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Biomaterials 174 (2018) 1e16

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Biomaterials

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Precisely controlled delivery of magnesium ions thru sponge-likemonodisperse PLGA/nano-MgO-alginate core-shell microspheredevice to enable in-situ bone regeneration

Zhengjie Lin a, b, Jun Wu b, Wei Qiao c, Ying Zhao d, Karen H.M. Wong a, Paul K. Chu e,Liming Bian f, i, Shuilin Wu g, Yufeng Zheng h, Kenneth M.C. Cheung a, Frankie Leung a, b,Kelvin W.K. Yeung a, b, i, *

a Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong, Chinab Shenzhen Key Laboratory for Innovative Technology in Orthopaedic Trauma, The University of Hong Kong Shenzhen Hospital, 1 Haiyuan 1st Road, FutianDistrict, Shenzhen, Chinac Dental Materials Science, Applied Oral Sciences, Faculty of Dentistry, The University of Hong Kong, 999077, Hong Kong, Chinad Centre for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Chinae Department of Physics, Department of Materials Science and Engineering, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, Chinaf Department of Mechanical and Automation Engineering, Chinese University of Hong Kong, Shatin, Hong Kong, Chinag Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Green Preparation andApplication of Functional Materials, Hubei Province Key Laboratory of Industrial Biotechnology, Faculty of Materials Science & Engineering, HubeiUniversity, Wuhan, Chinah State Key Laboratory for Turbulence and Complex System and Department of Materials Science and Engineering, College of Engineering, Peking University,Beijing 100871, Chinai China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China

a r t i c l e i n f o

Article history:Received 31 January 2018Received in revised form5 May 2018Accepted 5 May 2018Available online 8 May 2018

Keywords:Core-shell microspheresMicrofluidic capillary devicePrecisely controlled magnesium ion releaseBiocompatibilityBone regeneration

* Corresponding author. Department of OrthopaeUniversity of Hong Kong, Hong Kong, China.

E-mail address: wkkyeung@hku.hk (K.W.K. Yeung

https://doi.org/10.1016/j.biomaterials.2018.05.0110142-9612/© 2018 Elsevier Ltd. All rights reserved.

a b s t r a c t

A range of magnesium ions (Mg2þ) used has demonstrated osteogenic tendency in vitro. Hence, wepropose to actualize this concept by designing a new system to precisely control the Mg2þ delivery at aparticular concentration in vivo in order to effectively stimulate in-situ bone regeneration. To achieve thisobjective, a monodisperse core-shell microsphere delivery system comprising of poly (lactic-co-glycolicacid) (PLGA) biopolymer, alginate hydrogel, and magnesium oxide nano-particles has been designed byusing customized microfluidic capillary device. The PLGA-MgO sponge-like spherical core works as areservoir of Mg2þ while the alginate shell serves as physical barrier to control the outflow of Mg2þ at~50 ppm accurately for 2 weeks via its adjustable surface micro-porous network. With the aid ofcontrolled release of Mg2þ, the new core-shell microsphere system can effectively enhance osteoblasticactivity in vitro and stimulate in-situ bone regeneration in vivo in terms of total bone volume, bonemineral density (BMD), and trabecular thickness after operation. Interestingly, the Young's moduli offormed bone on the core-shell microsphere group have been restored to ~96% of that of the surroundingmatured bone. These findings indicate that the concept of precisely controlled release of Mg2þ maypotentially apply for in-situ bone regeneration clinically.

© 2018 Elsevier Ltd. All rights reserved.

1. Introduction

Bone healing is a biological process that can take up to weeks ormonths to complete, and patients suffering from bone fractures, in

dics and Traumatology, The

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particular, those with osteoporosis, usually require extended timeoff from work or school and other normal activities [1]. Hence, at-tempts have been considered by applying growth factors, such asrecombinant human bone morphogenetic proteins 2 (rh-BMP2) asdrugs to accelerate bone formation [2]. Despite reports of suc-cessful cases using rh-BMP2 [3,4], rapid degradation and cost andsafety are still the major concerns. High concentration of BMP-2 isharmful to cell proliferation and causes to increasing cell apoptosis

Z. Lin et al. / Biomaterials 174 (2018) 1e162

for human primary periosteal cells [5]. Additionally, anti-catabolicdrugs (e.g., bisphosphonates [BPs]) are common medications forosteoporosis due to their ability to suppress osteoclast activity andreduce overall bone turnover rate [6]. Moreover, BPs can induceapoptosis of osteoclasts by suppressing the mevalonate pathwayfor cholesterol synthesis [7]. Long-term medication of BPs, how-ever, may result in a brittle, hyper-mineralized bone associatedwith atypical bone fractures recently [8e11].

Therefore, an alternative strategy of promoting bone regenera-tion with high treatment efficiency and low-cost needs to be pro-posed. The magnesium in bone tissues is essential to metabolism,and it stabilizes structures of some proteins and nucleic acids andplays an indispensable role in human physiology and mineralmetabolism [12]. The role of Mg2þ in bone growth has demon-strated that it significantly enhances the adhesion of human bone-derived cells and improves bone healing by increasing the attach-ment and differentiation of osteoblastic cells [13]. Moreover, it is animportant bivalent ionwith the formation of biological apatite, andactively involved in the mineralization process to control boneformation and resorption [14]. However, high concentration ofMg2þ (e.g. >5.0mM) has detrimental effects on human osteoblastdifferentiation, osseous metabolism, and homeostasis, which maylead to bone mineralization defects, osteomalacic renal osteodys-trophy, and correlated bone disease [15,16]. In addition, magnesiumor magnesium alloy rods implanted in animal models leach largeamounts of magnesium ion combined with hydrogen gas, leadingto severe bone resorption around the implants [17,18]. Hence, atight control of extracellular magnesium concentration is crucial interms of bone formation and bio-safety issues. Indeed, the tech-niques on regulating the release of Mg2þ concentration to stimulatebone regeneration have been recently reported. Although a dipcoating (Mg/Epoxy resin-ZnO/PCL-Ibuprofen) on magnesium alloyhas been reported by Dong et al. [19] to realize bi-directional sus-tained release of Mg2þ and Ibuprofen in PBS for 22 days, the eval-uations of in vivo animal studies have yet to be investigated. Li et al.[20] has also designed a sustained-delivery platform to simulta-neously release strontium and magnesium on a titanium surface toachieve the enhanced bone-implant integration. The effectivedosage of magnesium ions used and its mechanism of sustainedrelease of Mg ions, however, are rarely mentioned. In our previousstudies, we found that the magnesium ions at a concentration of~50e200 ppm can effectively promote the proliferation and dif-ferentiation of pre-osteoblasts and the up-regulation of osteogenicgenes in vitro as well as significant new bone formation underin vivo tissue microenvironment [21,22]. The present study pro-poses to realize this concept via a specially designed magnesiumion delivery system that offers precise control delivery of magne-sium ions at ~50 ppm over two-weeks in order to trigger in-situbone regeneration.

To achieve the proposed objective, a monodisperse PLGA/MgO-alginate coreeshell microsphere system has been fabricated by aspecially designed microfluidic capillary device. The delivery sys-tem is a type of monodisperse core-shell microsphere comprised ofan FDA-approved polymer, namely poly (lactic-co-glycolic acid)(PLGA), alginate, and magnesium oxide nano-particles. A fixedamount of surface modified magnesium oxide (MgO) nano-particles is embedded within the PLGA porous matrix, and thealginate is chosen to fabricate the shell structure of the core-shellmicrosphere. We hypothesize that microfluidic-fabricated mono-disperse PLGA/MgO-alginate core-shell microsphere delivery sys-tem can significantly enhance osteoblastic activities in vitro andpromote new bone formation via the controlled release of mag-nesium ions in-situ. Our results demonstrate that this new deliverysystem can accurately regulate the delivery of magnesium ions at~50 ppm for two weeks in vitro. Additionally, the new PLGA/MgO-

alginate coreeshell microsphere system also exhibits excellentcyto-compatibility and in-situ new bone formation in rat models.

2. Materials and methods

2.1. Chemicals and reagents

Poly(lactic-co-glycolic acid)(PLGA, 50:50, ester terminated,Mw¼ 7000e17000,Aldrich), alginic acid sodium salt and n-octa-decyltrimethoxy silane (Sigma) were used in this study. The mag-nesium oxide nano-particles with size of～50 nm (PDF3973, Wako,Japan), poly-(vinly alcohol) (PVA, 87e89% hydrolyzed,Mw¼ 13,000e23,000), and sorbitanemonooleate (Span 80, Sigma-aldrich) were prepared and 3-(Trimethoxysilyl) propylmethacry-late (TMSPM, Sigma, USA) was adopted as a silane coupling agentfor surface modification of magnesium oxide nano-particles.Phosphate-buffered saline (PBS, pH¼ 7.4) was used to investigatethe magnesium ion release of microspheres in vitro.

2.2. Surface coating of magnesium oxide nano-particles

To allow the nano-particles to be uniformly suspended in thePLGA solution, surface coating was first carried out to MgO nano-particles that aimed to reduce their surface energy. In brief, 5 gMgO nano-particles, 2.5ml TMSPM, and 1ml propylamine wereadded into the cyclohexane (50ml) solution, respectively. Themixed solution was magnetically stirred (300rpm/min) at 80 �C for5 h and then the nano-particles containing the cyclohexane solu-tion was filtered by a buchner funnel followed by overnight drying.The TMSPM-treated MgO nano-particles were kept in a desiccatoruntil used.

2.3. Preparation of PLGA and PLGA/MgO microspheres

The monodisperse PLGA/MgO microsphere system was firstfabricated by an oil/water single emulsion method via a speciallydesigned microfluidic capillary device (Fig. 1a). The PLGA micro-sphere system without the incorporation of MgO nano-particlesserved as the control.

In brief, the PLGA/MgOmicrospheres were prepared by using anoil/water single emulsion approach. Specifically, the PLGA granuleswere dissolved in a dichloromethane (DCM) solution (10% w/v).Then, the TMSPM-treated MgO nano-particles were added into thePLGA solution (PLGA:MgO w:w¼ 1:0.2), which was defined as theinner phase (oil phase). In contrast, 3% (w/v) PVA dissolved in thedeionized water was defined as the outer phase (water phase).During fabrication, the PLGA/MgO inner phase flowed through theinjected capillary while the PVA outer phase flowed through thechannel of the square capillary to form a PLGA/MgO oil/water singleemulsion droplet in the collected capillary. The flow rates of theinner phase (500ml/h) and outer phase (2000 ml/h) were preciselycontrolled by two separate syringe pumps (LSP01-2A, Longer PumpInc., China), respectively. The collected PLGA/MgO droplets flowedinto a petri dish containing 0.1% (w/v) PVA aqueous solution, andthe PLGA/MgO microspheres were obtained after DCM solventevaporated overnight at ambient temperature. The obtained PLGA/MgO microspheres were rinsed with deionized water and lyophi-lized for 48 h. The PLGA microspheres were fabricated by the samemethod, serving as the control.

2.4. Preparation of PLGA/MgO-alginate core-shell microspheres

To demonstrate the significance of the alginate shell structureon regulating magnesium ion release, the PLGA/MgO-alginate core-shell microspheres were prepared by the oil/water/oil double

Fig. 1. Schematic diagram of (a) PLGA/MgO single emulsion droplets and (b) PLGA/MgO-alginate double emulsion droplets fabricated by the microfluidic technique; (c) Opticalimage of the microfluidic capillary device and (d) Microscopic image of PLGA/MgO-alginate core-shell droplets.

Z. Lin et al. / Biomaterials 174 (2018) 1e16 3

emulsion method via the modified microfluidic capillary device.Specifically, two cylindrical capillaries (Inner/Outer diameters:1mm/0.75mm,World Precision Instrument Inc., USA)were taperedby a flaming/Brown Micropipette Puller (P-97, Sutter InstrumentInc., USA). The tip diameters of the injected and collected capillarieswere 50 mm and 150 mm, respectively. Afterwards, the injected andcollected capillaries were coaxially aligned inside a square capillary(inner/outer diameters: 1.5mm/1.05mm, AIT precision glass tech-nology, USA) for fabrication. The outer surface of the injectedcapillary became hydrophilic after wiped with 2% hydrofluoric acidby a cotton swab for 15s while the inner surface of the collectedcapillary was turned into hydrophobic by 1% w/v n-octadecyl-trimethoxy silane immersion for 1min. After completely dried, themodified injected capillary and collected capillary were coaxiallyaligned inside a square capillary.

The synthesis of PLGA/MgO-alginate core-shell microsphereswas illustrated in Fig. 1b. In brief, three kinds of fluids flowed intothe modified microfluidic capillary device from different channels.As previously mentioned, the TMSPM-treated MgO nano-particleswere suspended in 10% (w/v) PLGA solution (dissolved in DCM)named the inner phase (oil phase). 10% (w/v) Span 80 in toluenewas defined as the outer phase (oil phase) while 5% (w/v) PVAaqueous solution containing 3% (w/v) alginate was defined as themiddle phase (water phase). The PLGA/MgO inner phase flowedthrough the injected capillary, and the alginate middle phaseflowed through the channel of the square capillary while the outerphase flowed through the channel between the collected andsquare capillaries to produce PLGA/MgO-alginate core-shell drop-lets. The flow rates of inner, middle, and outer phases were accu-rately controlled at 500 ml/h, 800 ml/h, and 2000 ml/h, respectively.After collected, the PLGA/MgOe alginate core-shell droplets weredropped into a petri dish containing 0.1% (w/v) PVA calcium chlo-ride aqueous solution for alginate shell cross-linking. The PLGA/MgOe alginate core-shell microspheres were obtained after DCMsolvent evaporated at room temperature. Finally, the core-shellmicrospheres were rinsed with deionized water and lyophilizedfor 48 h.

2.5. Morphological and chemical characterizations

The molecular structures of PLGA, PLGA/MgO, and PLGA/MgO-alginate core-shell microspheres were characterized by Fourier

transforminfrared (FTIR) transmission spectra (Perkin Elmermodel16 PC). The microspheres combined with KBr powder werepressed into pellets for FTIR test. The morphology of PLGA/MgO-alginate core-shell droplets were examined by an optical micro-scope (OM) (Nikon Eclipse 80i). The scanning electron microscopy(SEM, Hitachi S-3400N, Electron Microscope Unit, The University ofHong Kong) was applied to characterize the surface morphology ofall PLGA-based microspheres at 5 kV. Prior to the SEM observation,the microspheres were subject to gold coating for 40e50s in orderto overcome the poor conductivity of the PLGA polymer. Further-more, the size distribution and mean diameters of PLGA, PLGA/MgO, and PLGA/MgO-alginate core-shell microspheres weredetermined by measuring 300 microspheres randomly in SEM. Themicro-porous structures of alginate shell were observed at �21 �Cby a scanning electron microscopy (SEM, Hitachi S-3400N) equip-ped with a cooling stage.

2.6. In vitro release of magnesium ions

Themagnesium ion release profiles of each groupwere obtainedby immersing 10mg microspheres into 1ml PBS (pH¼ 7.4) andincubated at 37 �C for four weeks, respectively. The pure PLGAmicrospheres without MgO nano-particles served as the control.The release of magnesium ions was examined each day by aninductively coupled plasma optical emission spectrometry (ICP-OES, Perkin Elmer, Optima 2100DV, USA). At each time point pro-posed, the PBS solution containing microspheres was centrifuged,and, then, 600 ml supernatants were aspirated and filtered forevaluating the actual concentration of magnesium ions released.The PBS solutionwas refreshed each day. The total concentration ofmagnesium ions was then determined by lysing 10mg micro-spheres into 1ml aqua regia and measuring the lysates by the ICP-OES machine.

2.7. In vitro cell study

2.7.1. Cell cultureThe mouse MC3T3-E1 pre-osteoblasts were adopted for the

biocompatibility evaluations of all sample groups. The pre-osteoblasts were cultured in the Dulbecco's modified Eagle's me-dium (DMEM) containing 100 U/ml of penicillin, 0.1mg/ml ofstreptomycin, and 10% fetal bovine serum (FBS) and incubated

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under an atmosphere of 5% CO2 at 37 �C. The cell passages tookplace when cells proliferated to more than 80e90% confluence andfourth passage of MC3T3-E1 pre-osteoblasts was used in theexperiment.

2.7.2. Cell attachment assay10mg microspheres were subjected to gamma-ray sterilization

for 30min prior to the cell attachment test. The sterilized micro-spheres were added into the a 24-well plate and then the sus-pension of MC3T3-E1 pre-osteoblasts at a density of 2� 104 cells/well were co-cultured with microspheres under an atmosphere of5% CO2 at 37 �C for 3 and 7 days. At each time point proposed, thecells were rinsed by PBS for 3 times and subjected to gradualdehydration by a series of ethanol solutions (30%, 50%, 70%, 80%,90%, and 100% ethanol) for 10e15min each. The samples were driedby a critical point dryer for 48 h, and the cell attachment assay wasthen investigated by an SEM at 5 kV.

2.7.3. Cell viability by using 3-(4,5-dimethylfthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) bromide assay

The MTT assay was employed to investigate the cyto-toxicity ofthe PLGA-based microspheres (PLGA microspheres, PLGA/MgOmicrospheres, and PLGA/MgO-alginate core-shell microspheres) byusing direct method. Specifically, the MC3T3-E1 pre-osteoblastswith a density of 1� 104 cells/well were cultured with DMEMcontaining 100 U/ml of penicillin, 0.1mg/ml of streptomycin, and10% FBS into a 96-well plate under an atmosphere of 5% CO2 at 37 �Cfor cell attachment. After 1 day of culturing, the cells were rinsed byPBS for three times and then refreshed with DMEM. Afterwards,0.1 g/ml sterilized PLGA-based microspheres were added into eachwell and co-cultured with MC3T3-E1 pre-osteoblasts under a hu-midified atmosphere of 5% CO2 at 37 �C for 1 and 3 days, respec-tively. The MTT solution was prepared by dissolving the thiazolylblue tetrazolium bromide powder into PBS solution and filtered bya 0.22 mm membrane. At each designated time point, a 20 ml 5mg/ml of MTT solutionwas added into each well and incubated under ahumidified atmosphere of 5% CO2 at 37 �C for 4 h in order to allowthe formation of formazen. Then, the formazen was dissolved byusing 200 ml dimethyl sulfoxide. The absorbance was recorded by amicro-plate spectrophotometer (Thermo Scientific, USA) at awavelength of 570 nm and 640 nm (reference point).

2.7.4. Cell proliferation evaluated by 5-Bromo-2-deoxyUridine(Brdu) incorporation assay

The Brdu incorporation assay was used to analyze the cell pro-liferations of MC3T3-E1 pre-osteoblasts cultured directly with thePLGA, PLGA/MgO, and PLGA/MgO-alginate core-shell microspheres,respectively. The MC3T3-E1 pre-osteoblasts at the concentration of1� 104 cells/well were cultured on a 96-well plate with use of thesame approach as what the MTT assay performed. The ELISA Brdukit (Roche, USA) was used to evaluate the cell proliferation capa-bility. At each time point, the cells were rinsed by the PBS solutionfor 3 times and 100 mM Brdu labeling solution was added to labelthe cells for 2 h. Then, the cells were fixed at ambient temperaturefor 0.5 h followed by adding the anti-Brdu-POD working solutionfor 2 h. The 100ml/well substrate solution was used for photometricdetection, and the substrate reactionwas ceased by using 25ml/well1M H2SO4. The absorbance was recorded by a micro-plate spec-trophotometer (Thermo Scientific, USA) at a wavelength of 450 nmand 590 nm (reference point).

2.7.5. Alkaline phosphatase(ALP) activityThe osteogenic differentiation activity of PLGA-based micro-

spheres was initially examined by using an ALP assay. In brief, adensity of 2� 104 cells/well MC3T3-E1 pre-osteoblasts was

incubated with DMEM containing 100 U/ml of penicillin, 0.1mg/mlof streptomycin, and 10% FBS into a 24-well plate under a humid-ified atmosphere of 5% CO2 at 37 �C for 1 day. On the second day, thecells were rinsed by PBS 3 times and cultured with 0.1 g/ml steril-ized PLGA-based microspheres under a humidified atmosphere of5% CO2 at 37 �C for 3,7, and 14 days. From day 4, the differentiatedDMEM, including 50mg/ml ascorbic acid (Sigma, USA) and 10mM b-glycerol phosphate (Sigma, USA), were refreshed every 3 days ofculturing. At each time point, the cells were rinsed by the PBS so-lution for 3 times and lysed by 0.1% Triton X-100 (Sigma, USA) at4 �C for 0.5 h before the cell lysates were subjected to 574 gcentrifugation at 4 �C for 10min. Afterwards, the 10 ml supernatantof each specimen was transferred to the 96-well plate. The ALPactivity of MC3T3-E1 pre-osteoblasts was measured by a colori-metric assay via an ALP reagent kit (Stanbio, USA) containing p-nitrophenyl phosphate (p-NPP). The absorbance was recorded at awavelength of 405 nm on a micro-plate spectrophotometer(Bechman Coulter DTX880, USA). The ALP activity was thennormalized to the total protein level of the specimens evaluated bythe Bio-Rad Protein Assay. In the alizarin red staining (ARS) assay,the cells were directly cultured with PLGA-based microspheres atthe same condition of the ALP assay for 21 days. Afterwards, theMC3T3-E1 pre-osteoblasts were rinsed with PBS 3 times and fixedby 4% Paraformaldehyde for 15min. The cells were stainedwith ARS(2%, pH 4.2) for 30min. After washed by DI water for several times,the cells were observed by an optical microscopy. For the quanti-tative analysis of deposited calcium nodules, 10% cetyle-pyridiniumchloride was added to dissolve the calcium deposits and culturedfor 1 h. Then, the absorbance was measured by a micro-platespectrophotometer (Thermo Scientific, USA) at 570 nm.

2.7.6. Osteogenic gene expressions analyzed by real-timequantitative RT-PCR assay

To further investigate the osteogenic expression capability ofthe PLGA-based microspheres, 4 osteogenic-related gene expres-sions, such as type I collagen (Col I), alkaline phosphatase (ALP),runt-related transcription factor 2 (Runx2), and osteopontin (OPN)were included The MC3T3-E1 pre-osteoblasts with a density of1� 105 cells/well were cultured with DMEM containing 100 U/mlof penicillin, 0.1mg/ml of streptomycin, and 10% FBS into a 6-wellplate under a humidified atmosphere of 5% CO2 at 37 �C for 1 day.The cells were rinsed with the PBS solution 3 times and thendirectly incubated with 0.1 g/ml PLGA-based microspheres under ahumidified atmosphere of 5% CO2 at 37 �C for 3, 7, and 14 days. Fromday 4 of culturing, 50mg/ml ascorbic acid and 10mM b-glycerolphosphatewere added into the DMEM,which were refreshed every3 days. The gene expressions of MC3T3-E1 pre-osteoblasts culturedwith the PLGA-based microspheres were assessed by the real-time,reverse-transcriptase polymerase chain reaction (Real-time RT-PCR). The forward and reverse primers of the 4 related genes inconjunction with house-keeping geneglyceraldehyde-3-phosphatedehydrogenase (GAPDH) were listed in Table S1 (Supportinginformation).

At each designated time point, the cells were rinsed 3 times byPBS and lysed with a Trizol reagent (Invitrogen, USA). The chloro-form was added in order to extract the total RNA of osteoblasts tothe upper-aqueous phase and then transformed into a new 1.5mlRNase-free centrifuge tube followed by the addition of an equalvolume of isopropanol (biochemical grade) for RNA precipitation.Afterwards, the RNA precipitates were rinsed with 80% ethanol anddissolved into the diethypyrocarbonate (DEPC)-treated RNase-freeddH2O(30e50 ml). The concentration of isolated RNA wasmeasured by a nano-drop 1000 spectrophometer (Thermo Scien-tific, USA). In total, 1 mg isolated RNA was reverse-transcribed intothe complementary DNA (cDNA) using the RevertAid First Strand

Z. Lin et al. / Biomaterials 174 (2018) 1e16 5

cDNA Synthesis Kit (Thermo Scientific, USA) in accordance with therecommended protocol. In the cDNA synthesis, the reverse tran-scription reaction occurred at 42 �C for 1 h andwas then terminatedby heating at 70 �C for 5min. For quantification, the RT-PCR assaywas performed on the Bio-Rad C1000 Touch™Thermal Cycler, usingthe SYBR Green PCR Master Mix (Applied Biosystems, USA). Thetotal quantitative PCR reaction volume was 20 ml, containing 10 mlSYBR Green PCR Master Mix, 5 ml cDNA template, and 5 ml primers.The signal was amplified by setting 39 cycles for the reaction. Therelative mRNA's expressed levels of type I Col I, ALP, Runx2, andOPN were normalized by the house-keeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH). The normal DMEM group,without adding microspheres, was set as the control.

2.8. In vivo animal study

2.8.1. Surgical proceduresThe surgical procedures and post-operative care were approved

and fulfilled under the requirements of the Ethics Committee of theUniversity of Hong Kong and the Licensing Office of the Departmentof Health of the Hong Kong Government, respectively. A total of 3012-weeks-old Sprague-Dawley female rats (SD rats) were used inthe in vivo study. The rats provided by the Laboratory Animal Unit ofthe University of Hong Kong weighed 250e300 g and were dividedinto 4 groups, including 1) a sham control, 2) a PLGA microspheregroup, 3) a PLGA/MgO microsphere group, and 4) a PLGA/MgO-alginate core-shell microsphere group, respectively. Each PLGA-based microsphere group contained 8 rats while 6 rats were usedin the sham control. The bone defect model described in our pre-vious study [21,22] was used to evaluate the bone volume andquality of newly formed bony tissue in each group. The shamcontrol was defined as the bone defect without microsphere in-jection. In brief, the rats were first anaesthetized by using a com-bination of ketamine (67mg kg�1) and xylazine (6mg kg�1) viaintraperitoneal injection. A standard aseptic surgical procedurewascarried out on the surgical area and then a defect with 2mm indiameter and 3mm in depth was prepared at the lateral epicondyleof either the right or left femurs of the rats by a manual driller(Fig. S1, Supporting Information). Then, the sterilized PLGA-basedmicrospheres (0.1 g/ml) combined with 1ml 0.9% saline solutionwere injected into the defects. The rats were sacrificed at 8 weekspost-surgery.

2.8.2. Evaluation of new bone formation by real-time micro-computed tomography (micro-CT)

An in vivo micro-CT machine (SKYSCAN 1076, Skyscan Com-pany) was employed to evaluate new bone formation at post-operation 1, 2, 4, and 8 weeks. The percentage of new bone vol-ume, bone mineral density (BMD), trabecular thickness, andtrabecular number of all the rats were characterized by using aCTAn software (Skyscan Company), and the new bone formationpresented in 3D structure was reconstructed by the CTVol software(Skyscan Company). The grey threshold used for CT densitometricanalysis was 80e255 (�1000 to 9240 in Hounsfield units). Based onthe CTAn software, trabecular thickness was an estimate of theaverage thickness of all bone or tissue structures in a region of in-terest (2mm in diameter and 3mm in depth) and calculated byfilling maximal spheres into the 3D structure using distancetransformation. The trabecular number was determined by theinverse of themean distance between themid-axes of the structureto be examined. The mid-axes were assessed from the binary 3Dimage using the 3D distance transformation and extracting thecenter points of non-redundant spheres, which completely filledthe structure. For the quantitative analysis of bone mineral density(BMD), 2 standard BMD rods (0.25 g cm�3 and 0.75 g cm�3) were

used in the calibration, and the BMD was defined as the volumetricdensity of recognized bone and soft tissues in the region of interest.

2.8.3. Histological analysis and mechanical propertycharacterization of newly formed bone

The femurs of the rats operated on were harvested and pro-cessed for hard tissue sectioning after euthanization. The sampleswere fixed in a 10% buffer formalin solution for 3 days followed by adehydrating process starting from 70%, 95%, and 100% ethanol so-lution, respectively. In each dehydration step, the samples wereimmersed in various concentrations of ethanol solution for 4 days,according to the standard protocol. The samples were then trans-ferred into xylene as an intermedium for another 4 days and finallyembedded with methyl metharylate (MMA) for hard tissue cutting.A 4-stageMMA embedding (i.e., MMA I, MMA II, MMA III, andMMAIV) was involved. Specifically, the MMA I solution consisted of60mL of MMA (MERCK, Germany) and 35mL of butylmethacrylate(Aldrich, USA). TheMMA II and III solutions contained 100mL of theMMA I solution combined with either 0.4 g or 0.8 g benzoylperoxide (MERCK, Germany), respectively. MMA IV was made up of400 ml N,N-dimethyl-p-toluidine (Sigma, USA) and 100ml of theMMA III solution. The benzoyl peroxide was dried overnight beforebeing used, and the embedded specimens were cut onto slides witha thickness of 250 mm by a hard-tissue sliding microtome (EXAKT,Germany) and then ground to a thickness of 50e70 mm for eachslide. The sectioned specimens were stained with Giemsa solution(Giemsa(v): DI water(v)¼ 1:4, MERCK, Germany) at 57 �C for20min, and the tissue morphology was observed under opticalmicroscopy.

The mechanical properties of newly formed bone of Giemsa-stained slides in each group at the postoperative 8 weeks werecharacterized by a nano-indentation assay (Nano Indenter G200). ABerkovich tip with the radius of 20 nm was employed as anindenter to directly punch on the Giemsa-stained slides. Theloading axis was vertically aligned with the histology slides. Tworeference materials (fused silica and single-crystal tungsten) wereused to calibrate the tip shape and frame compliance of the Ber-kovich indenter [23]. The constant value of Poisson ratio was set at0.35 during the measurement while the maximum indentationdepth was 4000 nm. The applied maximum load, peak holding-time, and drift rate were 10mN, 120s, and 1.2 nm s�1, respec-tively. The Young's modulus of Giemsa-stained bone slides wascalculated by using Oliver and Pharr's method [24]. Specifically, themodulus was determined by fitting the first 45% of the unloadingcurve with a second-order polynomial, differentiating and evalu-ating the elastic recovery rate at maximum indention load tocalculate the contact depth. Each sample was indented 6 times invarious regions, and 6 samples in each group were analyzed forstatistical significance.

2.9. Statistical analysis

All the tests were independently triplicated, and 5 samples wereused at each time point for the in vitro and in vivo experiments. Thestatistical analysis was performed by a one-way analysis of varianceusing SPSS software. The p value< 0.05 was considered to be sta-tistically significant.

3. Results

3.1. The surface morphology and structure of PLGA-basedmicrospheres

Fig. 1aeb revealed the schematic fabrication process of thePLGA, PLGA/MgO microspheres, and PLGA/MgO-alginate core-shell

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microspheres via customized microfluidic devices. The PLGA andPLGA/MgO microspheres were produced by the single oil/wateremulsion method while a double oil/water/oil emulsion techniquewas employed to fabricate the PLGA/MgO-alginate core-shell mi-crospheres. The customized microfluidic device, composed of tworound capillaries and one square capillary, was specially designedby our teams, which could accurately control the size of mono-disperse core-shell microspheres (Fig. 1c). Fig. 1d exhibited that thesize of PLGA/MgO-alginate double emulsion droplet was approxi-mate 120 mm during fabrication.

The results of the FTIR spectrometery demonstrated that thepeak appeared on 1043 cm�1 and was attributed to the C-CH3stretching vibrations while another absorption peak at 1450 cm�1

was related to CeH stretching vibrations in methyl groups in thePLGA polymer (see Fig. 2). The C-O-C stretching vibrations and C]O stretching (in the ester group) peaks appeared on 1080 cm�1and1750 cm�1, respectively. With respect to the FTIR results of thePLGA/MgO microspheres, the pattern was similar to the spectra ofthe PLGA microspheres, indicating that they had almost the samefunctional groups in the polymer chains. For the specimen of PLGA/MgO-alginate core-shell microsphere, some peaks appeared to besimilar to that of the FTIR spectra of PLGAmicrospheres. In addition,according to the analysis of FTIR spectra of alginate hydrogel in theliteratures [25,26], a peak appearing on 3300-3400 cm�1 wasascribed to the stretching vibration of the O-H groups while a peakat about 1600 cm�1 was the asymmetric stretching of C]O groups.Therefore, we expected that an absorption peak at 1607 cm�1 in theFT-IR spectra of core-shell microsphere was related to C-O-Ocarboxyl group stretching in alginate, and the peak at 3300 cm�1

was attributed to the stretching vibrations of terminal hydroxylgroups in alginate. These characteristics qualitatively indicated theexistence of alginate shell on PLGA microspheres.

The SEM images of the PLGA-based microspheres and alginateshell with concentrations of 3% and 5% were depicted in Fig. 3. TheSEM images in Fig. 3aeg exhibited that the surface morphology ofthe PLGA and PLGA/MgO microspheres was porous while thealginate shell of the PLGA/MgO-alginate core shell microspheresshowed a smooth surface like a membrane. We believed that theformation of this smooth surface was due to the water evaporationunder a vacuum atmosphere during the SEM examination. How-ever, when examined through an environmental SEM equippedwith a cooling stage at �21 �C, it could be clearly seen that the

Fig. 2. Fourier transforminfrared (FTIR) transmission spectra of PLGA, PLGA/MgO andPLGA/MgO-alginate core-shell microspheres.

alginate shell presented with the pores in micron level over thesurface.When the concentration of alginate used increased from 3%to 5%, micro-pores over the surface tended to be of smaller size(Fig. 3d and h). Hence, the outflow of Mg2þ could be controlled byadjusting the size of surface pores of the alginate shell via theconcentration of alginate hydrogel used. Moreover, the innerstructure of all PLGA-based microspheres (Fig. 3iek) was sponge-like, and the PLGA cores loaded with magnesium oxide nano-particles possessed a uniform Mg element distribution(Fig. 3len). Hence, the PLGA/MgO core played as a reservoir of Mg2þ

whereas the alginate shell was adopted for the regulation of Mg2þ

outflow.Based on the random measurement of 300 microspheres in

SEM, the spherical size of the PLGA/MgO-alginate core-shell mi-crospheres was 115.41± 3.84 mm while the size of the PLGA andPLGA/MgO microspheres was 110.79 ± 3.25 mm and113.73± 3.31 mm, respectively (Fig. 3oeq). In general, all the PLGA,PLGA/MgO microspheres, and PLGA/MgO-alginate core-shell mi-crospheres were similar in dimension with a narrow size distri-bution, indicating that the PLGA-based microspheres fabricated viaa microfluidic capillary device exhibited to be homogenous andmonodisperse. The dimension of the PLGA-basedmicrosphereswasabout 15% smaller than that of PLGA-basedmicrospheres fabricatedby single emulsion or core-shell droplets (Fig. 1d) due to the DCMsolvent evaporation [27,28].

3.2. In vitro measurement of magnesium ion release

To characterize the magnesium ion release kinetics of the PLGA-based microspheres incorporated with MgO nano-particles in vitro,the initial burst release, release rate profile, and accumulativerelease profile of magnesium ions against number of days weresystematically investigated (Fig. 4). Fig. 4a revealed that the initialburst release of magnesium ions of PLGA/MgO-alginate core-shellmicrospheres at day 1 was significantly suppressed to ~250 ppm ascompared to PLGA/MgO microspheres where the burst release wasapproximately 550 ppm (p< 0.001). It was indicated that the ho-mogenous alginate shell effectively reduced the burst release ofmagnesium ions. Referring to the magnesium ion release rate ofPLGA-based microspheres as shown in Fig. 4a, the release rate ofPLGA/MgO microspheres presented a sharp downward trend from550 ppm per day to 30 ppm per day during the first 2 weeks, and,then, the release rate was almost 0 from days 16e28. Nevertheless,the PLGA/MgO-alginate core-shell microspheres exhibited pro-grammed release of magnesium ions at ~50 ppm per day from days2e16 followed by the release rate controlled between 100 and200 ppm per day during last two weeks. In brief, the PLGA/MgO-alginate core-shell microsphere system effectively controlledmagnesium ion delivery at 50 ppm per day during the first 16 daysand then regulated the release rate between 100 and 200 ppm perday from days 17e28. In fact, the Mg2þ release profile presentedwas within the effective range of new bone formation according toour previous studies [21]. Fig. 4b presented the accumulativemagnesium ion release profile of PLGA-based microspheres. Theresults suggested that the PLGA/MgO microspheres exhibited aparabolic release pattern during the first 2 weeks prior to theappearance of a long plateau at a total concentration of 3500 ppmuntil day 28, indicating that the PLGA/MgO microspheres deliveredmagnesium ions completed within 14 days. However, the PLGA/MgO-alginate core-shell microsphere system demonstrated atwo-staged, near zero-order release pattern. A near-linear releasetrend with a low slope of magnesium ions was attained from days1e16 while another, higher slope near-linear release pattern wasobtained from days 17e28. The accumulative release concentrationof the PLGA/MgO-alginate core-shell microspheres was less

Fig. 3. The surface morphology, inner structure, Mg element distribution (red dots) and size distribution of PLGA, PLGA/MgO and PLGA/MgO-algiante core-shell microspheres. TheSEM images of (a and e) PLGA microspheres, (b and f) PLGA/MgO microspheres, (c and g) PLGA/MgO-alginate core-shell microspheres and the surface microporous structure of (d)3% and (h) 5% alginate shell observed under environmental SEM equipped with cooling stage at �21 �C. All the fabricated microspheres are presented in monodisperse shape. Theinner structure of (i and l) PLGA microsphere, (j and m) PLGA/MgO microsphere and (k and n) PLGA/MgO-alginate core-shell microsphere was obtained by rupturing the mi-crospheres with tweezers. The inner structures of all cores were sponge-like and magnesium oxide nano-particles were uniformly distributed. The size distribution of (o) PLGAmicrosphere, (p) PLGA/MgO microsphere and (q) PLGA/MgO-alginate core-shell microsphere was determined by the calculation of diameter of 300 microspheres randomly in eachgroup. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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~1000 ppm than that of the PLGA/MgO microspheres at 4 weeks,indicating that the PLGA/MgO-alginate core-shell microspheresystem could achieve sustained magnesium ion delivery over aperiod of time. Based on the results of magnesium ion release ki-netics, the PLGA/MgO-alginate core-shell microsphere systempresented greater superiorities than that of PLGA/MgO micro-spheres. In fact, this specific morphology was significant inreducing initial burst release, prolonged release time, and accuratecontrol of magnesium ions release.

3.3. In vitro studies

3.3.1. Direct cell attachment on microspheresThe biocompatibility of PLGA, PLGA/MgO microsphere, and

PLGA/MgO-alginate core-shell microsphere systems was evaluatedby the direct assay approach in which the MC3T3-E1 pre-osteoblasts were directly co-cultured with the PLGA-based micro-spheres. Fig. 5a demonstrated that all three PLGA-based

microsphere groups exhibited no cyto-toxicity to the MC3T3-E1pre-osteoblasts regardless of the incorporation of nano MgO par-ticles. The MC3T3-E1 pre-osteoblasts were relatively reluctant toattach to the surface of PLGA and PLGA/MgO microspheres due tothe intrinsic hydrophobicity of PLGA biopolymer. However, moreMC3T3-E1 pre-osteoblasts were found when cultured with thePLGA/MgO-alginate core-shell microspheres. The cells were wellspread, and the F-actins evenly flattened on the surface of the core-shell microspheres due to the lure of stable release of magnesiumions and excellent hydrophilicity of the alginate shell as comparedto the PLGA control and PLGA/MgO groups. After cultured for 7days, a large amount of MC3T3-E1 pre-osteoblasts attached near tothe bottom of core-shell microsphere as compared with the othergroups.

3.3.2. Cell viability and proliferation cultured with microspheresReferring to the MTT assay results, the cell viability of the core-

shell microsphere groups was about the same as the PLGA/MgO

Fig. 4. The Mg ion release kinetics of PLGA, PLGA/MgO and PLGA/MgO-alginate core-shell microspheres immersed in PBS. The Mg ion release kinetics in vitro was characterized by(a) magnesium ion release rate per day until 28 days and (b) accumulative release of magnesium ions.

Fig. 5. The in vitro performance of PLGA, PLGA/MgO and PLGA/MgO-alginate core-shell microspheres subject to the cell culture of MC3T3-E1 pre-osteoblasts. (a) Cell attachmentassay of MC3T3-E1 pre-osteoblasts co-cultured with PLGA-based microspheres for 3 and 7 days, respectively. High number of cells attached and even flattened in the group of PLGA/MgO-alginate core-shell microspheres as compared with the PLGA microsphere control and the PLGA/MgO microsphere group at day 3. (b) The cell viability and proliferation ofMC3T3-E1 pre-osteoblasts cultured with PLGA-based microspheres for 1 and 3 days, respectively. (c) The differentiation and mineralization of MC3T3-E1 pre-osteoblasts co-cultured with PLGA-based microspheres after 3,7,14 days and 21days of culturing, respectively. (d) Osteogenic expressions of MC3T3-E1 pre-osteoblasts co-cultured with PLGA-based microspheres after incubated in DMEM at 37 �C for 3, 7 and 14 days assessed by RT-PCR assay. The osteogenic expressions were determined by relative mRNA expressedlevels of type collagen I (Col I), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteopontin (OPN) normalized to the house-keeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH). *denotes the significant difference (p < 0.05); **(p < 0.01); ***(p < 0.001).

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group at the first day of culturing, and both groups were about 10%higher when compared to the PLGA group (Fig. 5b). When culturedfor 3 days, the cell viability observed on the core-shell microsphere

group was 30% higher than the PLGA control (p< 0.05), indicatingthat the leaching of stable magnesium ions could promote the cellviability of MC3T3-E1 pre-osteoblasts in vitro. Fig. 5b also revealed

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the results of cell proliferation of PLGA-based microspheres by theBrdu incorporation assay. The absorbance of the core-shell micro-sphere group was exhibited to be 53% and 12% higher than that ofthe PLGA and PLGA/MgO microsphere groups at day 1, respectively.It was found that the PLGA/MgO group presented a 30% increase,compared with the PLGA control at day 3 (p< 0.05). Moreover,when compared to the PLGA and PLGA/MgO microsphere groups,the PLGA/MgO-alginate core-shell microsphere group approxi-mately exhibited a 1.6 times fold (p< 0.001) and 1.3 times fold in-crease (p< 0.01) after being incubated for 3 days, respectively.These results implied that the controlled release of magnesium ionsbenefited cell proliferation as well.

3.3.3. ALP activity and mineralization of cells cultured withmicrospheres

To examine the osteogenic differentiation properties, the ALPassay was carried out and the result was shown in Fig. 5c. In gen-eral, the ALP activity in each group, firstly, increased towardspeaking at day 7 and then dropped back after 14 days of incubation.At day 3, the ALP activity was relatively low, and the differencebetween these groups was insignificant since the pre-osteoblastswere still in the stage of proliferation rather than differentiation.After 7 days of culturing, when compared to the PLGA control andPLGA/MgO microsphere groups, the ALP expressions on the core-shell microsphere group increased by 70% (p< 0.01) and 52%(p< 0.01), respectively. At day 14, the ALP expression in the PLGA/MgO group exhibited 37% higher (p< 0.05) as compared to thePLGA control. However, the ALP activity on the core-shell micro-sphere group was 78% (p< 0.01) and 30% (p< 0.05) higher than thePLGA and PLGA/MgOmicrosphere groups, respectively. Referring tothe results of cell mineralization, the core-shell microsphere groupdemonstrated a higher number of calcium nodules deposited dur-ing mineralization and the total amount of calcium nodules shownin Fig. 5c was significantly higher (p< 0.001) than the PLGA group.The results proposed that a precisely controlled release of magne-sium ions from the core-shell microspheres contributed to thedifferentiation and mineralization of MC3T3-E1 pre-osteoblasts.

3.3.4. Osteogenic gene expressions measured by real-time RT-PCRassay

The RT-PCR assay was conducted to further elucidate the ex-pressions of osteogenic gene markers (e.g., type I Col I, ALP, Runx2,and OPN) of MC3T3-E1 pre-osteoblasts when directly cultured withthe PLGA-based microsphere systems for 3, 7, and 14 days. Theresult of a post-culture of 3 days proposed that no significant dif-ference was found on each group. However, there was a positivecorrelationwith all the genes being highly expressed alongwith theincrease of incubation time (Fig. 5d). In general, the ALP, type I Col Iand OPN expressions in the PLGA/MgO microsphere group were80% (p< 0.01), 67% (p< 0.01), and 67% (p< 0.05) higher than that ofthe PLGA control group at day 7, respectively. At 14 days, thosegenes increased by 67% (p< 0.01), 60% (p< 0.05), and 76%(p< 0.01), respectively, implying that magnesium ions delivery wasalso beneficial to osteogenic expressions. In comparison with thePLGA group, the expressions of type I Col I and ALP on the core-shellmicrosphere group were up-regulated 2.5e3 times (p< 0.001)while the OPN expression exhibited 2.3 times (p< 0.001) higher atdays 7 and 14, respectively. With respect to the Runx2 geneexpression, the core-shell microsphere group presented about a 1.7times up-regulation (p< 0.01) on days 7 and 14. Moreover, the ALPand type I Col I expressions on the core-shell microsphere groupwere nearly 2 times higher when compared to the PLGA/MgOgroup at day 7 (p< 0.001) and day 14 (p< 0.01), respectively. TheOPN and Runx2 expressions of the core-shell microsphere groupwere also about 1.5 times (p< 0.01) up-regulated at days 7 and 14.

The RT-PCR results evidenced that fixed amount of magnesium iondelivery from the core-shell microsphere system favored the up-regulations of those osteogenic gene markers as compared withthe non-regulated release of Mg2þ by the PLGA/MgO microspheresystem and the PLGA control group.

3.4. In vivo studies

3.4.1. In situ new bone formation examined by micro-CT analysisIn the animal study, the reconstructed micro-CT 3D models

revealed that new bone began to form in the bone defect implantedwith the core-shell microsphere group even after post-surgery forone week. For the qualitative analysis, the newly formed bonevolume in the core-shell microsphere group was found higherwhereas the bone tissue was rarely found in the bone defectimplanted with the PLGA and PLGA/MgO groups and sham control(Fig. 6a). In the post-operation of 2 and 4 weeks, large amounts ofbony tissues were continuously generated in the defect injectedwith the core-shell microsphere group while small amount of thenew bony tissuewas observed on the groups of the PLGA and PLGA/MgO microsphere and the sham control groups. In general, thebone defect filled with the core-shell microspheres was almostcompletely healed after 8 weeks of surgery. However, the bonedefects on those PLGA and PLGA/MgO groups, as well as the shamcontrol, were still seen at the end point and only small amounts ofnew bone formation was observed in the reconstructed 3D models.

For the quantitative analysis of newly formed bony tissue, thepercentage of new bone volume, bone mineral density (BMD),trabecular thickness (Tb, Th), and trabecular number (Tb. N)calculated by the CTAn software were measured and presented inFig. 6b. The sham control presented a slow rise of bone regenera-tion and only achieved 22% of total bone volume after post-operative 8 weeks. The bone volume of the PLGA/MgO groupexhibited no significant difference when compared to the PLGAgroup regardless of the increase of postoperative time, and only33% of total bone volume was formed at week 8, indicating thatnon-regulated magnesium ion delivery could not significantlyconvince in situ bone regeneration. As compared with the PLGAmicrosphere group, the percentage of new bone volume on thecore-shell microsphere group was, significantly, 3 times higher atweek 2 (p< 0.01) and week 4 (p< 0.001) post-operation. The per-centage of total bone volume achieved ~75% on the core-shellmicrosphere group at week 8 post-operation whereas it was only30% for the PLGA microsphere group. When compared with thePLGA/MgO group, the total bone volume of the core-shell micro-sphere group was 75% (p< 0.05), 140% (p< 0.001), and 114%(p< 0,001) higher from post-op weeks 2e8, respectively. Theseresults indicated that the controlled release of Mg ions couldeffectively induce new bone formation in situ. Furthermore, theBMD, Tb,Th, and Tb.N of the PLGA/MgOmicrosphere group showedno significant difference to that of the PLGA group at post-operationweeks 4 and 8. However, the BMD, Tb,Th, and Tb.N in the groupinjected with the core-shell microspheres were significantlyincreased at post-operation weeks 4 and 8. In fact, the BMD, Tb,Th,and Tb.N of the core-shell microsphere group were approximately65% (p< 0.05), 55% (p< 0.05), and 59% (p< 0.05) higher than thePLGA control at week 8, respectively. When compared with thePLGA/MgOmicrosphere group, the Tb,Th, and Tb.N of the core-shellmicrosphere group were 56% (p< 0.05) and 40% (p< 0.05) higher,and the BMD was 45% higher (p< 0.01) at post-operation week 8,respectively. These observations illustrated that the newly formedbone was well mineralized in the PLGA/MgO-alginate core-shellmicrosphere group in addition to the new bone formation.

Fig. 6. Real-time micro-CT evaluations of newly formed bony tissues on the sham control, PLGA, PLGA/MgO and PLGA/MgO-alginate core-shell microsphere groups after post-surgery at various time points. (a) The micro-CT images of the lateral epicondyle and 3D reconstructed models of newly formed bone within the defect highlighted by red box;(b) The measurements of new bone volume in %, bone mineral density (BMD), trabecular thickness (Tb,Th) and trabecular number (Tb.N) of newly formed bone tissue calculated bythe CTAn software quantitatively.*denotes the significant difference (p < 0.05), **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the readeris referred to the Web version of this article.)

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3.4.2. Histological analysis and mechanical property measurementof newly formed bone

The newly formed bony tissue after 8 weeks of surgery was alsohistologically revealed with the Giemsa staining technique (Fig. 7a).It could be clearly seen that the bone formation started from theperipheral area of the defects and thenmigrated to the central area.Furthermore, the bone defects implanted with the core-shell mi-crospheres were already filled with new bony tissue whereas thebone defects on the femur of the sham control, PLGA, and PLGA/MgOmicrosphere groups were still obviously seen. Particularly, thecortical and cancellous structures of newly formed bone implantedwith the core-shell microspheres were clearly observed. In addi-tion, the Young's modulus of newly formed bone tissues at post-surgery week 8 was characterized by a nano-indentation test(Fig. 7b). The modulus of new bony tissue induced by the core-shellmicrosphere group was 12.5±1.2 GPa while the modulus of sur-rounding mature bone was 13.1±2.0 GPa in average. This impliedthat the mechanical property of new bone induced by core-shellmicrospheres restored 96% as compared with the adjacentmature bony tissue. However, the moduli of newly formed boneinduced by the sham control, PLGA, and PLGA/MgO microspheregroups were 7.9±1:0 GPa; 8.5±1.1 GPa, and 9.3±1:5 GPa, whichwere only equivalent to ~60%, 65%, and 71% of the mechanicalproperty of mature bone, respectively. Furthermore, whencompared to the modulus induced by the core-shell microspheregroup against to that of the sham control, PLGA group, and PLGA/MgO group, it was 58% (p< 0.01), 47% (p< 0.05), and 34% (p< 0.05)higher, respectively. These promising results illustrated that thePLGA/MgO-alginate core-shell microsphere group could not onlystimulate new bone formation significantly but also convince therestoration of original mechanical property of newly formed bone.

4. Discussion

4.1. The role of magnesium ions on in-situ bone regeneration

Magnesium ions have demonstrated a positive effect on osteo-genesis by few literatures [29e31] in which the mechanism iseither the enhancement of osteoblastic activities or the suppressionof osteoclastic activities. For instance, Mg2þ can apparently pro-mote the adhesion of human bone-derived cells and differentiationof osteoblastic cells or improve bone healing by the deactivation ofosteoclast resorption activity [32].

In terms of underlying mechanisms, previous reports haveexplained how Mg2þ works on osteogenesis. It is believed that anMg2þ enriched microenvironment is able to stimulate in situ boneregeneration through the promoted proliferation of mesenchymalstem cells (MSCs) and osteogenic differentiation of pre-osteoblasts.Through the enhancement of BMP-receptor recognition and theactivation of Notch signaling pathways, Mg2þ is shown to bebeneficial in the proliferation of MSCs [33,34]. Moreover, Mg2þ fa-vors the differentiation of MSCs in osteoblasts through the upre-gulation of osteogenic expression of Col I, ALP, OPN [35,36]. Mg2þ

can also induce an increase of neuronal calcitonin gene-relatedpolypeptide-a (CGRP) in both the peripheral cortex of the femurand the ipsilateral dorsal root ganglia (DRG) in a rat model, whichfacilitates osteogenic differentiation of periosteum-derived stemcells [37]. However, a high concentration of Mg2þ may occupy thecalcium-related signaling pathways and then disrupt the mineral-ization process of bone formation during the bone remodelingprocess [38]. Additionally, the TRPM7 channel [39], which is a kindof transient receptor potential ion channel on cell membrane, alsoserves as an important regulator to magnesium ion and otheressential metal ions (e.g., calcium, zinc, manganese, and cobalt) inorder to maintain cellular metabolism [40,41]. It is reasonable to

believe that high concentrations of extracellular Mg2þ can poten-tially suppress the TRPM7 expression induced by other metal ionsthereby altering the intracellular balance of those cations andinterrupting normal cellular functions.

Additionally, studies on the utilization of magnesium ions forin vivo bone regeneration have been reported recently. Galli et al.[42] have adopted the use of mesoporous TiO2 coatings effectivelyincorporated with magnesium to enhance the osseointegration ofbone-implant interface in the rabbit models. Zhang et al. [43] havedeveloped an MeHA nanocomposite hydrogel stabilized bybisphosphonate-magnesium nanoparticles to regulate the sus-tained release of magnesium ions in vivo, and this specific hydrogelsystem facilitates the healing of calvarial defects in rats within 8weeks. Unfortunately, the effective dosage of magnesium ionreleased from the nanocomposite hydrogel has not beenmentioned. In our previous studies, we designed a functionalizedpolycaprolactone (PCL) polymeric membrane to control thedegradation of magnesium alloy (AZ91) [44,45]. We thus observedthat the membrane can mediate the delivery of magnesium ions soas to enhance the bone-implant integration. Additionally, anothersurface approach with the use of aluminum and oxygen plasmaimmersion ion implantation has demonstrated that this surfacetreatment can effectively regulate the magnesium ion release froma magnesium alloy surface. The regulated release of magnesiumions may incite in vivo bone regeneration without any issue oftissue inflammation, necrosis, or hydrogen gas accumulation [22].Most importantly, we have, therefore, identified that the addition ofMg2þ at the concentration of 50e200 ppm can significantly in-crease the proliferation, differentiation, and osteogenic expressionsof osteoblasts in vitro. Interestingly, it is observed that in vivo boneregeneration is suppressed when the implanted PCL/Mg bio-composites are incorporated with high amounts of magnesiummicro-particles [21]. This observation has illustrated that a highconcentration of Mg2þ in the local tissue microenvironment mayslowdown newbone regeneration. Therefore, the precise control ofmagnesium ion concentration in the local tissuemicroenvironmentis crucial if the use of magnesium ions to promote new bone for-mation is clinically adopted.

4.2. In vitro and in vivo performances of core-shell microspheres

In the cell attachment assay, the objective is to observe thechange of cell morphology, viability, and proliferation when theyare subject to an attraction of the stable release of magnesium ionsfrom those microspheres. In fact, the cells tend to instantly sinkdown to the bottom of the culture plate. When the incubation timeis prolonged to 7 days, it is observed that more pseudopodia arefirmly extended near the bottom of the core-shell microspheres ascompared to the PLGA and PLGA/MgO groups. It is believed that theenhanced viability and proliferation of MC3T3-E1 pre-osteoblastscan be attributed to the stable release of magnesium ions fromthe core-shell microspheres. Furthermore, a sustained and stableMg2þ release can up-regulate the osteogenic expressions of ALP,Type I Col I, Runx2, and OPN genes as well as cell mineralization.However, the MC3T3-E1 pre-osteoblasts used in this study were amatured pre-osteoblastic cell line in which their osteogenic dif-ferentiation tendency is more prominent while comparing to thatof primary human osteoblasts or bone marrow mesenchymal stemcells [46]. Hence, this cell line expresses osteoblastic markers atmRNA levels and may somehow exaggerate the osteogenic ex-pressions of the cells [47]. In future studies, primary human oste-oblasts or bone marrow mesenchymal stem cells should be theprimary choice for in vitro assays.

With respect to the in vivo study, it is found that the PLGA/MgO-alginate core-shell microsphere system is able to precisely control

Fig. 7. Histological evaluation and mechanical property assessment of newly formed bone on the sham control, PLGA, PLGA/MgO and PLGA/MgO-alginate core-shell microspheregroups after post-surgery 8 weeks. (a) Giemsa-stained images of newly formed bone tissues in the longitudinal section of femur (Green box: the bone defect; NB: new bone; Redcross: the area of interest for the measurement of Young's modulus of newly formed bone; Yellow cross: the area of interest of Yong's modulus of the surrounding mature bonemeasured by nano-indentation technique); (b) Young's moduli of newly formed bone tissues induced by the sham control, PLGA, PLGA/MgO and PLGA/MgO-alginate core-shellmicrosphere groups measured by nano-indentation. *denotes the significant difference (p < 0.05), **p < 0.01. (For interpretation of the references to color in this figure legend, thereader is referred to the Web version of this article.)

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the delivery of magnesium ions in vivo thereby effectively stimu-lating in situ bone regeneration. The current animal model of bonedefect on femur aimed to investigate whether the stable release ofmagnesium ions in local tissue microenvironment could regulatein-situ bone regeneration at early stage. As adopted in our previousstudy [44], this model is easy to operate surgically and can effec-tively compare the rate of new bone formation of new biomaterialsand its quality at an early time point although the defect is not incritical size (i.e., 5 mm defect on rat femur) [48]. It is thereforeexpected that the defect will eventually heal by itself but will healslowly. For instance, only 22% of total bone volume was restored inthe sham control at post-operation 8 weeks, and the BMD and Tb,Th presented were also sub-optimal. In contrast, the core-shellmicrosphere group induced new bony tissue formation even after1 week of surgery due to the stable release of magnesium ions insitu. The bone regeneration accelerated at post-op weeks 2 and 4,and the defect was almost completely healed after 8 weeks ofoperation. This observation implies that the precisely controlleddelivery of magnesium ions is able to shorten the bone defecthealing time in this animal model. However, it has also been re-ported that excessive release of magnesium ions can disorder the

proliferation, differentiation, and mineralization process of osteo-blasts [16,49], resulting in limited new bone regeneration andlower BMD and Young's modulus after surgery. In comparisonwithrecent research reported by Zhang et al. [43], this kind ofmagnesium-based core shell microsphere system is not only able toaccelerate bone healing process at an early stage in vivo but alsoeffectively restores the mechanical property of newly formed boneclose to the adjacentmature bony tissue. Moreover, this approach issimple and cost-effective to achieve bone regeneration in situwithout the aids of bone morphogenic proteins and reagents aswell as bioactive methacrylated hyaluronic acid hydrogels [50]. Infact, the core-shell microsphere system can be applied togetherwith bone allografts for the treatment of large segmental bonedefects when the effective dosage of magnesium ions in a human isidentified. If needed, the duration of magnesium ion release fromthis core-shell microsphere system can be regulated by adjustingthe molecular weight, concentration, and viscosity of the PLGAcore. The therapeutic concentration of magnesium ions can beprecisely mediated by regulating the thickness and the concen-tration of the alginate shell used. Hence, it is expected that thisPLGA/MgO-alginate core-shell microsphere system can be tailored

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for application in various clinical conditions.

4.3. Mechanism of the precise control of magnesium ion release

The customized microfluidic capillary device provides a goodtemplate to produce microspheres in specific sizes through theadjustment of fluid flow rate and capillary diameter. Due to theuniform size of each PLGA core particle, the degradation rate andthe diffusion kinetics of Mg2þ in the tissue microenvironment canbe calculated. Hence, the release rate of Mg2þ can be estimated. Theregulation of accurate delivery of magnesium ions from themicrosphere system depends on the PLGA sponge-like porous corestructure and its alginate shell. In general, the PLGA sponge-likecore encapsulated with MgO nano-particles functions as a reser-voir of magnesium ions. The delivery of magnesium ions is drivenby the hydrolysis of the PLGA porous core, andmagnesium ions are,therefore, released from encapsulated nano-magnesium oxide. Themagnesium ions then diffuse into the in vivo microenvironmentthrough the microporous network on the alginate shell. The shellmicroporous structure serves as a physical barrier to regulate therelease rate of Mg2þ via the control of the diameter and number ofmicropores over the surface. In order to explain the degradationmechanism, the release profile of the magnesium core-shellmicrosphere system has been divided into three stages (Fig. 8).Specifically, the first stage is the initial burst release of the PLGA/MgO core-shell system at day 1, which can be attributed to theremnant MgO nano-particles on the microsphere surface afterfabrication. The second stage is the stable release period from days2e16 at the targeted rate of ~50 ppm per day. It is believed that theformation of a micro-porous network on the alginate shell isattributed to the involvement of calcium ions from PVA calciumchloride aqueous solution. The multivalent Ca ions are able to formionic bridges between the chains of alginate hydrogel during thePLGA/MgO core-shell microsphere fabrication, leading to a three-dimensional micro-porous network structure [51,52]. When MgOnano-particles diffuse into the alginate shell, Mg2þ will then sub-stitute the Ca2þ in an ionic bridge and subsequently form a diffusivebond [53]. The degradation rate of the alginate shell formed bydiffusive bonds is expected to be quicker than that of establishedionic bridges, owing to a relatively brittle cross-linking effect.Therefore, this particular micro-porous alginate shell can becompletely degraded within 16 days. In addition, when the con-centration of alginate hydrogel is increased, the number and size ofmicro-pores generated will decrease. This phenomenon may beattributed to the fewer formation of ionic bridges, assuming thatthe amount of Ca2þ used is constant. Hence, it helps reduce theoutflow of Mg2þ. The last stage of Mg2þ release includes theaccelerated release of Mg ions from 50 to 200 ppm per day (days17e21), and the decelerated release of Mg ions from 200 to 0 ppmper day (days 22e28), respectively. At this stage, the alginate shelldegrades completely and leads to a rapid hydrolysis of PLGAsponge-like core. Therefore, the Mg ion delivery increases from50 ppm per day at day 17 and reaches to 200 ppm at day 21. Af-terwards, the Mg ions reserved in the PLGA porous core have beenalmost exhausted, and, then, the Mg ion concentration graduallydrops from 200 ppm to 0 ppm until the end point. Hence, the re-sults of in vitro release demonstrate that the alginate shell is able tophysically block the diffusion of MgO nano-particles into the so-lution and allows the diffusion of Mg2þ under a controlled manner.In fact, the Mg ions delivered from the PLGA core will substitute theCa ions in ionic bridge gradually and then the diffusive bond withthe alginate shell will be formed [43]. Therefore, the outflow ofmagnesium ions will be dramatically reduced and result in theregulation of Mg2þ released at ~50 ppm from days 2e16. In

contrast, when the PLGA porous core is not protected by the shellstructure, the Mg2þ release of the PLGA/MgO microsphere appearsto exponentially decrease from ~500 ppm to ~50 ppm within 2weeks. Additionally, the SEM images shown in Fig. 8 also reveal thedegradation process of the alginate shell (pink color) on the surfaceof the core-shell microsphere. In the first week, the alginate shellonly slightly degrades but maintains its integrity due to the rela-tively large proportion of the Ca ionic bridge found in the alginateshell cross-links.When Ca ions are gradually substituted byMg ionsupon degradation, the alginate shell starts to degrade in terms ofthickness and shape approximately from days 7e14. Finally, thedegradation of the alginate shell is almost completed at day 17.

4.4. The advantages of PLGA core-shell microsphere system

The microfluidic technique has been widely used in the fabri-cation of microspheres, sub-microspheres, and microbeads that aremainly for cell or protein encapsulation, drug delivery, and tissueengineering [54e56]. The PLGA, a kind of FDA-approved biode-gradable polymer, is usually chosen as the carrier for microspherefabrication because of its superior biocompatibility, adjustabledegradation rate, and the flexibility for chemical modifications[57,58]. The degradation rate of PLGA can be tailored through theadjustment of molecular weight or the lactic acid to glycolic acidratio in order to enable the controlled delivery of a “targeted sub-stance” (i.e., drugs, proteins, mRNA, or even mammalian cells).However, a typical PLGA microsphere fabricated by a conventionalemulsion method is unable to attain the monodisperse size,reduction of initial burst release, and sustained and zero orderrelease [59]. Hence, the microfluidic approach with the use of aspecially designed microfluidic capillary device is adopted so as toavoid the heterogeneous size and batch-to-batch variation duringmicrosphere fabrication. It is believed that these two variations arethe primary uncertainty of varied degradation rate and releasestability of the encapsulated “targets” [60]. Furthermore, a core-shell structure has been introduced in this study in order to ach-ieve a sustained and zero order release of magnesium ions within aperiod of time. The core-shell structure has been widely used towrap encapsulated “targets” into two separate components (coreand shell). The shell can be tailored to regulate the “targets” fromdissolution or hydrolysis in order to regulate the release period andconcentration consistent with the therapeutic time-window anddosage required [61,62]. Moreover, the core-shell structuredmicrosphere can be simply fabricated by a customized microfluidiccapillary device, and its yield is high and efficient. Therefore, thecore-shell system can also be used to precisely deliver the func-tional growth factors, proteins, or even cells to the targeted organs.In fact, our previous study demonstrated the controlled release ofrifampicin for the treatment of bacterial infections with the use ofthis fabrication technique [63,64].

4.5. The bone regeneration effect subject to local pH change

Lastly, the change of pH in the local tissue microenvironmentdue to the release of magnesium ions is a non-negligible factor onosteogenesis [65]. The present study reveals that the pure PLGAmicrosphere group exhibits undesired new bone formation in ananimal model in which this phenomenon can be attributed to theacidic by-products upon PLGA degradation. Literatures have shownthat a low pH value in the local microenvironment impacts newbone formation andmay induce undesired inflammatory responses[66e68]. Hence, magnesium ions released from the PLGA/MgOalginate core-shell microspheres are able to neutralize the acidiclocal tissue microenvironment induced by PLGA degradation

Fig. 8. Illustration of the mechanism of magnesium ion releases profiles of PLGA/MgO-alginate core-shell microspheres at different stages in vitro. The SEM images exhibited thesurface morphology of PLGA/MgO-alginate core-shell microsphere immersed in PBS at various time points observed under cooling stage at �21 �C (scale bar:50mm). The alginateshell in SEM image was false colored in red by the Photoshop CS6 software. (For interpretation of the references to color in this figure legend, the reader is referred to the Webversion of this article.)

Z. Lin et al. / Biomaterials 174 (2018) 1e1614

[69,70]. In fact, the elevation of local pH exerts a beneficial factor toosteoblastic activity, osteoblastic collagen synthesis, reduction ofosteoclastic function, and calcium efflux [71e74]. During the boneremodeling process, the pH of the local microenvironment is vitalto the regulation of bone resorption and formation as well asmineralization [73,75e77]. Arnett et al. [78,79] have reported thatthe chronic acidosis in the local microenvironment tends to pro-mote bone resorption while alkalosis (an elevated pH level) is ableto facilitate bone formation and mineralization. Hence, when thePLGA/MgO-alginate core-shell microsphere system is injected, itwill locally induce a sustained and controlled release of magnesiumions and simultaneously elevate the local pH level in the tissuemicroenvironment. We believe that these co-factors may signifi-cantly enhance osteoblastic bioactivity and subsequently stimulatein situ bone regeneration.

5. Conclusion

A specially designed microfluidic capillary device has beenemployed to fabricate sponge-like monodisperse PLGA/MgO-alginate core-shell microspheres in order to achieve effective insitu bone regeneration through a precise control of magnesium iondelivery at ~50 ppm per day. The homogenous alginate shell func-tions as a physical barrier to precisely regulate the outflow ofmagnesium ions into the local tissue microenvironment. Themagnesium ions precisely released not only contributed to theenhanced attachment and growth of pre-osteoblastic cells but alsoup-regulated themineralization and osteogenic differentiation (i.e.,ALP, type I Col I, Runx2, and OPN) of the cells. Furthermore, large

amounts of newly formed bony tissue (75% bone volume) with awell-mineralized structure was observed when the PLGA/MgO-alginate core-shell microspheres were injected in the bone defectin the rat model at post-operation 8 weeks. However, in respect tobone regeneration rates of volume on the sham control, the PLGAand PLGA/MgO microsphere groups were slowed down with only22%, 30%, and 33% bone volume at 8 weeks. Interestingly, theYoung's modulus of newly formed bony tissue induced by thePLGA/MgO-alginate core-shell microsphere had been restored to~96% compared with that of the surrounding mature bone. Incontrast, the moduli of the bone induced by the PLGA and PLGA/MgO microsphere groups were only 65% and 71%, respectively.With these promising results, it is believed that the concept of insitu bone formation locally induced by the sustained and controlledrelease of magnesium ions is feasible. This injectable PLGA/MgOcore-shell microsphere system has the potential to be commerciallytranslated for bone defect healing in a clinical environment.

Author contributions

Z. Lin performed the experiments, interpreted the data andwrote the manuscript. K.W.K.Yeung conceived the experiments andinterpreted the data. J. Wu, Y. Zhao, K.H.M.Wong and W. Qiaocontributed to the PLGA/MgO-alginate core-shell microspheresystem fabrication and experiments. P.K.Chu, L.M.Bian, S.L.Wu andY.F.Zheng contributed and interpreted the in vitro cell experiments.K.M.C.Cheung and F. Leung contributed and interpreted the vivo ratexperiments.

Z. Lin et al. / Biomaterials 174 (2018) 1e16 15

Competing interests

The authors declare no competing financial interests.

Acknowledgements

This work was financially supported by the General ResearchFund of Hong Kong Research Grant Council (#17214516,#N_HKU725/16), Hong Kong Innovation Technology Fund (#ITS/147/15), Hong Kong Health and Medical Research Fund(#03142446), HKU Seed Fund for Translational and AppliedResearch (#201611160006), Sanming Project of Medicine inShenzhen “Team of Excellence in Spinal Deformities and SpinalDegeneration Disease” (SZSM201612055), National Natural ScienceFoundation of China No. 31370957 and Shenzhen Science andTechnology Funding (JCYJ20160429190821781 &JCYJ20160429185449249) and Guangdong Scientific Plan(2014A030313743).

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.biomaterials.2018.05.011.

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1

Supporting information

Zhengjie Lin, Jun Wu, Wei Qiao, Ying Zhao, Karen H. M. Wong, Paul K. Chu,

Liming Bian, Shuilin Wu, Yufeng Zheng, Kenneth M. C. Cheung, Frankie Leung,

Kelvin W.K. Yeung*

Title: Precisely Controlled Delivery of Magnesium Ions thru Sponge-like

Monodisperse PLGA/nano-MgO-alginate Core-shell Microsphere Device to Enable

In-situ Bone Regeneration

Supporting Figures S1:

Figure S1. Intraoperative images of (a) bone defects established at the end of lateral

epicondyle of femur by a hand driller and (b) the post-injection of PLGA or

PLGA/MgO-alginate core-shell microspheres into the defect.

Table S1. The forward and reverse primers of gene used in the RT-PCR assay

Gene Forward primer Reverse primer

ALP 5′-CCAGCAGGTTTCTCTCTTGG-3′ 5′-GGGATGGAGGAGAGAAGGTC-3′

Col 1 5′-GAGCGGAGAGTACTGGATCG-3′ 5′-GTTCGGGCTGATGTACCAGT-3′

OPN 5′-TCTGATGAGACCGTCACTGC-3′ 5′-AGGTCCTCATCTGTGGCATC-3′

Runx2 5′-CCCAGCCACCTTTACCTACA-3′ 5′-TATGGAGTGCTGCTGGTCTG-3′

GAPDH 5′-ACCCAGAAGACTGTGGATGG-3′ 5′-CACATTGGGGGTAGGAACAC-3′

Supporting video:

The video demonstrated the fabrication process of PLGA/MgO-alginate core-shell

microspheres by a specially-designed microfluidic capillary device is uploaded in a

separate file.