Vancomycin- and Strontium-Loaded Microspheres with Multifunctional

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Biological and Medical Applications of Materials and Interfaces

Vancomycin- and Strontium-Loaded Microspheres with Multifunctional Activities in Antibacteria, Angiogenesis and Osteogenesis for Enhancing Infected Bone Regeneration Pengfei Wei, Wei Jing, Zuoying Yuan, Yiqian Huang, Binbin Guan, Wenxin Zhang, Xu Zhang, Jianping Mao, Qing Cai, Dafu Chen, and Xiaoping Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10219 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 3, 2019

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ACS Applied Materials & Interfaces

Vancomycin- and Strontium-Loaded Microspheres with Multifunctional Activities in Antibacteria, Angiogenesis and Osteogenesis for Enhancing Infected Bone Regeneration

Pengfei Weia, Wei Jinga, Zuoying Yuana, Yiqian Huanga, Binbin Guanb, Wenxin Zhangc, Xu Zhangc, Jianping Maod, Qing Caia,*, Dafu Chene,*, Xiaoping Yanga

a

State Key Laboratory of Organic-Inorganic Composites; Beijing Laboratory of Biomedical

Materials; Beijing University of Chemical Technology, Beijing 100029, P.R. China.

b

Department of Stomatology, Tianjin Medical University General Hospital, Tianjin 300052, P.R.

China

c

Department of Endodontics, School and Hospital of Stomatology, Tianjin Medical University,

Tianjin 300070, P.R. China.

d

Department of Spine Surgery, Beijing Jishuitan Hospital, Beijing 100035, P.R. China.

e

Laboratory of Bone Tissue Engineering, Beijing Research institute of Traumatology and

Orthopaedics, Beijing Jishuitan Hospital, Beijing 100035, P.R. China.

*Corresponding authors: [email protected] (Q.Cai); [email protected] (D.Chen)

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Keywords: microsphere; antibacterial; angiogenesis; osteogenesis; bone regeneration

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ABSTRACT

Biomaterials that simultaneously have capacities in inducing bone regeneration and in killing bacteria, are in demand when bone defects face risks of severe infection in clinical therapy. To meet the demand, a kind of multifunctional biodegradable microspheres are fabricated, which contain vancomycin to provide anti-bacterial activity, and contain strontium-doped apatite to provide osteocompatibility. Moreover, the strontium component shows activity in promoting angiogenesis, which further favors osteogenesis. In producing the microspheres, vancomycin is loaded into mesoporous silica and embedded in polylactide-based microspheres via the double emulsion technique, and the strontium-doped apatite is deposited onto the microspheres via biomineralization in strontium-containing simulated body fluid. Sustained release behaviors of both vancomycin and Sr2+ ion are achieved. The microspheres exhibit strong antibacterial effect against S.aureus, while demonstrate excellent cell/tissue compatibility. Studies of differentiation confirm that the introduction of strontium element strengthens the angiogenic and osteogenic expressions of mesenchymal stromal cells. Subcutaneous injection of the microspheres into rabbit back confirms their effectiveness in inducing neovascularization and ectopic osteogenesis. Finally, infected rabbit femoral condyle defect model is created with S.aureus infection and the multifunctional microspheres are injected, displaying significant antibacterial activity in vivo and achieve efficient new bone formation in comparison with biomineralized microspheres without vancomycin loading. The vancomycin and strontium-loaded microspheres, being biomineralized, injectable and biodegradable, are attractive for their flexibility in integrating multifunction into one design, whose potentials in treating infected bone defects are highly expected.

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1. INTRODUCTION

Achieving the effective regeneration of infectious bone defects remains a tough challenge in clinical therapy. The severe contamination in the sites will not only retard the process of bone healing, possibly, but also cause serious adverse consequences such as tissue necrosis and sepsis.13

The treatments for these situations include antibiotics injection intravenously and locally,

however, both of them are not so satisfactory for relatively low concentration of antibiotics reaching the infected sites or short drug durable time.4 To induce efficient regeneration of severely infected bone defects, dual functionalized biomaterials with controlled release of both antibiotics and osteo-inductive factors are highly expected to meet the demand.5

Many attempts have been reported by loading various osteo-inductive factors (e.g. growth factor, bioceramic) into scaffolds developed for bone tissue engineering, displaying significantly promising outcomes in enhancing osteogenesis.6-11 To these scaffolds, in recent years, some studies have tried to incorporate antibacterial components (e.g. silver nanoparticles, antibiotics) for the purpose to treat infected bone defects. For instance, S. Zhang et al. fabricated a composite hygrogel containing both nanosilica and nanosilver to against infection and to enhance osteogenesis in infectious bone defects.12 M.K. Pierchala et al. electrospun a multilayered polylactide/halloysite porous membrane with gentamicin being embedded to prevent infection and favor bone regeneration.13 In our previous work, a kind of bioresorbable microspheres covered with Ag nanoparticles and hydroxyapatite was reported to regenerate S.aureus contaminated calvarial defects in rat model.14 For the critical-sized ( = 8 mm) defect, promising outcomes were obtained in new bone formation in comparison with microspheres without nanosilver loading.15

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S.aureus is found as one of the most popular bacteria that causes infection problems in injured bone tissues.16 Though nanosilver is effective in against both Gram-positive and Gram-negative bacteria, being non-cytotoxic at optimized doses,17,18 it is reported that the efficiency of nanosilver in killing S.aureus is inferior to antibiotics like vancomycin.19 At the meantime, there are concerns about clinical applications of nanosilver because it may linger in the body for a long time and bring potential adverse effect.20,21 In this study, therefore, a kind of vancomycin-containing biomineralized microspheres were proposed as infilling for severely infected bone defect and investigated on their antibacterial and osteogenic activities.

Vancomycin is a common antibiotics available in clinic, being known for its strong activity in killing S.aureus.22,23 Controlled release of vancomycin at the target site is strongly suggested for the purpose to obtain a sustaining effect against infection.5,24,25 For the water soluble feature of vancomycin, significant initial burst release is not avoidable if it is directly encapsulated into microspheres via emulsion technique.26 This has challenged the following biomineralization on microspheres by using simulated body fluids (SBF), because significant loss of vancomycin would occur during the preparation. Before its encapsulation, it is proposed to adsorb vancomycin into mesoporous silica nanoparticles (MSN), a kind of well-known drug carrier,27,28 to increase the loading efficiency of vancomycin. Additionally, MSN itself was reported having the potential in promoting bone regeneration.29

In addition to the apatite coating, the osteo-bioactivity of the microspheres can be further improved by incorporating other bioactive factors.30 Strontium is one of those microelements doping in bone mineral, which plays roles in regulating structure and strength of bone tissue.31-34 Studies have confirmed that strontium salts are unusual for their capacities in enhancing new bone 5



deposition and decreasing bone resorption by modulating osteoblasts and osteoclasts.35 For instance, a dual action bone agent, strontium ranelate (Servier, France), has been approved by European Union as a medication for osteoporosis.36 What’s more, the advantage of strontium is also known for its capacity in enhancing angiogenesis.37-39 Accordingly, the osteo-inductive activity of repairing materials for bone defects was improved with the incorporation of strontium.40,41 In our previous works, SBF biomineralization was applied to prepare microspheres with surface apatite coating, and thus it is possible to dope strontium alongside the apatite deposition taking the advantage of the affinity of strontium to calcium phosphates.14,15

From what have been discussed, biomineralized microspheres loaded with both vancomycin and strontium components were investigated in this study, targeting the regeneration of severely infected bone defects. The microspheres were made of a block copolymer consisting of poly(Llactide) (PLLA) and poly(ethyl glycol) (PEG) blocks. Vancomycin was adsorbed into a mesocellular foam (MCF-26) type MSN and encapsulated into the microspheres via emulsion method. Apatite deposition was carried out in SBF containing strontium chloride. For these microspheres, in vitro characterizations including release behaviors of vancomycin and Sr2+ ions, antibacterial activity, cytotoxicity and capacity in inducing differentiation (angiogenic, osteogenic) of bone marrow mesenchymal stromal cells (BMSCs), were performed. In vivo evaluations were conducted via injecting the multifunctional microspheres both subcutaneously and into infectious femoral condyle defect in rabbit models to identify their potentials in treating severely infected bone defects. The expectable positive outcomes can provide useful guidance in designing and preparing multifunctional bone repairing materials via flexible combinations of both different functional components and different preparation techniques. 6


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2. MATERIALS AND METHODS

2.1. Materials

L-lactide (> 99%) was obtained from Pilot plant of Shandong, Academy of Pharmaceutical Sciences (China). Poly(vinyl alcohol) (PVA), surfactants (Tween 60 and Span 80), vancomycin, strontium chloride and ingredients for the preparation of SBF were all supplied by Aladdin (China). PEG6000 (MW= 6000, from Sigma-Aldrich, USA) and stannous octoate (Sn(Oct)2, from Alfa Aesar, USA) were used directly. MCF-26 type MSN was kindly provided by professor Yunming Fang from Beijing University of Chemical Technology (China), which was synthesized in referring to reference.42 Dichloromethane and HCl were bought from Beijing Chemical Plant (China).

As previously reported,43 PLLA-PEG-PLLA tri-block copolymer was synthesized in lab. Briefly, the L-lactide polymerized using the PEG6000 with two end hydroxyls as the initiator and the Sn(Oct)2 as the catalyst, by setting L-lactide to PEG6000 at the molar ratio of 9:1. The molecular weight and polydispersity of the obtained copolymer were determined ~ 60000 and ~1.3 by gel permeation chromatography (GPC, Waters 1515).

2.2. Preparation of Vancomycin-Loaded Microspheres

Vancomycin (abbreviated as V) was dissolved in deionized water (12 mg/mL), and MCF-26 MSN powders (abbreviated as Si) were dispersed into the solution at different vancomycin/MSN weight ratios (1:1, 2:1 and 3:1). The suspensions were gently stirred at R.T. for 24 h to allow adsorption balance, and then the vancomycin-loaded MSN powders (termed as V@Si) were obtained by centrifugation, followed by deionized water rinsing and lyophilization. 7



Next, the V@Si powders (200 mg) were re-dispersed in deionized water (2 mL). This suspension was dropped into 20 mL PLLA-PEG-PLLA dichloromethane solution (5%, with Span 80 being previously dissolved at 0.1% concentration) under sonication. The formed W/O emulsion was slowly poured into 200 mL PVA aqueous solution (1%, with Tween 60 being previously dissolved at 0.1% concentration) under steady stirring (300 rpm). Allowing solvent evaporation at R.T. for 4 h, the microspheres (simplified as V@Si+M) solidified, which were collected, rinsed and lyophilized. In parallel, blank microspheres (simplified as M) or blank MSN encapsulated microspheres (simplified as Si+M) were similarly prepared, by replacing the V@Si suspension with 2 mL deionized water or a suspension containing blank MCF-26 MSN, respectively.

2.3. Biomineralization and Strontium Incorporation

Biomineralization was performed by immersing microspheres (M or V@Si+M) in a five-times SBF (5SBF) at 37oC for 24 h, accordingly, the obtained microshperes with apatite coating were termed as M+B or V@Si+M+B, respectively. The 5SBF was prepared by dissolving required salts in deionized water at determined concentrations, and Tris-HCl was used to stabilize the solution pH approximate to 6.5.44 To prepare vancomycin and strontium co-loaded microspheres (simplified as V@Si+M+B@Sr), strontium chloride was dissolved in the 5SBF at molar ratios of SrCl2 to CaCl2 being 5:95 or 10:90, and the same biomineralization process was performed for V@Si+M microspheres. For simplification, V@Si+M+B@Sr microspheres resulting from different SrCl2 concentrations were termed as V@Si+M+B@Sr(5) and V@Si+M+B@Sr(10), with the numbers referring to the feeding doses of SrCl2 in the 5SBF. For all the preparations, the biomineralized microspheres were ready for further use after lyophilization.

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2.4. Characterizations

The morphology and particle size of MCF-26 MSN were examined using transmission electron microscope (TEM, Tecnai G2 F20 U-TWIN, USA) and dynamic light scattering (DLS, Brookhaven 90Plus PALS, USA), respectively. The specific surface area of MCF-26 MSN was determined on an ASAP2460 analyzer (Micromeritics, Atlanta, USA) following the multipoint N2 adsorption Brunauer-Emmett-Teller (BET) method. Biomineralized microspheres were analyzed using X-ray diffraction (XRD, Rigaku, Japan) and thermogravimetry analysis (TGA, TA instruments, USA). For TGA analysis, the heating rate was 10oC/min, and the measurement was conducted from R.T. to 800oC in air. X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, VG Scientific) was applied to analyze elemental components and acquired at 20 eV pass energy and 300 W anode power under 3×10-9 mbar.. Microspheres were observed on scanning electron microscope (SEM, JSM-7500F, Japan) after being sputter-coated. For chemical composition analysis, elemental mapping images were obtained (exposure time: 180 s), and Xray energy-dispersive spectroscopy (EDS, Inca X-Max, UK) was conducted. Basing on multiple SEM images, size and size distribution of microspheres were analyzed with ImageJ.

Release behaviors of Ca2+, Sr2+and PO43- ions from biomineralized microspheres were determined by ICP-OES (ICPS-7500, Shimadzu, Japan). In brief, suspensions of microspheres (30 mg) in 10 mL of deionized water were kept at 37oC for 21 days under gentle shaking (50 rpm). At pre-determined intervals, the suspensions were centrifuged and the liquids were submitted to ICP measurements. Meanwhile, the microspheres were re-suspended in 10 mL fresh deionized water and kept at 37oC continuously. Three parallel measurements were conducted for average.

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2.5. In vitro Release of Vancomycin and Antibacterial Tests

Vancomycin concentration in aqueous solution was measured at 280 nm using UV-vis spectrophotometer. In brief, microspheres (30 mg) were dispersed in deionized water (10 mL) to carry out the release test of vancomycin. Liquids were submitted to UV−vis spectrophotometer measurements at pre-determined intervals, and the release was continued by re-dispersing the microspheres in 10 mL fresh deionized water. Three parallel measurements were conducted for average.

Antibacterial analysis of vancomycin-loaded microspheres (V@Si+M+B) was evaluated using culture of S.aureus by both live/dead staining assay and inhibition zone test. S.aureus suspension with a density of 108 colony-forming units (CFU) per milliliter was prepared by incubating the bacteria in brain heart infusion broth (BHI, Solarbio) at 37oC. To each well of 96-well tissue culture plate, microspheres (5 mg) were fitted, and S.aureus suspension (100 L) was added. After 24 h of incubation at 37oC, the systems were examined with acridine orange / ethidiumbromide (AO/EB) staining to judge the live/dead state of S.aureus with fluorescent photos being taken on a confocal laser scanning microscope (CLSM, TCS SP8, Leica). To conduct the inhibition zone test, microspheres (50 mg) were shaped into circular discs ( = 13 mm, h = 1 mm). These discs were put on the surface of agarose gel (1.5%) with ~1×107 CFU S.aureus per milliliter being embedded, followed by keeping the systems at 37°C for one day.

2.6. In vitro Tests

BMSCs, from Sprague Dawley (SD) rat (Cyagen Biosciences, China), were cultured under 5% CO2 and saturated humidity at 37°C. The culture medium was α-MEM (Hyclone) with fetal 10


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bovine serum (Gibco, 10%), penicillin (Sigma-Aldrich, 100 IU/mL) and streptomycin (SigmaAldrich, 100 g/mL) being added. Suspensions of passages 4 BMSCs were prepared for further use by being digested with trypsin/EDTA (Gibco) at 80% confluence. Microspheres for cell culture were sterilized by 75% ethanol and ultraviolet exposure (4 h), followed by removing the residual ethanol by PBS washing and culture medium soaking.

2.6.1. Cytotoxicity Assay

To determine the effects of soluble components (e.g. vancomycin, Sr2+ ions) on cell viability, Transwell assay was applied. To assess the cell affinity of microspheres, BMSCs were seeded onto the microspheres. In both cultures, 1×104 cells and 0.6 mg microspheres were placed into each well, and the cells grew on tissue culture polystyrene (TCPS) were set as control. The culture was continued for 7 days with the medium being refreshed every 2 days. Alamar blue (Invitrogen, USA) assay method was used to determine cell proliferation following the manufacturer’s guidance, the excitation λex = 530 nm and the emission λem = 590 nm were applied to read fluorescence densities on fluorescence microplate reader (CytationTM5, Biotek, USA), which correlated to cell numbers positively.

Seven days after the cells were seeded on microspheres, live/dead assay was conducted using calcein-AM/PI double staining kit (Sigma, USA). To observed cell morphology with SEM, the cell/microsphere complexes were prepared by being fixed with 2.5% glutaraldehyde and soaked in gradient ethanol solutions to complete dehydration. Visualization of cytoskeleton was performed on CLSM by staining the cell/microsphere complexes with phalloidin and Hoechst 33528 for F-actin and nucleus staining, respectively. 11



2.6.2. Differentiation Study

One day after BMSCs (1×105) were seeded onto microspheres (3 mg), osteoinductive medium was used to replace the culture medium in the well. The osteoinductive medium contained 50 μM ascorbate, 100 nM dexamethasone and 10 mM β-glycerophosphate. It was refreshed every 3 days during the differentiation culture. Markers for osteogenic differentiation including alkaline phosphatase (ALP) and collagen type I (COL-I) were detected using ALP assay kit (Thermo Fisher, USA) and COL-I ELISA kit (Thermo Fisher, USA), respectively. Cell lysates were prepared as previously reported by adding cell lysis buffer and applying freezing-thawing treatment.7 For comparison, normalization was conducted on all the measured ALP and COL-I data by using the total protein content that was measured with BCA protein assay kit (Thermo Fisher, USA). Staining on ALP expressions were conducted on cellular samples after 14 days culture, which were treated with 4% paraformaldehyde (1 h) and stained with ALP color development kit (Beyotime, China). Photographs were acquired by optical microscope (Nikon). To evaluate calcium deposition, the cellular samples were collected after 21 days of osteoinductive culture, soaked 30 min in alizarin red working solution (1%, w/v) and observed under optical microscope.

The procedures for quantitative reverse transcription real-time PCR (qRT-PCR) analysis were briefly described as follow. Cell/microsphere complexes were crushed under frozen state using liquid nitrogen, and thenTrizol RNA extract kit (Invitrogen, Thermo Fisher, USA) was applied to extract the total RNA. Genes including BMP2, osteopontin (OPN), COL-I, bone sialoprotein (BSP) VEGF and angiopoietin-1 (Ang-1) were chosen for analysis, among them, the formal four genes were in relation to osteogenesis and the latter two were in relation to angiogenesis. Basing on 12


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those primers shown in Table S1, analysis was performed using qTOWER RT-PCR system (Toyobo, Japan) and data was processed using 18S-rRNA as the normalization standard by a ΔΔCt method.

2.7. In vivo Tests

All animal experiments were performed at Tianjin Medical University (China), which was approved by the University Animal Care and Use Committee and followed the international standards on animal welfare. New Zealand rabbits (2.5-3 kg in weight) were used in the tests.

2.7.1. Subcutaneous Evaluation

In this evaluation, animal surgery was performed by injecting 50 mg V@Si+M, V@Si+M+B or V@Si+M+B@Sr(10) microspheres into subcutaneous pockets on the backs of six rabbits. Twelve weeks post-operation, the implanted microspheres were carefully retrieved together with surrounding tissues. After being fixed with paraformaldehyde (4%) and dehydrated, the tissues were embedded in paraffin and sliced (5 μm in thickness). Histological analysis were conducted by staining the sections with Hematoxylin-eosin (H&E) and Masson’s trichrome (Senbeijia, China), followed by being observed on a digital slice scanning equipment (Nanozoomer, Hamamatsu, Japan). Similarly, kidney and liver sections were also prepared for H&E staining.

Expressions of COL-I and osteocalcin (OCN) were evaluated using immunohistochemical analysis as described in our previous report.7 Briefly, sections were treated with hydrogen peroxide and horse serum in sequence to block endogenous peroxidase and to inhibit nonspecific binding. Primary antibodies (Abcam, UK), i.e. anti-COL-I (No. ab6308) and anti-OCN (No. ab13420), were then applied to treat the sections, followed by being visualized with DAB 13



detection kit (GBI, USA). Images were taken on the Nanozoomer equipment using control sections as references, which were treated in parallel by omitting the use of primary antibodies.

2.7.2. Generation of Infected Bone Defects

To evaluate the efficiency of using V@Si+M+B@Sr microspheres in regenerating infected bone defect, a cylindrical defect (diameter: 6 mm; depth: 8 mm) in rabbit lateral femoral condyle was created and infected with S.aureus for 2 weeks, followed by microspheres being implanted for another 12 weeks. The defects being left unfilled or filled with Si+M+B@Sr microspheres were set as controls. Accordingly, the total 27 rabbits were divided randomly into three groups with 9 rabbits in each group: 1) blank infected bone defect (the control group); 2) filled with Si+M+B@Sr(10) microspheres (the Si+M+B@Sr(10) group); 3) filled with V@Si+M+B@Sr(10) microspheres (the V@Si+M+B@Sr(10) group).

One week before surgery, all rabbits were moved to the site, allowed them to acclimate. Blood analysis was carried out for all the rabbits before the anesthesia by taking blood samples from marginal ear veins. The analysis included levels of white blood cell (WBC), percentage of neutrophile granulocyte and C-reaction protein, as well as interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) being analyzed with ELISA kit. After the rabbits were anesthetized with pentobarbital sodium (3%, 30 mg kg-1) through an intraperitoneal injection, a vertical defect (diameter: 6 mm; depth: 8 mm) was generated in the femoral condyle of each lateral leg by a dental drill. The schematic process of generating infected bone defects was shown in Figure S1. To each defect, a resorbable collagen sponge (Bicon, USA) was pre-soaked with S.aureus suspension (107 CFU) and implanted, and then the incised skin was closed with a 4.0 nylon suture. 14


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Two weeks later, the femoral condyle was re-exposed to remove abscess and implant microspheres. The infected defects were left unfilled for the control group, and the incisions were closed again. After the surgery, no restriction was applied on rabbits in movement and food/water uptaking. During the 2 weeks of defects being infected with S.aureus, as well as, three days after the second surgery (i.e. 3 days after microspheres being filled), blood samples were collected from each rabbit for assessments as above mentioned.

2.7.3. Anti-infection Evaluation in vivo

Three days after the second surgery, three rabbits from each group were euthanized and the femoral condyle was re-exposed using the same incision to collect the formed granulation tissues. A patch of granulation tissue (1 mm3) was vibrated 5 min in sterile normal saline (10 mL), subsequently, 100 L liquid was transferred to a agar plate and spread evenly. The agar plate was placed at 37°C for 24 h and bacterial quantity was counted. In another evaluation, retrieved granulation tissues (1 mm3) were extracted for qRT-PCR test. The extraction of total RNA and corresponding tests were conducted similarly as aforementioned. The primers designed for genes as TNFα, IL-6 and interleukin-1 (IL-1) are listed in Table S2.

2.7.4. Evaluation of New Bone Formation

At 4 and 12 weeks after the microspheres being filled, three rabbits from each group were euthanized to harvest the femoral condyles. After being fixed 24 h in neutral buffered formalin (10%), the femoral condyles were scanned with micro-CT (Bruker micro-CT, Belgium). After standardized reconstruction, three-dimensional images were obtained by data conversion with the aid of the micro-CT system software package. A cylinder ( = 5.8 mm, h = 8 mm) was put in the 15



defect area and the region of interest was analyzed to evaluate new bone formation. Data of new bone volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp) were obtained using CT an software version 1.14 (Bruker micro-CT, Kontich, Belgium).

Next, the femoral condyles were decalcified at R.T. for 7 days using a rapid decalcifier (RapidCal. Immuno, ZS-Bio, China). After being dehydrated and paraffin embedded, the samples were sliced and analyzed both histologically and immunohistochemically as described above in the subcutaneous evaluation.

2.8. Statistical Analysis

All quantitative data were presented as mean ± standard deviation (SD), which were averaged from at least triplicate tests. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Turkey’s test. Differences between groups were considered statistically significant as p < 0.05 and highly significant as P < 0.01.

3. RESULTS

3.1. Preparation and Identification of Various Microspheres

The MCF-26 type MSN used in the present study demonstrated an irregular porous shape with an average particle size of 276±21 nm via ImageJ (Figure S2A and S2E). As the N2 adsorptiondesorption isotherms shown in Figure S2C, the SBET value and the Barrett−Joyner−Halenda (BJH) desorption cumulative volume (VP) of the MCF-26 MSN were measured 287.36 m2 g−1 and 2.47 cm3 g−1, respectively. The average BJH desorption pore size of the MCF-26 was 20~40 nm (Figure 16


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S2D). After vancomycin being loaded (Figure S2B), taking V@Si(Si:V=1:1) as representative, the morphology of the MSN did not show much difference from the pristine one, while the hydrodynamic diameter of the vancomycin-loaded MCF-26 MSN was 286 ± 25 nm, displaying an increase in comparison with that of the pristine MSN (273 ± 12 nm) (Figure S2E). By dispersing the MSN with or without vancomycin in deionized water (1 mg mL−1), the liquid containing vancomycin-loaded MSN showed a distinct UV absorption peak at ~280 nm, while no peak was detected for the liquid containing the pristine MSN (Figure S2F). From these findings, it was confirmed the vancomycin was successfully loaded into the MSN by simple adsorption.

Then, the vancomycin-loaded MSN (i.e. V@Si) was introduced into the inner aqueous phase to prepare the V@Si+M microspheres via the double emulsion method. From SEM observations (Figure 1), the surface of the blank PLLA-PEG-PLLA microspheres (i.e. the M sample) was porous and rough (Figure 1f and 1k). When the vancomycin-loaded MCF-26 MSN was embedded, the resulting V@Si+M microspheres displayed even rougher surface (Figure 1g and 1l), while the pore structure was not so obvious as the M microspheres. For the V@Si+M microspheres, Si element was detected with even distribution by elemental mapping (Figure 1q), confirming the successful encapsulation of MSN powders. After the biomineralization in 5SBF (with or without SrCl2) for 24 h, the V@Si+M+B and V@Si+M+B@Sr microspheres were covered with mineral particles (Figure 1h-1j and 1m-1o). The mineral depositions contained abundant calcium component (Figure 1r), and the V@Si+M+B@Sr microspheres contained strontium component as confirmed by the elemental mapping images (Figure 1s and 1t). It could be seen the strength of strontium signal was stronger in the case of V@Si+M+B@Sr(10) than that in V@Si+M+B@Sr(5), indicating higher concentration of SrCl2 in SBF leading to higher incorporation amount of 17



strontium into the mineral deposition. From EDS analysis (Figure S3), the Sr/(Sr+Ca) molar ratio is measured about 4.50% in the V@Si+M+B@Sr(5) microspheres, and about 7.29% in the V@Si+M+B@Sr(10) microspheres. All these microspheres were in good spherical shapes, with similar average particle sizes. As summarized in Figure S4, the microspheres were mainly in diameters of 100 - 150 μm.

Figure 1. SEM photos (a-o) to display morphology of various microspheres prepared in this study and mapping photos (p-t) to identify the characteristic elements existing in corresponding microspheres: (a, f, k, p) M; (b, g, l, q) V@Si+M; (c, h, m, r) V@Si+M+B; (d, i, n, s) V@Si+M+B@Sr(5); (e, j, o, t) V@Si+M+B@Sr(10).

For the V@Si+M+B and V@Si+M+B@Sr microspheres, further analysis using TGA, XPS and 18


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XRD were conducted. For all biomineralized microspheres, the TGA analysis showed that the minerals deposited in similar amounts (~30%) though presenting a slight decrease alongside the increasing concentration of SrCl2 in the 5SBF (Figure S5A). The XPS results confirmed the existence of strontium component (Sr3d at 132.95eV) in both the V@Si+M+B@Sr(5) and V@Si+M+B@Sr(10) microspheres (Figure S5B), The XRD profiles displayed two broad diffraction peaks around 26° and 32°, revealing the formation of weakly crystallized HA (Figure S5C). The incorporation of strontium did not change the XRD patterns, indicating the doped strontium having not influenced the crystalline structure of HA.

3.2. In vitro Release of Vancomycin and Antibacterial Activity

Biomineralized microspheres containing V@Si were prepared by changing the Si/V weight ratios as 1:1, 1:2 and 1:3. Accordingly, the released concentrations of vancomycin from these microspheres were different, showing apparent dependence on the initially amounts of embedded vancomycin (Figure 2A). However, the release trends were similar for the microspheres, independent on the amounts of embedded vancomycin. They all showed fast release within the first day, then the release rate leveled off gradually. Using the leveled off concentrations, the loading amounts of vancomycin in corresponding V@Si+M+B microspheres were estimated ~32 g, ~56 g and ~70 g/mg microspheres. respectively, for Si/V feeding ratios being set as 1:1, 1:2 and 1:3.

By incubating S.aureus with these V@Si+M+B microspheres for 24 h, live/dead staining was performed to show their antibacterial effects. As shown in Figure 2B, red fluorescence spots representing dead bacteria were observed for all the three V@Si+M+B microspheres, without 19



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showing apparent green fluorescence. The Si+M+B microshperes without vancomycin were also tested as control, from the figure, green fluorescence representing live bacteria was detected in majority. Thus, it was clear that V@Si+M+B microspheres had strong antibacterial activity, while Si+M+B microshperes did not show such activity.

Figure 2. Evaluating vancomycin release behaviors (A) and antibacterial activities of vancomycin-loaded microspheres with different contents of vancomycin in vitro (B-D). (B) Live/dead staining assay of S.aureus co-incubated for 24 h with (a) Si+M+B, (b) V@Si+M+B(Si:V=1:1),

(c)

V@Si+M+B(Si:V=1:2)

and

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(d)

V@Si+M+B(Si:V=1:3)

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microspheres. (C) S.aureus co-incubated with Si+M+B and different V@Si+M+B microspheres for (a) 6 h and (b) 24 h to show the inhibition zones. (D) Statistics on the sizes of inhibition zones of (a) 6 h and (b) 24 h basing on panel (C). ★ Significant (p < 0.05).

In the inhibition zone experiments using V@Si+M+B microsphere discs, as shown in Figure 2C(a) and 2D(a), inhibition zones were able to be detected after 6 h of incubation, though no significant difference being identified between samples at this time point. After 24 h of incubation, the inhibition zones in all the cases were enlarged, with the size of the inhibition zone increasing in the order of V@Si+M+B(Si:V=1:1) < V@Si+M+B(Si:V=1:2) < V@Si+M+B(Si:V=1:3) (Figure 2C(b) and 2D(b)). This was in accordance with the release amounts of vancomycin from the microspheres, showing a stronger antibacterial activity at a higher loading amount of vancomycin in the microspheres.

3.3. Cytotoxicity Assay of Vancomycin-Loaded Microspheres

Since all the prepared V@Si+M+B microspheres demonstrated vancomycin release and antibacterial activity, there were concerns about their potential cytotoxicity against BMSCs. Thereby, these microspheres were co-incubated with BMSCs in Transwell chambers or by seeding cells on the microspheres for 7 days. From Figure S6, cells were seen proliferating continuously in all the groups, however, the growth rate displayed a significantly negative trend as the amounts of loaded vancomycin being raised. Particularly, V@Si+M+B(Si:V=1:2) and V@Si+M+B(Si:V=1:3) microspheres displayed remarkable adverse effects on cell growth, suggesting their significant cytotoxicity. Only V@Si+M+B(Si:V=1:1) microspheres presented 21



comparable cell growth behaviors with those cells being cultured on TCPS. Thus, V@Si+M+B(Si:V=1:1) was used to carry out the following experiments by incorporating strontium at different values during the biomineralization procedure.

Figure 3. (A) Growth of BMSCs in the presence of microspheres using Transwell. (B) Growth of BMSCs seeded on microspheres. Biocompatibility detection of BMSCs and microspheres via Images of (C) SEM observation, (D) phalloidin-Hoechst staining and (E) live/dead staining for cells cultured 7 days on microspheres.

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Then, V@Si+M+B@Sr(Si:V=1:1) microspheres were co-incubated with BMSCs in Transwell chambers or by seeding cells on the microspheres for 7 days. From Figure 3A and 3B, continuous cell growths were identified for both the cases of V@Si+M+B@Sr microspheres being present, sharing similar trends to those cells cultured on TCPS or co-incubated with V@Si+M+B(Si:V=1:1) microspheres. It demonstrated that the incorporation of strontium components had not caused adverse effect on cell proliferation. BMSCs could attach tightly and spreading widely on biomineralized or unbiomineralized microspheres (Figure 3C). The cytoskeleton was clearly visible for all the cells on different microspheres, exhibiting the normal spindle morphology (Figure 3D). All the tested microspheres demonstrated good cell affinity and noncytotoxicity as those green fluorescence representing live cells shown in the live/dead staining images (Figure 3E).

3.4. Ions Release and their Effects on Differentiation

For biomineralized microspheres, release behaviors of ions including Sr2+, Ca2+ and PO43- were detected. From Figure S7, it could be seen that the three kinds of ions were released in a similarly sustained way, while their released amounts varied from case to case. In summary, more Sr2+ ion was released if higher amount of strontium has been incorporated (i.e. V@Si+M+B@Sr(10)) (Figure S7A). The release rates of both Ca2+ and PO43- ions displayed dependence on the incorporation of strontium component. As seen from Figure S7B and S7C, the doping of strontium component would cause a slowdown in the release rates of both Ca2+ and PO43- ions. The higher level the strontium was doped, the slower the release rates were detected for the both Ca2+ and PO43- ions. Theoretically, these differences in ion release behaviors should influence the differentiation of BMSCs when they were co-incubated with the microspheres. 23



The ability of microspheres in inducing osteogenic differentiation was primarily determined by the expressions of general marker proteins as ALP and COL-I. The ALP activity, an early marker indicating the occurrence of osteoblastic differentiation, achieved the highest expressions at the 14th day of osteoinductive culture (Figure 4A). Compared with the control group, higher ALP expression was identified for cells cultured on all the tested microspheres, among them, those biomineralized microspheres displayed even stronger ability in enhancing the ALP expression. The highest ALP expression was found for cells cultured on V@Si+M+B@Sr(10) microspheres, followed by V@Si+M+B@Sr(5) and V@Si+M+B microspheres, showing significant difference between groups. For COL-I synthesis, its content gradually increased alongside the inductive culture, and displayed a similar ascending trend between groups as the ALP expression (Figure 4B). Noticeably, both the strontium-containing microspheres demonstrated remarkably higher COL-I synthesis capacity than other cases. From Figure 4C and 4D, the ALP expression and the mineral synthesis were vividly shown via staining, which clearly illustrated that biomineralization modification, particularly the strontium co-deposition, could promote BMSCs to differentiate osteogenically, showing dependence on the amount of incorporated strontium. The richest ALP staining and calcium deposition were found for cells cultured on V@Si+M+B@Sr(10) microspheres.

Expression of genes (BMP2, OPN, COL-I, BSP) in relation to osteogenesis were then measured to further identify the strong ability of V@Si+M+B@Sr(10) microspheres in inducing osteogenic differentiation. The expressions of the four genes for cells cultured on microspheres were in the order of V@Si+M+B@Sr(10) > V@Si+M+B > V@Si+M (Figure 5(A-D)). In most of these characterizations, the V@Si+M case shared similar results with the TCPS control, suggesting the 24


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incorporated MSN having negligible effect on cell activities. To assess the potential of strontium in enhancing angiogenesis, expressions of two angiogenic genes (VEGF, Ang-1) were also evaluated using PCR alongside the osteogenic differentiation study. From Figure 5E and 5F, the expressions of both the angiogenic genes were detected showing the highest levels for cells cultured on the V@Si+M+B@Sr(10) microspheres, being remarkably higher than cells in other groups including the V@Si+M microspheres.

Figure 4. Extent of osteogenic differentiation being evaluated with the expressions of (A) ALP activity and (B) COL-I content for cells cultured on microspheres, as well as, illustrated by (C) ALP staining and (D) alizarin red staining for cells having been cultured for 14 days. Comparison between experimental groups and TCPS control: ★ Significant (p < 0.05), ★★ highly significant (p < 0.01); comparison between experimental groups: ▲ significant (p < 0.05). 25



Figure 5. Evaluating osteogenic and angiogenic expressions for BMSCs being seeded and inductively cultured on microspheres by detecting gene expressions of (A) BMP2; (B) OPN; (C) COL-I; (D) BSP; (E) VEGF; (F)Ang-1. ★ Significant (p < 0.05), ★★ highly significant (p < 0.01).

3.5. Subcutaneous Angiogenesis and Osteogenesis with Microsphere Implantation

To evaluate the biocompatibility of vancomycin-loaded microspheres, to look into the promotion effects of strontium on angiogenesis and osteogenesis, three kinds of microspheres (V@Si+M, V@Si+M+B, V@Si+M+B@Sr(10)) were injected into the back of rabbits for 3 months. From Figure S8, all the implanted microspheres showed excellent biocompatibility, and no obvious inflammatory reaction could be identified by the gross observation. No abnormality was identified for the liver and kidney tissues basing on H&E staining results (Figure S9). From histological 26


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images of slices retrieved from the implantation sites (Figure 6A), tissues could be seen having grown into the spaces between the microspheres and integrated tightly with the microspheres in all the cases. Promisingly, neovascularization was clearly detected within the implantation. The quantitative analysis on the numbers or the areas of the newly formed vessels per unit area (mm2) revealed that the V@Si+M+B@Sr(10) group displayed a distinct capacity in promoting the neovascularization in comparison with V@Si+M and V@Si+M+B microspheres (Figure 6B and 6C). To evaluate the capacities of the microspheres in inducing ectopic osteogenensis, characterizations including Masson’s trichrome and immunohistochemical (COL-I, OCN) staining were also performed. As shown in Figure S10, the staining color for collagen components became deeper in the sequence of V@Si+M < V@Si+M+B < V@Si+M+B@Sr(10). The COL-I and OCN expressions were also verified richer in the same order. These results verified that biomineralized microspheres were superior than unbiomineralized ones in enhancing the ectopic osteogenesis. Particularly, an even stronger enhancement on the ectopic osteogenesis could be achieved when the strontium component was incorporated into the mineral coating during biomineralization.

27



Figure 6. (A) Histological evaluations (H&E staining) to evaluate the angiogenesis at 12 weeks after the microspheres being subcutaneously injected into rabbit back, and the red arrows indicate the location of neovascularization. Quantitative analysis on the numbers (B) and the areas (C) of vessel-like tissues estimated within selected rectangular areas (1.5 mm × 1 mm) basing on panel A.

3.6. In vivo Antibacterial Activity Against Iinfected Bone Tissue

As above stated, only the microspheres prepared from a relatively low Si/V weight ratio (i.e. 1:1)

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did not display significant adverse effect on cell growth, then it was important to know if the resulting microspheres had enough activity to kill bacteria in the infected bone defects. The defects were created in rabbit femoral condyle and infected with S.aureus for 2 weeks, during which, blood samples were taken and tested. Levels of five indicators in relation to the occurrence of inflammation, including WBC, neutrophile granulocyte, C-reaction protein, TNFα and IL-6 were measured and compared between groups. From Figure 7A, it could be seen the levels of all the five indicators in blood had been elevated within the initial 7 days due to the infection. Within the next 7 days (i.e. 8-14 days), however, a descending trend was found for all the indicators, which was probably ascribed to the autoimmune response. These changes in blood items indicated the success in generating the infected defects. At the end of 14 days, the infected region was visualized with micro-CT and compared with the normal lateral femoral condyle. As presented in Figure S11, the effect of infection on the defect could be distinguished easily by the morphology. After being debrided, micropsheres were filled and 3 days later, blood samples were taken again for analysis. As shown in Figure 7A, the five indicators in relation to inflammation changed differently in different groups. In both the control group and the Si+M+B@Sr(10) microspheres filled group, except the WBC, the other four indicators generally remained the levels. However, they were detected decreasing in the group with V@Si+M+B@Sr(10) microspheres being filled. This blood analysis primarily proved the in vivo antibacterial activity of V@Si+M+B@Sr(10) microspheres.

29



Figure 7. Confirmation of the success in creating infected bone defects and in vivo antibacterial activity of V@Si+M+B@Sr(10) microspheres by: (A) blood analysis on WBC numbers (a), neutrophile granulocyte (b), C-reactive protein (c), IL-6 (d) and TNFα (e); (B) bacterial cloning assay on granulation tissues retrieved from control group (a), Si+M+B@Sr(10) group (b) and V@Si+M+B@Sr(10) group (c), and corresponding bacterial counts (d); (C) qPCR analysis on expressions of genes reflecting inflammatory for retrieved granulation tissues including IL-1β (a), IL-6 (b) and TNFα (c). ★ Significant (p < 0.05), ★★ highly significant (p < 0.01). 30


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At the 3rd day of micropsheres being filled, the defect regions were re-opened to collect granulation tissues for in vitro incubation. The granulation tissues were homogenized and plated for 24 h at 37oC. As the photos in Figure 7B(a, b) shown, bacterial colonies vigorously formed in both the control and the Si+M+B@Sr(10) group, while there was only sporadic bacterial colonies presenting in the V@Si+M+B@Sr(10) group (Figure 7B(c)). By counting the numbers of bacterial colonies, an overwhelming in vivo antibacterial activity of V@Si+M+B@Sr(10) microspheres was illustrated in comparison with other two groups. The expressions of inflammatory-related genes as IL-6, IL-1β and TNFα were further analyzed using qPCR on the extracts from the collected granulation tissues (Figure 7C). The results were in accordance with aforementioned findings that the lowest expressions of these genes were found for the V@Si+M+B@Sr(10) group, showing significant difference from the the control and the Si+M+B@Sr(10) group.

3.7. Regeneration of Infected Femoral Condyle Defect

Next, the osteogenesis of the infected sites was evaluated. The femurs were collected at 4 and 12 weeks post-operation and observed with micro-CT. From Figure 8A, almost no newly formed bone was able to be identified for the control group even at 12 weeks post-operation, and the BV/TV value was estimated quite low (8.36%) (Figure 8B). In both the groups that the defects were filled with Si+M+B@Sr(10) or V@Si+M+B@Sr(10) microspheres, bone regeneration was detected and the defect regions had been partly filled. In comparison, the V@Si+M+B@Sr(10) group demonstrated significantly higher BV/TV values than the Si+M+B@Sr(10) group at both 31



time points post-operation. Further analysis on Tb.Th (trabecular thickness), Tb.N (trabecular number) and Tb.Sp (trabecular spacing) also confirmed that the V@Si+M+B@Sr(10) microspheres had the strongest ability in promoting the new bone formation due to the facts that Tb.Th and Tb.N. values were the highest, while the Tb.Sp value was the lowest in the V@Si+M+B@Sr(10) group among all the measurements. The promotion of Si+M+B@Sr(10) microspheres on osteogenesis was not so strong because they lacked the activity against bacteria and the infection delayed the new bone formation.

The results of H&E, Masson and immunohistochemical (COL-I, OCN) staining were all consistent with the conclusions drawn from micro-CT analysis. From Figure 9, new bone formation was identified being the most significant at 12 weeks post-operation in the V@Si+M+B@Sr(10) group, while both the Si+M+B@Sr(10) group and the control group displayed inferior results. Also, the expressions of COL-I and OCN in the V@Si+M+B@Sr(10) group were identified the strongest among all the groups (Figure 10). The Si+M+B@Sr(10) microspheres could enhance the osteogenesis in comparison with the control group, while they were much inferior to the V@Si+M+B@Sr(10) microspheres. From these staining images, the degradation and resorbability of the microspheres were observed, providing spaces for newly formed bone tissues alongside their degradation. No abnormal response deriving from the degradation products of the microspheres could be detected.

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Figure 8. (A) Reconstructed three-dimensional micro-CT images of rabbit lateral femoral condyle at 4 and 12 weeks post-operation. The regenerated bone being quantitatively evaluated by analyzing (B) BV/TV, (C) Tb.Th (trabecular thickness), (D) Tb.N (trabecular numbers) and (E) Tb.Sp (trabecular space) for the control group and groups filled with Si+M+B@Sr(10) or V@Si+M+B@Sr(10) microspheres. ★ Significant (p < 0.05), ★★ highly significant (p < 0.01).

33



Figure 9. H&E and Masson’s trichrome staining to illustrate the osteogenesis of infected bone defects filled with Si+M+B@Sr(10) or V@Si+M+B@Sr(10) microspheres at 4 and 12 weeks post-operation in comparison with the control group. MS indicates the microspheres, and NB represents the newly formed bone.

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Figure 10. Illustrating the expressions of COL-I and OCN via immunohistochemical staining to evaluate the osteogenesis of infected bone defects filled with Si+M+B@Sr(10) or V@Si+M+B@Sr(10) microspheres at 4 and 12 weeks post-operation in comparison with the control group. MS indicates the microspheres, and NB represents the newly formed bone.

4. DISCUSSION

In facing severely infected bone defects, several issues are essential to achieve efficient regeneration and functional reconstruction.45,46 The first thing is that the repairing materials should have the activity to kill bacteria such as S.aureus, because S.aureus is an ubiquitous bacterial in infected injuries.47 In many published works, nanosilver or antibiotics are common and effective choices in treating infections.48,49 But S.aureus is more sensitive to antibiotics like vancomycin

35



than to the nanosilver.19 The skeleton of vancomycin can bind to the terminal D-alanyl-D-alanine portion of gram-positive bacteria via hydrogen bonding interactions.22 The next issue is that the repairing materials should have the capacity to promote osteogenesis. To this end, bioceramic components are usually incorporated into biodegradable scaffolds in view of the facts that natural bone tissues are intrinsically composited with both organic and inorganic components.50,51 Biomineralization using SBF is an easy and mild way to coat apatite on polymeric scaffolds, and the resulting substrates have demonstrated positive effects in inducing osteogenesis.15 In designing the bone repairing materials, however, there is the third consideration of introducing components that can enhance angiogenesis like strontium component.37,38 because the importance of angiogenesis in accelerating tissue regeneration has been widely accepted.52.53 Therefore, these considerations have put forward the present study to develop a kind of vancomycin and strontium co-loaded microspheres with multifunctional activities in antibacteria, angiogenesis and osteogenesis for the purpose to enhance the regeneration of severely infected bone defects.

Microsphere-type injectable materials are selected for the purpose due to the considerations of their easy implantation into defect sites with diverse shapes and sizes.54 And for a long time, biodegradable microspheres have been studied as drug carriers.55,56 Thus, controlled release of vancomycin from microspheres is envisioned as a ready and feasible design to meet the antibacterial requirement in this study. To encapsulate the water-soluble vancomycin, double emulsion evaporation method was utilized. Due to amphiphilic feature of PLLA-PEG-PLLA copolymer, the fabricated microspheres demonstrated rough and porous surface, which was proved favoring cell attachment.57 In pilot attempts, however, it was found the encapsulated vancomycin dissolved out within hours (data not shown) when the microspheres were soaked in 36


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aqueous solution like SBF for biomineralization. The hydrophilic feature of vancomycin itself and the porous structure of the microspheres both contributed to the rapid release of the encapsulated vancomycin. Apparently, it was not practical to encapsulate vancomycin directly inside the microspheres. A proper carrier to obtain a high loading and controlled release of vancomycin was necessary, and MSNs turned out to be the proper choice, whose porous structure provides spaces to load drugs. Additionally, silicates (including MSNs) have been widely investigated both in cell culture and for in vivo applications.58,59 MSNs are reported able to enter cells via endocytosis, and dissolve under the intracellular acidic environment.60,61 The released Si ion displays positive effect in promoting osteogenic differentiation and new bone formation.29,62 Inspired by these reports, a kind of MCF-26 type MSN with interconnected porous structure and large pore size (20-40 nm) was selected to load vancomycin. By encapsulating the V@Si powders into the microspheres, followed by 5SBF soaking, the resulting biomineralized V@Si+M+B microspheres displayed sustained release behaviors of vancomycin in relation to the amounts of the loaded vancomycin (Figure 2A).

All the V@Si+M+B microspheres demonstrated strong activity against S.aureus in vitro (Figure 2), while only the V@Si+M+B(Si:V=1:1) microspheres showed non-cytotoxicity to the proliferation of BMSCs (Figure S6). The V@Si+M+B(Si:V=1:1) microspheres were proved safe for in vivo applications by both the subcutaneous and femur defect implantation (Figs. 6, 9 and S9). By introducing SrCl2 into the SBF, V@Si+M+B@Sr microspheres were further developed via biomineralization. The prepared V@Si+M+B@Sr(5) and V@Si+M+B@Sr(10) microspheres demonstrated excellent biocompatibility and cell affinity (Figure 3), releasing Sr2+, Ca2+ and PO43+ ions dependent on the amounts of incorporated strontium component (Figure S7). 37



In our previous work, it was proved that biomineralized PLLA-PEG-PLLA microspheres could promote BMSCs to differentiate osteogenically and favor the osteogenesis of critical-sized rat calvarial defects.15 The incorporation of strontium was expected to further strengthen their capacity in promoting osteogenesis.31,34 Strontium is identified as an essential trace element in human body, which has the potential to substitute the calcium element in apatite,40,41 playing roles in inhibiting osteoclastic activity and stimulating bone formation. The doping of strontium into bioceramic like HA, calcium silicate, and bioactive glass, had been identified able to promote the osteoblastic differentiation of BMSCs.32,39 One explanation for the promotion effects of strontium on osteogenesis is ascribed to its ability in promoting angiogenesis in connection with related cytokines. X. Wang et al.37 found that the release of Sr ions in tissue-engineered bone had stimulated the expressions of proangiogenic factors such as VEGF, basic fibroblast growth factor (bFGF) and matrix metalloproteinase-2 (MMP-2). Z. Gu et al.38 suggested that Sr-doped calcium polyphosphate had the potential to enhance neovascularization due to its ability in stimulating de novo synthesis of VEGF and bFGF. S. Zhao et al.39 discovered that Sr ions were able to enhance angiogenesis by promoting osteoblasts to secrete vascular related cytokines. In other published works, it was reported that the strontium component promoted osteogenesis via stimulating angiogenesis through MAPK/ERK1/2 and PI3K/Akt signaling pathways,63,64 which were thought closely relating to events that are able to initiate both osteogenic and angiogenic activities.65 Hif1α is an important mediator in coupling angiogenesis and osteogenesis, and its expression would be enhanced by activating the ERK1/2.66 And PI3K/Akt can act as the downstream signaling of hif-1α to increase relative gene expressions in replying to the angiogenic and osteogenic responses induced by hypoxia.67,68 Accordingly, the V@Si+M+B@Sr(5) and V@Si+M+B@Sr(10) 38


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microspheres both demonstrated greater capacities in inducing the osteoblast differentiation of BMSCs than the V@Si+M+B microspheres, showing a positive relation to the amount of incorporated strontium. And noticeably, the expressions of genes (VEGF, Ang-1) in relation to angiogenesis were also identified being the highest when BMSCs were cultured with V@Si+M+B@Sr(10) microspheres. This promotion of Sr-doped microspheres on angiogenesis was further confirmed via subcutaneous implantation. Vigorous angiogenesis was detected in the V@Si+M+B@Sr(10) group, while not so extensive in both the groups without the presence of strontium. The occurrence of ectopic osteogenesis was also found most likely in the V@Si+M+B@Sr(10) case, followed by the V@Si+M+B and V@Si+M groups. Higher doping content of strontium was not tested in the present study because some studies reported adverse effect on cell activities being identified when the amount of strontium in HA or calcium silicate was more than 10%.63,69

When the V@Si+M+B@Sr(10) microspheres were filled into the S.aureus infected bone defects in rabbit femoral condyle, they were able to kill the bacteria and promote osteogenesis efficiently. In comparison with the unfilled control group, the Si+M+B@Sr(10) group resulted in more hints of osteogenesis, however, its new bone formation was much inferior to that in the V@Si+M+B@Sr(10) group due to the presence of infection. In summary, the developed V@Si+M+B@Sr microspheres were multifunctional, possessing effective capacities in against infection and in promoting biological activities (angiogenesis, osteogenesis), which displayed bright potential in bone tissue engineering, especially when facing infection crisis, to effectively improve regeneration efficiency and functional reconstruction.

5. CONCLUSION 39



Targeting the efficient regeneration of severely infected bone defects, in this work, a success attempt in developing a kind of multi-functionalized bioresorable microspheres has been achieved. The amphiphilic PLLA-PEG block copolymer based microspheres contain vancomycin inside and have strontium-doped apatite deposition on surface. These designs allow the microspheres displaying both antibacterial activity and biocompatibility at proper vancomycin loading amount, remarkable promotion on angiogenesis and bone regeneration in relation to bioactive components as Sr2+, Ca2+ and PO43+ ions. All the evaluations convinced that V@Si+M+B@Sr microsphere is a potential and promising treatment to enhance the regeneration of infectious bone defect. Due to the flexibility in design and fabrication, injectable microsphere-type repairing biomaterials with multifunction is highly expected to have broad applications in the field of tissue engineering.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Contains schematic process of generating infected bone defect, characterizations on MCF-26 type MSN, particle sizes of prepared microspheres, characterizations on biomineralized microspheres, cell proliferation in the presence of micrpshperes, macroscopic observation on subcutaneously implanted microspheres, HE staining on rat liver and kidney sections, ectopic osteogenesis, microCT on infected defect, primers for various genes.

AUTHOR INFORMATION

Corresponding Authors 40


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*E-mail: [email protected] (ORCID: https://orcid.org/0000-0001-6618-0321); [email protected] Notes

The authors declare no competing financial interests. ACKNOWLEDGEMEMTS

The authors acknowledged the financial support from National Key R&D Program of China (2017YFC1104302/4300), National Natural Science Foundation of China (51873018, 51873013), and Beijing Municipal Natural Science Foundation (7182068, 7161001).

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Vancomycin- and Strontium-Loaded Microspheres with Multifunctional

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