release is observed from Sr-HA-PBLG microcarriers by adjusting the grafting rate of nanocomposite. Cells could infiltrate and survive in the inner regions of the microcarriers, and the release of Sr2+ could enhance the osteogenic differentiation of ADSCs. The injected specimens of cell-seeded Sr-HA-PBLG microcarriers resulted in new bone-like tissue formation in vivo at the 8th weeks after injection. This work explored a novel approach for bone tissue engineering by developing injectable microcarriers with osteoinductive capacity to treat bony nonunion problems. 4. Experimental Section


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4.1. Materials Dioxane, dichloromethane purchased from Sinopharm Chemical reagent Co., Ltd. were dried by refluxing with Na and CaH2, respectively. BLG-NCA was synthesized according to literature procedure.γ-benzyl-L-glutamate, APS and Strontium hydroxide (Sr(OH)2) were of the analytical grade and used without further purification. 4.2. Synthesis of Sr-HA-PBLG 4.2.1 Preparation of Sr-HA NPs Sr-HA(Sr10(PO4)6(OH)2) NPs were synthesized using a chemical precipitation technique at 70℃ for 3 hours by adding H3PO4 solution drop-wise into Sr(OH)2 solution with Sr/P ratio of 1.67 (equal to the ratio of Ca/P of HA). Ammonium hydroxide solution was adopted for keeping titration-period pH at 10.5±0.5. Wash the precipitate with water and ethanol for 3 times, and dry it in vacuum oven at 45℃ overnight. 4.2.2 Amino-functionality of Sr-HA NPs 0.01 mol APS was added into an aqueous alcohol solution containing 50mL of deionized water and 450mL of alcohol. The mixture was stirred at room temperature for 30 minutes and then 5.0g Sr-HA was added. After stirring for another 3 hours, the pH of the mixture was adjusted to 9-10 with ammonium hydroxide, and then the reaction was continued for additional 3 hours. After washing, filtration and freeze-drying, Sr-HA-NH2 NPs were obtained. 4.2.3 Synthesis of Sr-HA-PBLG Composite NPs


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Sr-HA-PBLG NPs were synthesized by a Grafting-From Approach. Sr-HA-NH2 wre used as solid initiator, and a general surface-initiated ROP procedure for synthesizing Sr-HA-PBLG NPs. Add Sr-HA-NH2 solid initiator, BLG-NCA and anhydrous dioxane to a 100mL dry eggplant-shaped flask in which the air was replaced with water-free nitrogen for five times. The mixture was ultrasonically treated for 30 minutes and then stirred for 3 days. After precipitating in ethanol, filtration, and washing with dioxane, Sr-HA-PBLG composite particles were obtained. 4.2.4 Characterization of Sr-HA-PBLG Composite NPs The surface chemical composition of Sr-HA and functionalized Sr-HA NPs were determined by X-ray photoelectron spectroscopy (XPS), using a Thermo ESCALAB 250 (VG Scientific Co., UK) . The morphology of the Sr-HA-PBLG was investigated by transmission electron microscopy (TEM) and atomic force microscope (AFM). TEM observation was performed on a JEOL JEM200CX (Tokyo, Japan), operating at an acceleration voltage of 120kV. A drop of Sr-HA or SrHA-PBLG solution in methylene chloride (0.5g/L) was placed onto a 200-mesh copper grid coated with carbon. The samples were air-dried before measurement. AFM image was obtained by AFM (5500 AFM/SPM, Agilent, USA) in tapping mode. Sr-HA/Sr-HA-PBLG NPs were dispersed in methylene chloride (0.05 g/L), then dropped onto surface of the mica sheet. After the solvent was evaporated, the surface morphology of the films was analyzed. X-ray diffraction (XRD) patterns of the nanocomposite were obtained on a Rigaku D/MAX2550 diffractometer equipped with Cu Kα radiation and a curved crystal graphite monochomator operating at a voltage of 40 kV and 30 mA.


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The thermal gravimetric analysis (TGA) was monitored by thermogravimetry (TA Q-500 ,TA instruments, USA) from 20℃ to 750℃ with a heating rate of 10℃·min-1 under nitrogen atmosphere. And the grafting rate was calculated as follows:

×100%

where RSr-HA , RPBLG and R are the weight-loss ratio of Sr-HA, PBLG and Sr-HA-PBLG samples from 200℃ to 700℃ measured by TGA analysis, respectively. Number-average molecular weight of PBLG grafted on Sr-HA was measured by Gel permeation chromatography (Waters GPC e2695, Waters, Co., USA). The grafted PBLG was stripped from the Sr-HA cores in a rational method. Firstly, the Sr-HA-PBLG NPs were dissolved in dichloroacetic acid (DCA) and then precipitated by abundant alcohol after full etching. Finally, Sr-HA-PBLG were obtained through washing, filtration, and vacuum drying. 4.3 Formation of Microcarriers 4.3.1 Preparation of the microcarriers At first, 0.2gram Sr-HA-PBLG was dissolved in dichloromethane (20mL) followed by gelatin solution (3.5mL, 6.5 wt%). And then, the mixed solution was emulsified with a homogenizer at 20,000 rpm for 2 minutes. Subsequently, microcarriers was prepared by adding the water-in-oil emulsion to aqueous PVA solution (1 wt%) while being stirred with a cylindrical magnetic dipole at 500 rpm for 60s. After the anhydrous dichloromethane was volatized, microcarriers were transferred to water at 50℃ for 3hours to remove gelatin and porous microcarriers were obtained after freeze-drying.


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4.3.2 Characterization of Sr-HA-PBLG porous microcarriers Surface morphology of the microcarriers was observed using a thermal field emission scanning electron microscopy (Apollo 300, CamScan, UK). The diameter and pore diameters of microcarriers were measured SEM images of 300 microcarriers by Image J soft program (National Institutes of Health). The porosity of microcarriers was obtained by a mercury porosimeter (Pore-Master-60, USA). The distribution of the strontium element was measured by Energy Dispersive X-ray Spectroscopy (EDS) maps. The overall density of the microcarriers was determined by the equation ρ=4m/(πd2h), where m is the mass, d is the diameter, and h is the thickness of the microcarrier aggregation in a disc shaped container. 4.3.3 In vitro degradation of microcarriers and Sr2+ release At first, Sr-HA-PBLG porous microcarriers (100.0 mg) were immersed in 50mL PBS (0.1M, pH 7.4) and in an orbital shaker at 37℃. The PBS was renewed every two days. At indicated time point, microcarriers were taken out and dried to constant weight. The remaining weight of the microcarriers was carefully gravimetrically. At the same time, The solution containing released Sr2+ was analyzed by inductively-coupled plasma atomic emission spectrometry (ICP-AES, ICAP 6300, USA). This process last for 8 weeks to generate solutions at indicated time points in order to determine the Sr2+ release time profile. 4.4 Seeding, growth and osteogenic of ADSCs within microcarriers The animal experiment was approved by the Animal Care and Experiment Committee of Tongji University School of Medicine. Subcutaneous adipose tissue was harvested from groin of 8-


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week-old C57 mice. Samples were cut into small pieces and followed by digestion with 0.075% type I collagenase (Washington Biochemical Corp., USA) at 37 ℃ for 30 minnutes. After centrifugation at bottom were resuspended and cultured in low glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in 100 mm diameter culture dishes (Falcon, USA) at 37℃ in a 5% CO2 atmosphere. When subconfluence reached, cells were passaged with trysinization (0.05% trypsin, 0.02% EDTA), and ADSCs prior to passage 3 were used in the following study. Before seeding, the microcarriers were sterilized by

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Co γ irradiation at 5 mrad. Microcarriers

(30mg) suspended in 10ml culture medium was mixed with 5×106 ADSCs in flasks which was subjected to beshaken at 75 rpm for 6 h in an incubator. At the1st,3rd,5th,7th and 14th days postseeding, cell numbers within the microcarriers were quantified by Hoechst 33258 dye DNA assay as previously reported28. ADSCs were labeled before seeding with fluorescent 3,30-dioc tadecyloxacarbocyanine perchlorate (DiO) dye (Molecular Probes, USA) at 37°C for 20 min and then mixed with the microcarriers as described above. Cells growth and distribution within the microcarriers were observed by confocal laser microscopy (Nikon Y-FL, Japan) 1, 3, 5, and 7days after seeding. Expression of osteogenic specific markers including ALP, Runx-2, COL I and OCN by ADSCs within microcarriers was determined by real-time PCR analysis. Briefly, total RNA was extracted from cells using an RNAprep Micro Kit (TianGen Biotech, Beijing, People’s Republic of China). After determination of RNA concentration, and cDNA synthesis, Real-time PCR was carried using a quantitative real time amplification system (MxPro-Mx3000P, Stratagene, La Jolla, CA).


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Relative expression levels for each gene of interest were calculated by normalizing the quantified cDNA transcript level (cycle threshold) to that of β-actin. 4.5 Healing of nonunion bone defect with injection of ADSCs /microcarrier complex C57BL6/J mice with a mean age of 10 weeks and average weight of 24g were used in this study. Nonunion of femoral fractures were induced with modification to previous reports39. Under anesthetization with intraperitoneal injection of 400 mg/kg chloral hydrate, the femur patella was dislocated laterally and , a flattened 24 G needle was inserted from the trochlear groove into the femoral canaza. A 2mm critical-sized defect was created in middle femur with periosteum around the fracture site was cauterized. To maintain the nonunion space of defect, a customized U-shape clip made of 27G needle in length of 8mm was inserted through medullary cavity. At the 4th week after surgery, nonunion was examined by X-ray imagination. Mice developed nonunion received injection of microcarriers loaded with ADSCs(1×106) in 50µl PBS. Mice injected with PBLG microcarrieers loading by ADSCs were servered as the control group. 8 weeks after injection, micro-computed tomography (µCT) scan (Bruker micro CT, Belgium) were performed. Bone samples containing nonunion segment were fixed, decalcified and subjected to with hematoxylin-eosin (H&E) staining. 4.6 Statistical analysis The experimental data from all the studies are expressed as means ± standard deviations (SD). Single factor analysis for variance (ANOVA) was used to assess the statistical significance of the results. To test the significance of observed differences between the study groups, an unpaired Student’s t-test was applied. Statistical significance was set at P ≤ 0.05.


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Long Gao carried out Sr-HA-PBLG synthesis, fabrication of microcarriers and structural characterization. Zhongyue Huang carried out the cell culture, animal studies and tissue analyses. Guifei Li carried out the γ-benzyl-Lglutamate Ncarboxyanhydride synthesis. Shenghua Xu carried out fabrication of PBLG microcarriers. Shifeng Yan and Kunxi Zhang contributed to assistance of data analysis. Jingbo Yin, Lei Cui were responsible for the overall project design and manuscript organization. Notes The authors declare no competing financial interest. Acknowledgments Funding for this study was provided by the Science and Technology Commission of Shanghai Municipality (No.15JC1490400), the National Natural Science Foundation of China (No. 51373094,81471798, 51473090) and Beijing Yangfan Project (XMLX201611 ) . References 1.

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TOC Graphic


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Figure.1 Schematic illustration for fabricating porous Sr-HA-PBLG microcarriers. At first, SrHA was amino-modified by APS, and PBLG was covalently bonded to Sr-HA nanoparticle surface through the ROP of BLG-NCA. Next, the double emulsion was prepared by emulsifying the mixed solution of Sr-HA-PBLG dichloromethane solution and aqueous gelatin using a homogenizer. Then, porous microcarriers was obtained by addition of the emulsion to aqueous PVA solution (1 wt%) while being stirred at 500 rpm for 60s and then remove the gelatin. Finally, adipose-derived stem cells (ADSCs) was seeded in microcarriers and injected into nonunion sites on the femur of rats to evaluate the new bone formation in vivo.


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Figure.2 Synthesis and characterization of Sr-HA-PBLG. (a) The XPS spectra of Sr-HA, Sr-HANH2, Sr-HA-PBLG. (b) 1H NMR spectra of Sr-HA-PBLG and PBLG (c) TGA curve of Sr-HA, Sr-HA-PBLG and PBLG. (d) Estimated grafting rate and calculated value from the TGA data. (e) Number-average molecular weight of PBLG grafted on Sr-HA. (f) XRD pattern of Sr-HA, Sr-HA-PBLG and PBLG.


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Figure.3 Morphology and colloidal stability of Sr-HA-PBLG nanoparticles. (a) TEM images of Sr-HA and Sr-HA-PBLG. (b) AFM images of Sr-HA, Sr-HA-PBLG, a three-dimensional perspective image and a height profile along the direction marked above. (c) Monitoring of timedependent colloidal stability of Sr-HA and Sr-HA-PBLG nanoparticals with grafting rate of 50, 60, 70, 80, 90% from left to right.


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Figure.4 SEM images of the Sr-HA-PBLG (grafting rate：50,60,70,80,90% from the bottom up ) and EDS images of hybrid microcarriers (green spots, red spots, white spots and blue spots were corresponded to the strontium, phosphorus, carbon and oxygen signal, respectively)


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Figure.5 Structure and properties of microcarriers. (a ) diameter , (b)porosity, (c) pore diameters and (d) overall density of Sr-HA-PBLG microcarriers with grafting rate of 50, 60, 70, 80 and 90%.


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Figure.6 Degradation of microcarriers and Sr2+ release. Incubated in PBS at 37℃ at the indicated time point: (a) the remaining weight of gradient grafting rate Sr-HA-PBLG porous microcarriers. (b) SEM images of degraded microcarriers. (c) Sr2+ release of microcarriers.


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Figure.7 (a) Confocal fluorescence images of ADSCs in Sr-HA-PBLG microcarriers at 1, 3, 5 and 7 days. And (b) the number of cells on microcarriers at 1, 3, 5, 7 and 14 days post-seeding (P < 0.01, n = 3 for each group). (c) SEM images of ADSCs in microcarriers at 5 and 7 days. (d) Confocal microscope images showed that ADSCs could infiltrate into the innermost regions of microcarriers. (The Sr-HA-PBLG used has a grafting rate of 90%.)


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Figure.8 The expression of osteogenic genes.(a) alkaline phosphatase (ALP).(b) Runx-2,(c) osteocalcin (OCN) and (d) type I collagen (COL I) after culture in vitro for 2 weeks. The values were quantified by real time PCR (The Sr-HA-PBLG used has a grafting rate of 90%. ***,P < 0.001, n = 3 for each group).


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Figure.9 Healing of bony nonunion. (a-b)Procedures for the femoral segmental defect model. After exposing the trochlear groove and inserting a 24 G needle in a retrograde manner, a 2mm critical-sized defect was created in the middle of the femur. (c)Quantitative measurement of bone volume(BV)/tissue volume (TV) at 8 weeks after local transplantation. (d,g)Histological evaluation at 8 weeks after local transplantation(HE staining). (e,f,h,i)Micro-computed tomography of bone nonunion at 8 weeks after local transplantation. (The Sr-HA-PBLG used has a grafting rate of 90%. **,P < 0.01, n = 5 for each group).


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Sr-HA-graft-Poly(Î³-benzyl-l-glutamate) Nanocomposite Microcarriers

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