CaCO3 Vaterite Microspheres and

Subscriber access provided by READING UNIV

Article

Fabrication of Uniform Casein/CaCO3 Vaterite Microspheres and Investigation on Its Formation Mechanism Yan Li, Ximei Li, Zhinan Cao, Yue Xu, Yihong Gong, and Xuetao Shi Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00306 • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on November 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Fabrication of Uniform Casein/CaCO3 Vaterite Microspheres and Investigation on Its Formation Mechanism Yan Li,a,b Ximei Li,a,b Zhinan Cao,a,b Yue Xu,c,d Yihong Gong,a,b* and Xuetao Shie a

Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument, Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China b Guangdong Provincial Engineering and Technology Center of Advanced and Portable Medical Devices, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China c Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China d Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China e School of Materials Science and Engineering, South China University of Technology, Guangzhou, P.R. China

Uniform Casein/CaCO3 microspheres were fabricated with well tuned properties and the formation mechanism based on the templating effect of casein micelles was proposed. In bone tissue engineering, vaterite microspheres were promising because of the biocompatibility and being easily fabricated. Here Casein/CaCO3 microspheres were precipitated by mixing CaCl2 and Na2CO3 solutions under stirring in the presence of casein. All samples were mainly in vaterite, composed of aggregates of nano-sized crystals. With the increase of casein concentration, the amount of calcite and microspheres size decreased while the loading content of casein increased, suggesting that casein induced the formation of vaterite and also stabilized the crystal phase. The formation mechanism was further investigated. With the increase of CaCl2 amount, the size of forming microspheres increased while the zeta potential was stabilized, the polycrystalline nature was shown and the presence of Ca inside microspheres was confirmed. Hence, the formation mechanism based on casein micelles which demonstrated the template effect was proposed. Casein/CaCO3 microspheres enhanced the deposition of hydroxyapatite crystals and sample II-8 was tested as the most cytocompatible on mesenchymal stem cells. The properties of Casein/CaCO3 microspheres could be well controlled; for better performance in bone tissue engineering, forming composite scaffolds may be preferred.

ACS Paragon Plus Environment 1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

*Corresponding author: Yihong Gong, Telephone: +86-20-39332146; Email: [email protected]


Page 2 of 33

+86-20-39332146;

Fax:

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fabrication of Uniform Casein/CaCO3 Vaterite Microspheres and Investigation on Its Formation Mechanism Yan Li,a,b Ximei Li,a,b Zhinan Cao,a,b Yue Xu,c,d Yihong Gong,a,b* and Xuetao Shie a

Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical

Instrument, Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China b

Guangdong Provincial Engineering and Technology Center of Advanced and

Portable Medical Devices, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China c

Department of Orthodontics, Guanghua School of Stomatology, Hospital of

Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China d

Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University,

Guangzhou, Guangdong, P.R. China e

School of Materials Science and Engineering, South China University of Technology,

Guangzhou, P.R. China

ABSTRACT Uniform Casein/CaCO3 microspheres were fabricated with well tuned properties and the formation mechanism based on the templating effect of casein micelles was proposed. In bone tissue engineering, vaterite microspheres were promising because of the biocompatibility and being easily fabricated. Here Casein/CaCO3 microspheres were precipitated by mixing CaCl2 and Na2CO3 solutions under stirring in the presence of casein. All samples were mainly in vaterite, composed of aggregates of nano-sized crystals. With the increase of casein concentration, the amount of calcite and microspheres size decreased while the loading content of casein increased, suggesting that casein induced the formation of vaterite and also stabilized the crystal phase. The formation mechanism was further investigated. With the increase of CaCl2 amount, the size of forming microspheres increased while the zeta potential was stabilized, the polycrystalline nature was shown and the presence of Ca inside microspheres was confirmed. Hence, the formation mechanism based on casein micelles which demonstrated the template effect was proposed. Casein/CaCO3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

microspheres enhanced the deposition of hydroxyapatite crystals and sample II-8 was tested as the most cytocompatible on mesenchymal stem cells. The properties of Casein/CaCO3 microspheres could be well controlled; for better performance in bone tissue engineering, forming composite scaffolds may be preferred.

KEY WORDS: vaterite microspheres, fabrication, characterization, formation mechanism, tissue engineering


Page 4 of 33

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Bone is a type of highly vascularized tissue which provides mechanical support for muscles and organs. Inorganic component such as hydroxyapatite composes 65%-70% of bone, hence it is also functioning as a mineral reservoir.1 The remaining amount belongs to collagen fibers which are around the hydroxyapatite nanocrystals to form the well-organized composite structure as extracellular matrix (ECM) in bone tissues.2 Bone usually has a unique capacity to repair and remodel its structure without leaving scars. However, its structure and remodel process may be impaired and inactivated by diseases or accidents. Thus, bone grafts are widely needed to repair these defects.

Bone defects can be repaired by transplanting bone grafts. However, surgical procedures are needed to remove grafts from donors. Such transplantations are limited by grafts availability, donor site infections and pain caused by second surgery.3 Tissue engineering which combines molecular and cell biology technology and biomaterials together to regenerate tissues,4 provides a new and better strategy.5 In tissue engineering, biomaterial plays an important role, which serves as a substrate for cells to attach and migrate, a delivery vehicle to transport some specific types of cells, and also a drug carrier to activate the local cell response.3

In bone tissue engineering, coral exoskeletons (mainly calcium carbonate in aragonite) were one kind of remarkable biomaterial of which the 3D morphology and pore interconnections are similar to human bones.6 Coral implants were reported to enhance space provision for guided tissue regeneration and alveolar bone formation to repair periodontal defects in adult beagle dogs,7 which demonstrated its osteoconductivity, showing that it was a suitable material for bone repair. However, corals for medical applications were limited to a select number of species: Porites, Acropora, Lobophyllia, Goniopora, Polyphyllia and Pocillopora, and have been overused.6 Thus researchers have been trying to develop a synthetic biomaterial to replace it.

Calcium carbonate (CaCO3), the main content in corals, is also a mineral with an extremely wide range of applications in health care, industry and environmental areas, which can be used as a substitution for coral implants. There were mainly three methods to fabricate CaCO3, fast precipitation by mixing calcium chloride (CaCl2)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and sodium carbonate (Na2CO3) solutions, diffusion route using CaCl2 and (NH4)2CO3, and carbonation route using Ca(OH)2 and CO2 as reagents.8 In addition, other modified method such as in situ generation of CO32− ions via hydrolysis of dimethylcarbonate in the presence of CaCl2 was reported.9 The fabrication process needed to be precisely controlled to obtain CaCO3 with desired morphological and crystallographic properties.

CaCO3 has three anhydrous crystal polymorphs, calcite, aragonite and vaterite. The thermodynamic stability is indicated by the solubility product Ksp, where Calcite is the most stable form (logKsp,calcite = -8.475), followed by aragonite (logKsp,aragonite = -8.36) and the most unstable one is vaterite (logKsp,vaterite = -7.913).10 Among these three crystal phases, vaterite has been reported to have advantages such as higher bioactivity regarding transformation into hydroxyapatite11, 12 and also better suitability as carriers for drug release purpose.13,

14

However, vaterite is the least

thermodynamically stable form and organic additives are usually needed to stabilize its crystals.

Various molecules have been applied to influence the crystallization of CaCO3, such as amino acids including alanine, lysine and glycine,15 acrylic acid polymers16 and poly(sodium 4-styrenesulfonate).10 When ethylene glycol and glycerol were included in CaCl2 and Na2CO3 solutions, the formation of vaterite was promoted and the precipitated spherical framboid particles were in the size range of 350 nm to 2 µm.14 These vaterite nanoparticles were used as sacrificial templates for assembly of polymer multilayer capsules.14 In the presence of small amounts of sodium polyacrylate chains, stable amorphous CaCO3 particles were fabricated. The formation process was initiated by an in situ generation of CO32− ions through hydrolysis of dimethylcarbonate in the presence of CaCl2 and analyzed with means of time-resolved small angle X-ray and light scattering. The results suggested that in the presence of sodium polyacrylate chains, calcium polyacrylate aggregates were first formed, and then followed by a modulated amorphous CaCO3 growth, during which the shape of particles was changed toward loose and less homogeneous entities.9 However, when sodium polyacrylate was added into the reaction system in a delayed manner (delaying the addition from 1 to 60 min), vaterite microparticles were successfully obtained. The formation mechanisms of CaCO3 particles were found to


Page 6 of 33

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


be different when the addition time of sodium polyacrylate was changed.17 Besides polyelectrolytes, the effect of egg-white protein ovalbumin on the formation of CaCO3 particles was investigated using time-resolved small-angle neutron scattering.18 The investigations revealed that without protein, CaCO3 particles were homogeneously crystallized, involving an initial formation of thin plate-shaped nuclei which subsequently reassembled to three dimensional particles. In the presence of ovalbumin, amorphous CaCO3 was first formed; Ca2+-mediated unfolding and cross-linking of protein monomers accompanied the formation and dissolution of amorphous CaCO3. During the formation of amorphous phase, the protein complexes acted as nucleation centers. The amorphous CaCO3 was subsequently transformed into crystalline polymorphs vaterite and aragonite.18 Moreover, self-assembled films were used to control CaCO3 morphologies and polymorphs.19 Yamamoto et al.20 reported that CaCO3 also nucleated under the induction of chitin matrix in chitin gels to form a three-dimensional hybrid structure. However, none of these additives have the potential to demonstrate osteoconductivity or to improve the bioactivity of CaCO3. Meanwhile, little is known about the fabrication parameters of fast precipitation on the properties of CaCO3 vaterite microparticles. Herein, casein which has a strong affinity with Ca2+ ions to form a unique calcium-phosphate transport complex in mammary glands21 was selected to stabilize the crystallization of CaCO3. In addition, casein was also reported to improve bone repairing22 and enhance the osteogenic differentiation of mesenchymal stem cells (MSC).23,

24

Here, the fast precipitation method was conducted to fabricate

Casein/CaCO3 microspheres using CaCl2 and Na2CO3 solutions in the presence of casein. To investigate the effects of fabricaion parameters on the properties of microspheres and also the formation mechanism, concentration of casein, mixing mode and the adding rate of solutions were varied. Several analytical techniques, including X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and BCA protein assay were applied to characterize the products. The in vitro bioactivity of Casein/CaCO3 microspheres were evaluated by soaking in simulated body fluid (SBF) and its cytotoxicity was investigated using human bone marrow mesenchymal stem cells (hBMSCs).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EXPERIMENTAL SECTION Material Casein (from bovine milk) was purchased from Sigma Aldrich (St, Louis, MO, USA). Na2CO3, CaCl2, dichloromethane (DCM, 99.5%), dimethyl formamide (DMF, 99.5%), dimethylsulfoxide (DMSO, 99.0%), and ethanol were obtained from Guangzhou Chemical Reagent Co. (Guangzhou, Guangdong, China) and used without further purification. SBF was prepared following procedures as reported elsewhere25 and all chemicals needed were from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Alpha minimum essential medium (α-MEM) and PS (penicillin/streptomycin antibiotics) were purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) and trypsin-EDTA were purchased from Gibco (Carlsbad, CA, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was from Amresco (Solon, OH, USA). Deionized (DI) ultrapure water was used throughout the experiment.

Synthesis of Casein/CaCO3 Microspheres Solutions of CaCl2 and Na2CO3 at a concentration of 50 mM were first prepared. Casein was dissolved in Na2CO3 solution at various concentrations. Under stirring at 600 rpm, either Na2CO3/casein solution (20 ml) was added into CaCl2 solution (20 ml) (mixing mode I) or CaCl2 solution was added into Na2CO3/casein solution (mixing mode II). After addition, the mixture was stirred for 20 min. Microspheres were collected by centrifugation, followed by rinse three times with DI water, freeze-dried and stored in a desiccator for characterizations.

In order to obtain Casein/CaCO3 microspheres with a high amount of casein and narrow size distribution, fabrication parameters including concentration of casein (2, 4, 6 or 8 mg/ml in Na2CO3 solution), mixing mode and adding rate of solutions (fast addition at ~50 ml/min or slow injection at 2 ml/min) were varied (summarized in Table 1). All fabrication processes were conducted at room temperature.


Page 8 of 33

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Fabrication and characterization of Casein/CaCO3 microspheres

NO

Sample name

1 2 3

I-2 I-4 I-6

4 5 6

I-8 I-2_2 II-2

7 8 9

II-4 II-6 II-8

Mixing mode

Ia

I_2

b

IIc

casein Particle (mg/ml) size (µm)

Loading Encapsulatio content of n efficiency casein (wt%) of casein (%)

2

3.08±0.68

15.7±0.1

54.8±0.3

4 6

2.13±0.42 1.09±0.39

16.3±2.2 18.5±4.5

36.7±5.0 33.9±8.2

8 2 2

1.14±0.43 4.40±1.40 1.67±0.43

24.0±1.2 13.1±0.8 15.3±0.4

38.8±1.9 45.7±2.9 53.6±1.2

4 6 8

1.19±0.18 1.11±0.18 1.30±0.47

20.0±0.5 20.3±0.5 23.9±1.0

45.1±1.2 37.3±0.9 38.9±1.6

a

CO32- solution was quickly added into Ca2+ solution at ~50 ml/min, mixing mode I

b

CO32- solution was injected into Ca2+ solution at 2 ml/min using a syringe pump

(KD100, KD Scientific, USA) c

Ca2+ solution was quickly added into CO32- solution at ~50 ml/min, mixing mode II

Characterization of Casein/CaCO3 Microspheres XRD analysis The crystal phase in Casein/CaCO3 microspheres was studied using XRD (D/Max-IIIA) with monochromatic Cu Kα radiation at 40 kV and 20 mA. The data was collected in a 2θ range of 20° to 60° at a rate of 0.05° sec−1. The average crystalline size was calculated based on Scherrer equation (Eq 1),26 where d represents the average crystalline size, k is the shape factor, λ is the X-ray wavelength, FWHM is the line broadening at half of the maximum intensity (FWHM) in radians, and θ is the Bragg angle. Here, a shape factor of 1.0 was chosen. FWHM of (112) peak at 27.1° for vaterite and (104) peak at 29.4° for calcite was relied on.

d=

k ×λ FWHM × cosθ

(1)

FTIR investigation The functional groups of samples were examined with a FTIR spectroscopy



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

(VERTEX 70, Bruker Optics, Germany) in the wavenumber range of 4000-400 cm−1 at room temperature with a resolution 4 cm−1. Samples were grounded with KBr and pressed into nearly transparent pellets.

SEM observation The shape and surface morphology of Casein/CaCO3 microspheres were observed using a SEM (JSM-5600, JEOL, Tokyo, Japan) at an accelerating voltage and a beam current at 15 kV and 10 mA, respectively. Before observation, the freeze-dried microspheres were sputter-coated with gold.

BCA protein assay The amount of casein in Casein/CaCO3 microspheres was quantified with a BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA). The microspheres were dissolved in EDTA solution (2 mM, pH 8.5) and manufacturer’s instructions were followed with bovine serum albumin (BSA) as the reference standard. The loading content (Eq 2) and encapsulation efficiency (Eq 3) was determined based on the following equations, where the theoretical loading content was calculated based on the assumption that all added casein was entrapped in microspheres and all added CaCl2 and Na2CO3 were precipitated as CaCO3 microspheres. Loading content (%) = (weight of casein/weight of microspheres) × 100%

(2)

Encapsulation efficiency (%) = (loading content/theoretical loading content) × 100% (3)

Mechanism of Casein/CaCO3 Microspheres Formation The crystallization of Casein/CaCO3 microspheres was very fast. In order to investigate the formation mechanism, all fabrication parameters were the same as that described in section “Synthesis of Casein/CaCO3 Microspheres” except the volume of CaCl2 solution. Here, a small volume (0.1, 0.25, 0.5, 0.75, 1 or 2 ml) of CaCl2 solution (50 mM) was rapidly added into 20 ml of Na2CO3 solution (50 mM) containing casein at 2 mg/ml or 8 mg/ml. After stirring for 20 min, suspensions were collected without centrifugation; since casein were in micelles, the size and zeta potential of forming microspheres in suspensions were first measured with a Zetasizer Nano-ZS90 (Malvern, UK) and further characterized using a transmission electron


Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microscope (TEM, FEI Tecnai G2 Spirit, USA) operated at 120 kV. Regarding the functional groups, suspensions were centrifuged and the precipitate was collected, rinsed with DI water, freeze-dried and analyzed with FTIR.

In Vitro Bioactivity Assessment To better investigate the in vitro bioactivity of Casein/CaCO3 microspheres, polycaprolactone (PCL) electrospun composite membrane was fabricated.24 I-8 was selected as the representative microspheres and the formed composite membrane was named as PV; while calcite was used for comparison and the formed sample was PC; the membrane without CaCO3 was named as P. Briefly, 100 mg I-8 or calcite microparticles were well-dispersed in 0.5 ml DMF and 200 mg PCL was dissolved in 1.5 ml DCM. For sample P, DMF was added into PCL/DCM solution. Then these two solutions were mixed and stirred for 24 hr at room temperature. The solution was electrospun under a voltage of 14 kV at a flow rate of 1.0 ml/hr and the electrospun membrane was collected on an aluminum foil which was 11 cm away from the injection needle tip. To expose the entrapped CaCO3, and also improve the hydrophilicity, the electrospun membranes were slightly hydrolyzed in NaOH solution (1.5 M) at 4°C for 1 hr, then washed extensively with DI water and dried in vacuum at room temperature for 24 hr. The membranes were soaked in SBF at 37°C for 21 days without changing the SBF. Then samples were gently rinsed with DI water and dried at room temperature. The surface morphology was observed using SEM. The concentration of calcium in SBF on day 1 and day 21 was measured using a Calcium-O-Cresolphthalein complexone method as reported elsewhere.26

In Vitro Cytotoxicity Tests In order to evaluate the cytotoxicity of Casein/CaCO3 microspheres, II-8 and II-2 were selected as the representatives and extraction assay was carried out using hBMSCs as model cells. For comparison, calcite was included. Microparticles were first sterilized by soaking in 75% ethanol for 3 hr, then incubated in medium at 20 mg/ml in an incubator for 24 hr, and the corresponding supernatant was collected after centrifugation. The supernatant was the extracted medium used for cytotoxicity test. HBMSCs (bought from Sciencell Research Laboratories, Carlsbad, CA, USA) were seeded onto a 48-well plate at a density of 6000 cell/well and cultured in α-MEM medium supplemented with 10% FBS and 1% PS solution in an incubator (Thermo



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

scientific) for 2 days. Then the medium was exchanged with extracts (0.5 ml/well) and cells cultured in medium containing Triton X-100 at 0.1% were as the control group. After cultured for 24 and 72 hr, MTT assay was conducted to evaluate the relative cellular viability.

RESULTS AND DISCUSSION Fabrication and Characterization of Casein/CaCO3 Microspheres In this study, fast precipitation method was carried out to fabricate Casein/CaCO3 microspheres at room temperature. In order to obtain small microspheres with a high loading content of casein, various fabrication parameters were widely investigated. The effects of casein concentration and mixing mode on the properties of Casein/CaCO3 microspheres were firstly analyzed. As shown in Figure 1, all diffraction peaks were well-resolved and sharp-shaped, indicating all Casein/CaCO3 microspheres were well crystallized. These strong peaks were assigned to (004), (110), (112), (114), (300), (118) and (224) planes of vaterite (JCPDS# 74-1867), showing all samples were mainly in vaterite phase. Furthermore, the width of these strong peaks was broad, indicating these vaterite microspheres were aggregates of nanoparticles. Besides the peaks belonging to vaterite, two small diffraction peaks of calcite (JCPDS# 86-0174) at 29.4° and 39.3° were also observed. The intensities of these two calcite peaks which represented the amount of calcite decreased with the increase of casein concentration, where at higher concentrations of casein (i.e., 6 and 8 mg/ml), there were nearly no calcite peaks present. Here, the change regarding the relative peak intensities of calcite phase in Casein/CaCO3 microspheres along with casein concentration was very different from the literature.27 In the literature, a peak of calcite at 29.4° was always present, and much higher than vaterite peaks of (112) and (114) planes even when the concentration ratio of CaCl2/Casein (i.e., 20 mM/(2 mg/ml)) was close to the value here (i.e., 50 mM/(4 mg/ml), sample I-4 and II-4). The difference was probably resulted from the fact that here casein was dissolved in Na2CO3 solution while in the literature casein was in CaCl2 solution. Furthermore, the intensities of calcite peak at 29.4° for II-2, II-4 were lower than that for I-2, I-4, respectively. Hence, when the same casein concentration (smaller than 4 mg/ml) was applied, calcite amount in Casein/CaCO3 microspheres was smaller by applying mixing mode II than that by mixing mode I.


Page 12 of 33

Page 13 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. XRD patterns of all Casein/CaCO3 microspheres synthesized at different casein 2-

2+

concentrations using (a) mixing mode I (CO3 solution was quickly added into Ca ~50 ml/min) and (b) mixing mode II (Ca

2+

solution was quickly added into

2CO3

solution at

solution at ~50

ml/min) under stirring. “C” represents peaks belonging to calcite phase.

The average crystalline size of vaterite and calcite phase in each sample was calculated based on Scherrer equation and XRD diffraction peaks of (112) and (104) planes, respectively (Table 2). The sizes of both vaterite and calcite phases decreased slightly with the increase of casein concentration (from 19.0 nm to 15.9 nm for vaterite, from 52.2 nm to 28.7 nm for calcite), where the trend was not affected by the mixing mode. At the same casein concentration, the average crystalline size of calcite was smaller when mixing mode II was adopted (i.e., the values for I-2 and II-2 were 52.2 nm, 43.7 nm, respectively).

Table 2. The calculated average crystalline size of vaterite and calcite phase in Casein/CaCO3 microspheres based on Scherrer equation. NO 1 2 3 4 5 6 7

Sample name I-2 I-4 I-6 I-8 I-2_2 II-2 II-4

Vaterite (nm) 18.7 18.4 15.9 16.1 20.6 19.0 16.7

Diffraction plane 112 112 112 112 112 112 112

Calcite (nm) 52.2 36.4

Diffraction plane 104 104

56.3 43.7 28.7

104 104 104



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8 9

II-6 II-8

16.6 17.6

112 112

The functional groups in all samples were analyzed using FTIR. As shown in Figure 2, for all samples, besides the CO32- adsorption bands at 1318 cm-1 and 870 cm-1,28, 29 the characteristic band of vaterite at 751 cm-1 was also present;30 due to the presence of casein, C-H band at 3400 cm-1 and -CONH- absorption band at 1650 cm-1 were both observed.31 For I-2 and II-2, the characteristic adsorption band of calcite at 706 cm-1 was also present. With the increase of casein concentration, the corresponding adsorption bands belonging to casein intensified, especially for -CONH- absorption band at 1650 cm-1, suggesting that the amount of casein in Casein/CaCO3 microspheres increased.

The morphology of Casein/CaCO3 microparticles were observed under SEM. As shown in Figure 3, all samples were in spherical morphology, which was very different from the reported shape, a spiky dumbbell-like superstructure.27 In the literature, casein was dissolved in Ca2+ solution27 while here casein was in CO32solution. The different fabrication routes probably resulted in the different morphologies. When casein concentration was low (i.e., smaller than 4 mg/ml) and the mixing mode was the same, the diameter of microspheres decreased with the increase of casein concentration. The diameter of all samples was summarized in Table 1 and the value decreased from 3.08±0.68 µm (for I-2) to 2.13±0.42 µm (for I-4). The trend was similar to Trushina et al14 and Wang et al’s10 results, where the size of vaterite particles decreased with the increase of organic additives in the reaction system when fast precipitation method was applied. In addition, when sodium polyacrylate was included into the reaction system in a delayed manner and diffusion route was used, the size of formed vaterite microparticles also slightly decreased with the increase of sodium polyacrylate concentration.17 Even though the trends were similar, the effects of reported additives on crystallization of vaterite were probably different from that of casein in this research. At the same concentration of casein (smaller than 4 mg/ml), the Casein/CaCO3 microspheres fabricated using mixing mode II had smaller diameters in narrower distributions (for II-2, the diameter was 1.67±0.43 µm and for I-2, the diameter was 3.08±0.68 µm) and the surface of microspheres appeared to be smoother. When casein concentration was high (i.e.,


Page 14 of 33

Page 15 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


larger than 6 mg/ml), the size of Casein/CaCO3 microspheres was not significantly affected by the mixing mode and casein concentration.

Figure 2. FTIR spectra of all Casein/CaCO3 microspheres synthesized at different casein 2-

2+


2+


CO32-

solution at

solution at ~50

ml/min) under stirring. “V” indicates the characteristic band of vaterite and “C” indicates the characteristic band of calcite.

The loading content of casein in Casein/CaCO3 microspheres was quantified using a BCA protein assay kit. Regardless of mixing mode, when casein concentration increased from 2 mg/ml to 8 mg/ml, the loading content of casein increased from ~15 wt% to ~24 wt% which was consistent with the intensified -CONH- absorption band at 1650 cm-1 (Figure 2); while the encapsulation efficiency decreased from ~55% to ~39%. When casein concentration was fixed, it seemed that more casein was encapsulated into these microspheres when mixing mode II was adopted (Table 1). Samples I-2, I-8, II-2 and II-8 were selected as representatives for measurement of practical yields which were 98.6±2.5 mg, 98.0±0.8 mg, 100.5±1.0 mg and 102.2±0.1 mg, respectively, for each batch as described in section “Synthesis of Casein/CaCO3 Microspheres”. If assuming that added casein, CaCl2 and Na2CO3 were all precipitated as microspheres, theoretical yields of samples fabricated at casein concentration 2 mg/ml, 8 mg/ml were 140 mg, 260 mg, respectively. Thus the percent



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

yields for I-2, I-8, II-2 and II-8 were 70.4% ± 1.8%, 37.7% ± 0.3%, 71.8% ± 0.7% and 39.3% ±0.0%, which for samples at casein concentration of 2 mg/ml were much higher than that at 8 mg/ml. Besides the fact that not all casein were encapsulated into microspheres, the smaller microspheres of I-8 and II-8 may also contribute to the difference as smaller microspheres may be easier to be lost during centrifugation and rinse. In addition, the percent yield for mixing mode II was slightly higher than that for mixing mode I.

Figure 3. SEM images of all Casein/CaCO3 microspheres synthesized at different casein 2-

2+


2+


CO32-

solution at

solution at ~50

ml/min) under stirring. Scale bar represents 5 µm.

Based on these investigations, it was found that in the presence of casein, the formed


Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microspheres were mainly in vaterite phase. With the increase of casein concentration, the amount of calcite in the samples was reduced while the amount of casein was increased, and the size of microspheres was decreased. When fabricated using mixing mode II (Ca2+ solution was quickly added into CO32- solution) at a small casein concentration (i.e., smaller than 4 mg/ml), it was easier to obtain vaterite microspheres in smaller size with narrower size distribution than fabricated under mixing mode I.

Figure 4. Fabrication of Casein/CaCO3 microspheres by slowly adding CO32- solution (the concentration of casein at 2 mg/ml) into Ca2+ solution using a syringe pump at 2 ml/min (mixing mode I). I-2_2 was characterized using (a) XRD, (b) FTIR and (c) SEM. “C” represents XRD diffraction peak belonging to calcite phase and “V” indicates the characteristic band of vaterite. The scale bar is 5 µm.

Investigation on the Formation Mechanism of Casein/CaCO3 Microspheres The properties of Casein/CaCO3 microspheres were influenced by casein concentration and the mixing mode, where the size and morphology was more sensitive to casein concentration under mixing mode I (CO32- solution was quickly added into Ca2+ solution). In order to investigate the formation mechanism of Casein/CaCO3 microspheres, under mixing mode I, the adding rate of CO32- solution (casein concentration at 2 mg/ml) was reduced to 2 ml/min (the sample was named as I-2_2). As shown in Figure 4, I-2_2 was mainly in vaterite phase, the characteiristic adsorption band of vaterite at 751 cm-1 was observed and samples were in spherical



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

morphology with an average size of 4.40±1.40 µm. When compared with I-2 (addition rate of CO32- solution was ~50 ml/min), the calcite diffraction peaks were smaller, representing that the calcite amount was smaller; the average crystalline sizes (Table 2) and average microsphere size were slightly larger; microspheres in the range of 1.07 µm to 7.33 µm were present in the SEM image, demonstrating that the size distribution was broadened; the loading content of casein was smaller (Table 1). Here, casein was dissolved in CO32- solution. When CO32- solution was slowly added into the reaction system, it seemed that both the nucleation rate and the crystal growth rate were slow, resulting larger average crystalline size and broadened microsphere size distribution.

Based on these studies, it was found that under mixing mode I, uneven microspheres were easily formed and calcite was inevitably precipitated at casein concentration of 2 mg/ml. Hence, to continue investigating the formation mechanism of Casein/CaCO3 microspheres, mixing mode II were then adopted and the adding rate of Ca2+ solution into CO32- solution (casein concentration at 2 mg/ml) was first reduced to 2 ml/min. As shown in Figure S1 (supporting information), the calcite diffraction peaks were significantly smaller than that in II-2. This along with the properties of I-2_2 suggested that even when the addition rate was slow, vaterite was preferred to be precipitated and stabilized due to the presence of casein.

Under mixing mode II, various volumes (e.g., 0.10, 0.25, 0.50, 0.75, 1.00 or 2.00 ml) of Ca2+ solution were then added into CO32- solution (20 ml, casein concentration at 2 or 8mg/ml) under stirring. When 0.10 ml of Ca2+ solution was added, the reaction solution was transparent and no precipitate was formed; more than 0.25 ml of Ca2+ solution was added, after stirring for ~1 min the transparent solution became cloudy. As casein was in micelles,32 the suspension was collected for size investigation. As shown in Figure 5(a), with the increase of CaCl2 amount, the size first increased and then stabilized. For 2 mg/ml system, the size increased from 275 nm and stabilized at ~1400 nm; for 8 mg/ml system, the size increased from 280 nm and stabilized at ~900 nm. When the added volume of Ca2+ solution was the same, the size at 8 mg/ml of casein was always smaller than that when casein concentration was 2 mg/ml. In addition, when a small volume (i.e., 0.10 ml) of Ca2+ solution was added, the size was nearly the same as that for blank micelles, which was also similar to the reported


Page 18 of 33

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


value. The diameter of bovine casein micelles was in the range of 80–400 nm with an average value of ~200 nm.33

The forming microspheres in 8 mg/ml system were further selected as representatives for zeta potential evaluation and TEM characterizations. As shown in Figure 5(b), the value was stabilized at ~ -17.0 mV which was not affected by the amount of added Ca2+. When observed under a TEM, blank micelle was ~200 nm (Figure 5c1); the forming microspheres gradually enlarged (~400 nm for Figure 5c4), became condensed and regular shaped with the increase of Ca2+ amount (Figure 5c2-c4 and Figure S2). No diffraction spots were detected for blank micelles (Figure 5d1). When a small amount of Ca2+ (i.e., 0.10 ml) was added into Na2CO3/casein solution, SAED pattern showed the polycrystalline nature of forming microspheres (Figure 5d2). When more Ca2+ was added into the reaction system, the diffraction became stronger and all demonstrated the polycrystalline characteristics (Figure 5d3-d4). Limited by resolution, only Na was detected in casein blank micelle; when 0.10 ml of Ca2+ solution was added, besides Na, the presence of C, O and Ca inside the forming microspheres was confirmed in the EDX spectrum (Figure S3). As shown in Figure S3, the amounts of these elements appeared to increase with the amount of Ca2+.

Sample II-8 was selected as the representative for the investigation of the surface composition based on X-ray photoelectron spectroscopy (XPS) analysis and Ca element was detected (Figure S4). The surface composition of II-8 was very similar to that of the as-received casein and based on XPS data, the calculated weight percent of casein in II-8 was 83.9% (Table S1) which was much higher than the loading content of 23.9% as shown in Table 1. This difference suggested that Casein/CaCO3 microspheres had a hierarchical structure with CaCO3 concentrated in the core.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 33

Figure 5. Investigation on the formation mechanism of Casein/CaCO3 microspheres based on (a) size, (b) zeta potential, (c) TEM and (d) selected-area electron diffraction (SAED) studies. 2+

Different volumes of Ca

2-

solution were added into 20 ml of CO3 solution containing casein at

2 mg/ml or 8 mg/ml under stirring (mixing mode II). After reaction for 20 min, samples were collected for characterizations without centrifugation. (c1, d1), (c2, d2), (c3, d3) and (c4, d4) were the representative TEM images and corresponding SAED patterns of forming microspheres when 0, 0.10, 0.25 and 0.50 ml of Ca2+ solution were added into 20 ml of CO32solution containing casein at 8 mg/ml under stirring, respectively.

The functional groups in the precipitate were investigated using FTIR. As shown in Figure 6, even when 0.5 ml of Ca2+ solution was added into CO32- solution containing casein at 8 mg/ml, the presence of casein and vaterite characteristic bands were observed in the very small amount of precipitate (i.e., -CONH- absorption band at 1650 cm-1, CO32- adsorption bands at 1410 cm-1 and 870 cm-1, and the characteristic vaterite band at 751 cm-1). When more Ca2+ solution was added, these adsorption


Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


bands were more dominant. Since the precipitate was not enough for XRD characterization, after reaction for 20 min, the suspension was collected immediately without centrifugation and freeze-dried. The representative XRD pattern of the sample fabricated by adding 1 ml of Ca2+ solution into CO32- solution (20 ml, casein concentration at 8 mg/ml) was shown in Figure S5. Besides the diffraction peaks belonging to Na2CO3 (JCPDS# 72-0628), several small peaks of vaterite (JCPDS# 74-1867) were present, indicating that vaterite was formed even when a very small amount of CaCl2 was added into the reaction system.

2+

Figure 6. Different volumes of Ca

2-

solution were added into 20 ml of CO3 solution containing

casein at 8 mg/ml under stirring. The precipitate was collected for FTIR analysis.

In bovine caseins, there are 4 different fractions, αs1-, αs2-, β- and κ-caseins at approximate ratios of 4:1:3.5:1.5. In micelles, αs- and β- caseins are extensively phosphorylated and probably bonded with calcium phosphate nanoclusters, forming the core of micelle; while κ-caseins are mainly located on the surface to inhibit the aggregation of micelles.33 During the addition of Ca2+ solution, the pH of reaction system was nearly stabilized at ~10.3. The isoelectric point of casein was ~ 4.6. Hence casein molecules were negatively charged throughout the reaction. Furthermore, the zeta potential of forming microspheres was stabilized at ~ -17.0 mV, indicating the negative surface of micelles was not affected by the increase of CaCl2 amount (Figure 5b). Hence, it was highly speculated that during CaCO3 crystallization the exterior of casein micelles might be rich of negatively charged functional groups



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

such as –COO-, and positively charged functional groups such as –NH3+ groups might be more concentrated in the interior. Due to the electrostatic attraction between –NH3+ and CO32- and the fact that casein was dissolved in Na2CO3 solution, CO32- ions were probably more located in the interior than on the exterior (Na was detected in blank casein micelle as shown in Figure S3). The calculated average crystalline size ranged from 15.9 nm to 19.0 nm for vaterite and from 28.7 nm to 52.2 nm for calcite (Table 2), which were much smaller than the blank micelle (~280 nm). When CaCl2 was added, Ca2+ ions were probably attracted into the interior of micelles due to CO32ions and nucleated in situ, forming vaterite crystals. Such speculation was supported by SAED patterns (Figure 5d2-d4), EDX spectra (Figure S3), FTIR (Figure 6) and XRD (Figure S5) results. When more vaterite crystals formed, these micelles were gradually enlarged (Figure 5). Some vaterite crystals were then probably also deposed on surface, eventually forming micro-sized Casein/CaCO3 microspheres. According to these speculations and results, the formation mechanism of Casein/CaCO3 vaterite microspheres based on templating effects of casein micelles was proposed and summarized in Figure 7. The mechanism proposed here was very different from that reported in the literature.27 When CO32- was added into casein/Ca2+ solution, amorphous CaCO3 nanoparticle was first formed; some amorphous nanoparticles were immediately transformed into nanocrystals; these amorphous nanoparticles and early formed nanocrystals aggregated into the dumbbell-like microparticles almost simultaneously; after this stage, the nanocrystals may gradually attach to the dumbbell-like microparticle forming spear-like branches. The fabrication method in the literature was different from that used in this research, which probably lead to the different formation mechanism and also different morphologies of CaCO3 microparticles.


Page 22 of 33

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Scheme demonstration for the formation mechanism of Casein/CaCO3 microspheres.

At higher concentration of casein, the formed microspheres were smaller than that at lower casein concentration (Table 1). This was consistent with the proposed formation mechanism of Casein/CaCO3 microspheres (Figure 7) as more nucleation sites were present in the reaction system at higher casein concentration. In solutions, it was probably that not all CO32- ions were located in interior of casein micelles, especially for the sample fabricated at low casein concentration. For those CO32- ions outside micelles, calcite might be precipitated (I-2, I-4, II-2 and II-4). The amount of calcite could be significantly reduced by lowering the addition rate of solutions (Figure 4a and S1).

Since vaterite was the least thermodynamically stable crystal phase, the stability of Casein/CaCO3 microspheres was then evaluated. Vaterite was reported to be stable at anhydrous conditions and in aqueous phase, the transition from vaterite into calcite was completed within 10 hr.34 Hence, II-2 and II-8 were selected as representatives and suspended in phosphate buffer solution (PBS) at 20 mg/ml for 24 hr at 37°C. Then microspheres were collected for characterization using XRD and SEM. As shown in Figure S6, for the same batch, the XRD patterns of as-prepared microspheres and that after immersion in PBS were nearly the same. In addition, the morphology of microspheres was not affected by the immersion in PBS and microsphere sizes were comparable with that of as-prepared samples (Figure S7). The additive casein here were suggested to have two functions: on the one hand, binding to the growth sites of the crystals during precipitation that can influence crystal



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

growth, on the other hand acting as a heterogeneous nucleator, which can control and stabilize the precipitated polymorph.

35

Hence, it can be concluded that the presence

of casein stabilized Casein/CaCO3 microspheres.

Figure 8. Representative SEM images of composite membranes after NaOH treatment and after soaking in SBF for 21 days for in vitro bioactivity evaluation. The NaOH treatment was aimed to improve the hydrophilicity and also expose the embedded microparticles. The insets were the enlarged images of the deposited crystals. The scale bar is 10 µm.

In Vitro Bioactivity Evaluation In vitro bioactivity of Casein/CaCO3 microspheres was evaluated after these

microspheres embedded into electrospun PCL composite membranes. Immersion of a material in SBF to examine apatite formation was concluded to be useful for predicting the in vivo bone bioactivity so as to reduce the number of animals used and the duration of animal experiments.25 The surface morphology of the as-electrospun membrane PV was shown in Figure S8. To improve the hydrophilicity and also expose Casein/CaCO3 microspheres or calcite, these composite membranes were first


Page 24 of 33

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


treated with NaOH. As shown in Figure 8, after NaOH treatment, the surface morphology was not significantly changed and the microparticles were still in fiber matrix. After soaked in SBF for 21 days, there were significant more crystals deposited on PV membrane than PC and P. The enlarged SEM image for sample PV showed that the crystals were needle-like, which was consistent with other reported results,36 demonstrating that significant more hydroxyapatite crystals were precipitated on PV. Ion concentrations of SBF were nearly equal to those of human blood plasma.25 For Ca2+ and HPO42-, the concentrations were 2.5 mM and 1.9 mM, respectively. To further compare the amount of hydroxyapatite crystals deposited on different samples, the calcium concentration in SBF was quantified (Figure 9). When compared with the values on day 1, Ca2+ concentrations on day 21 were all lower, indicating that certain amount of apatite formed on each sample. On day 21, the lowest amount of calcium was remained in SBF for sample PV showing that the largest amount of crystals were deposited. Based on these results, we can conclude that Casein/CaCO3 microspheres have better bioactivity than calcite. Due to the presence of Casein/CaCO3 microspheres, the in vitro bioactivity of PCL composite membrane was enhanced, indicating the composite membrane has the potential to be used for bone tissue engineering.

Figure 9. The concentration of Ca

2+

ions in SBF on the first and last day of in vitro bioactivity

test.

In Vitro Cytotoxicity Tests



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The cytotoxicity of Casein/CaCO3 microspheres was investigated using extraction assay and cell viability was evaluated using MTT method. As shown in Figure 10, after incubating hBMSC with extracts (prepared by immersion of microparticles in medium in an incubator for 24 hr and centrifugation to collect the supernatant which was the extract) for 24 hr, when compared with medium group (cells were cultured in normal medium), both II-8 and II-2 groups had slightly higher absorbance values; for calcite group lower cell viability was observed; only Triton X-100 group demonstrated significant smaller absorbance value and thus significant cytotoxic effect. After incubating for 72 hr, absorbance values for all groups except Triton X-100 group increased, indicating cells still proliferated. However when comparing with medium group, all groups showed significant lower absorbance values, where the relative cell viability was 89.5±3.5%, 50.7±10.9%, 41.8±18.7% and 7.5±0.4% for II-8, II-2, Calcite and Triton X-100 groups, respectively.

Figure 10. In vitro cytotoxicity of Casein/CaCO3 microspheres on hBMSC based on extraction assay. The extracts were prepared by first incubating microparticles/medium suspension (20 mg/ml) for 24 hr in an incubator and then centrifugation to collect supernatant for the test. Cells were cultured with these extracts for 24 hr and 72 hr. At the end of the culturing period, cells activity was determined with MTT assay. Data = mean ± SD; n = 3; * p

CaCO3 Vaterite Microspheres and

Recommend Documents