Dissolution behavior of biomimetic minerals on 3D PLGA scaffold

Surface & Coatings Technology 200 (2006) 6336 – 6339 www.elsevier.com/locate/surfcoat

Dissolution behavior of biomimetic minerals on 3D PLGA scaffold A. Champa Jayasuriya a,⁎, Malak Assad a , Ahalapitiya H. Jayatissa b , Nabil A. Ebraheim a a

Medical University of Ohio, Department of Orthopaedics, 3065 Arlington Avenue, Toledo, OH 43614-5807, USA b University of Toledo, Department of Mechanical Engineering, Mail Stop 312, Toledo, OH 43606-3390, USA Available online 9 January 2006

Abstract The bone-like carbonate apatite (BLCA) coatings have a great potential to apply in orthopaedic and dental implants due to their excellent biocompatibility and biodegradability. The BLCA layer can be coated biomimetically in the surfaces of polymers or inorganic materials using simulated body plasma. We have prepared 3D poly(lactic-co-glycolic acid) (PLGA) porous scaffolds by solvent casting/ particulate leaching method. The BLCA was coated on 3D PLGA scaffolds incubating them at 37 °C with modified simulated body fluid. We investigate the dissolution behavior of BLCA coated on PLGA scaffolds in two physiological medias such as Tris-buffered saline (pH = 7.4), 2-(N-morpholino)-ethanesulfonic acid (MES) (pH = 5.5), and cell culture media containing αMEM supplemented with 10% FBS and 1% penicillin–streptomycin. The BLCA dissolution and release ionic components were analyzed using FTIR, SEM and ICP. Approximately 70% of mineral was dissolved in Tris-buffered saline (8 ml) within 6 h relative to 48 h dissolution period. The investigation of mineral dissolution behavior is important when this BLCA use as a carrier for therapeutic agents in orthopaedic and dental implant applications. © 2005 Elsevier B.V. All rights reserved.

1. Introduction The BLCA layer can be coated biomimetically in the polymer surfaces by soaking in the simulated body fluid (SBF) [1–3]. This SBF contains similar ionic constituents to human blood plasma. The deposition of mineral on substrates using this biomimetic method provides many advantages over conventional methods, which utilize higher temperatures and pressures. This biomimetic technique can be applied to make carbonate apatite mineral in polymer films or scaffolds, which used as templates for bone tissue engineering. Advantage of this biomimetic method is that biologically active molecules can be co-precipitated with inorganic components, such as carbonate apatite to form organic–inorganic matrix due to gentle conditions throughout the process [4–6]. Therefore, these matrices can be used as carriers for organic molecules such as protein, drug or growth factors. In addition, creating a biocompatible surface for the newly forming bone reduces patient recovery time by facilitating rapid bone formation around the implant. Studies

have shown that this new coating binds more tightly to bone than mineral-coated implants produced by conventional methods [4–6]. In order to use BLCA for therapeutic applications, not only mineral formation but also mineral dissolution behavior in physiological media should be investigated. In this manuscript, we discuss the formation of BLCA layer on surfaces of 3D porous PLGA scaffolds and dissolution behavior of these mineral in two different physiological media, such as Trisbuffered saline (pH = 7.4), which mimic the pH conditions in human body, MES (pH = 5.5), and cell culture media containing αMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. These 3D biomimetic mineral/ scaffolds can be used as templates to seed the bone marrow stromal cells (BMSCs) for bone tissue engineering and, therefore, culture media has been selected one of the mineral dissolution media. 2. Experimental 2.1. Materials

⁎ Corresponding author. Tel.: +1 419 383 6557; fax: +1 419 383 3526. E-mail address: [email protected] (A. Champa Jayasuriya). 0257-8972/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2005.11.032

Poly(lactide-co-glycolide)-PLGA (85/15) copolymer Medisorb Alkerenes® amorphous with glass transition temperature

A. Champa Jayasuriya et al. / Surface & Coatings Technology 200 (2006) 6336–6339 Table 1 Ion concentrations (in mM) of blood plasma and simulated body fluids Na+

K+ Mg2+ Ca2+ Cl−

Blood 142.0 5.0 1.5 plasma 1× SBF 145.2 5.0 1.5 1× mSBF 141.0 6.0 1.5

HCO−3 H2PO−4 SO2− pH 4

2.5

103.0 27.0

1.0

0.5

7.2–7.4

2.5 5.0

152.0 157.0

1.0 2.0

0.5 0.5

7.4 6.8

4.2 4.2

(Tg = 55 °C) was used to fabricate the scaffolds used in this study. 2.2. Scaffold fabrication Microporous 3D PLGA scaffolds were fabricated by the solvent casting/salt leaching technique using chloroform. In this method, sieved sodium chloride particles were dispersed in a polymer/solvent solution, which was then cast to make a scaffold. The salt particles were then leached out by continuous soaking in deionized water for 2 to 3 days. This selective dissolution produces highly porous polymer. The sodium chloride was sieved to obtain granules between 250 and 425 μm; therefore, the pore sizes of the scaffolds were as same as the size of salt granules.

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2.5. Evidence of having BLCA layer and BLCA dissolution— FTIR A piece of scaffold (∼ 8 mg) was ground in to a fine powder and blends with KBr and then pressed to get a pellet which was used for FTIR measurements. The measurements performed with 4 cm− 1 resolution with FTIR Perkin Elmer spectroscopy. 2.6. BLCA dissolution The scaffolds coated with BLCA layer were placed in the glass vials containing 8 ml of each media, such as Tris-buffered saline (pH 7.4), MES (pH 5.5) and cell culture media containing αMEM supplemented with 10% FBS and 1% penicillin– streptomycin, and incubated at 37 °C for several time points up to 48 h to dissolve the minerals. At each time point, four scaffolds were taken out to perform SEM and FTIR analysis and

2.3. Biomimetically coated BLCA The BLCA layer can be deposited soaking the scaffolds in simulated body fluid (SBF) for 16 days. This simulated body fluid contains the similar salt ions to human body plasma (Table 1). We accelerated the deposition of mineral on scaffolds from 16 days to 5 days modifying the mineralization process as follows. 2.3.1. Surface hydrolysis Porous PLGA scaffolds were completely soaked in the deionized water to completely hydrolyzed them. Afterwards, these scaffolds were dipped in 0.5 M NaOH solution for 5 min per side to improve surface functional groups such as carboxyl acid (COOH) and hydroxyl (OH) groups. These scaffolds were rinsed thoroughly to remove any remaining NaOH from the surfaces and pores in the scaffold. 2.3.2. Modifying SBF Following surface hydrolysis, scaffolds were exposed to modifieSBF (mSBF) instead of 1× SBF (Table 1). Ca2+ and PO43− ion concentrations were doubled in mSBF. Surface modified PLGA scaffolds were placed in the 60 ml falcon tubes containing 50 ml of mSBF and incubated at 37 °C and mSBF was changed for every 24 h as necessary. 2.4. Mineral morphology—SEM Mineral morphology images of scaffolds were obtained using a JEOL 6100 SEM operating with 15 kV accelerating voltage under high vacuum. Conventional secondary electron scintillator detector was used with tungsten filament. Scaffolds were coated with a 20 nm gold layer using a sputter coater.

Fig. 1. SEM images showing (a) open-pore structure, (b) mineral on surfaces of scaffold and (c) mineral on surfaces of scaffold (higher magnification).

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A. Champa Jayasuriya et al. / Surface & Coatings Technology 200 (2006) 6336–6339

Transmittance (Arbitrary Units)

(a) PLGA control ∗

(b) Mineralized control ∗ ∗

(c) Tris saline - 6h

∗

(d) Tris saline -2days

∗ ∗

carbonate ∗ phosphate 2200

2000

∗ 1800

1600

1400

1200

1000

800

600

400

-1

Wavenumbers (cm ) Fig. 2. FTIR spectra for (a) control PLGA, (b) 5 days mineralized, (c) mineralized scaffold in Tris-buffered saline media for 6 h and (d) mineralized scaffold in Trisbuffered saline media for 48 h.

stored the solution in the refrigerator until use for ICP analysis. Ca concentrations were determined by ICP, Perkin Elmer Optima 3300DV OES, Sigma; Ca standard solution was dissolved in media and used for the calibration.

mineralized scaffold immersed in Tris saline was increased when incubated time increases. The amount of P release into the media was very low and could not detect the correct levels of P under ICP.

3. Results

4. Discussion

The open-pore structure of 3D PLGA (85/15) scaffolds was analyzed by SEM (Fig. 1a) and the pore size of these scaffolds is in the range of 250–425 μm, with a rectangular shape. This pore range was well established for osteoblast infiltration into a porous material [7,8]. The BLCA layer was coated biomimetically, in the surfaces of the PLGA scaffolds after 5 days incubation at 37 °C with mSBF (Fig. 1b and c). The surfaces of the scaffolds were fully covered with the mineral and similarly interior surfaces also covered with the mineral. Fourier transform infrared (FTIR) spectrum for the mineralized 3D PLGA scaffolds demonstrated the carbonated mineral structure information, giving the characteristic peaks for CO32− (1630 (ν3), 1551 (ν3) and 864 cm− 1 (í2)) and PO43− (1037 (ν3), 960 (ν1), 602 (ν4) and 563 cm− 1 (ν4)) compared with control PLGA (Fig. 2a and b). When mineralized scaffolds were immersed in Tris-buffered saline at 6 h and 48 h, CO32− and PO43− peak intensity decrease or disappear completely (Fig. 2c and d). Biomimetic mineral dissolution of surfaces in the PLGA scaffolds was observed with SEM at 48 h in three medias: Trisbuffered saline (Fig. 3a), MES (Fig. 3b) and cell culture media (Fig. 3c). The most mineral was dissolved within 48 h and barely dispersed mineral particles were observed in the surfaces of PLGA scaffolds, which immersed in both Tris saline (Fig. 3a) and culture media (Fig. 3c). The dispersed mineral particles in the surfaces of PLGA scaffold, which immersed in MES at 48 h, was negligible relative to other two medias (Fig. 3b). The incubation time dependence of relative Ca dissolution (%) was obtained using ICP for the mineralized scaffold immersed in Tris-buffered saline (Fig. 4). The Ca release from

The biomimetic mineralization involves nucleation and growth of apatite crystals from supersaturated ionic solutions under near-physiological conditions [1,2]. The dissolved Ca ions increase the degree of super saturation of the surrounding fluid with respect to apatite, while the hydrated substrate surface provides favorable site for apatite nucleation [9]. Once apatite nuclei form, they spontaneously grow, consuming Ca and PO4 ions from the surrounding fluid. The BLCA was formed in the surfaces of porous PLGA scaffolds within 5 days using prehydrolysis, NaOH treatment and incubating with mSBF. FTIR data has proved that this mineral contains the carbonated hydroxyl apatite similar to bone. The apatite layer on the surfaces of PLGA scaffold was not very thick, approximately 10–12 μm range according to SEM (Fig. 1c). Therefore, mineral layer does not interfere with pore structure of scaffold and it is important parameter for in-vitro BMSCs culture. Some studies have shown that biomimetic mineral coating can stimulate cellular activity and positively influence proliferation and differentiation of marrow stem cells [10,11]. Non-bioactive materials typically do not exhibit surface-dependent cell differentiation [12]. Therefore, an essential requirement for a biomaterial to bond to living bone is the formation of a biologically active BLCA layer. The investigation of mineral dissolution in physiological media is necessary if BLCA use as a carrier for biological molecules. Incorporation of biomolecules into BLCA by coprecipitation provides interaction with crystal lattice in the mineral instead of surface adsorption [4]. Therefore, release of biological agents from the mineral occurs upon dissolving the mineral layer in the implant. In order to provide the exact

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Relative Dissolution (%)

physiological environment similar to the body, mineralized scaffolds were not vortexed using the stir bars on the vials. If mineralized scaffolds stirred, the mineral dissolution rate would be even faster than unstirred ones. We have selected three medias to dissolve the BLCA in scaffolds, depending on the pH and biological components of them. pH of Tris-buffered saline is 7.4 and, therefore, mimic the physiological pH environment in the body and MES has lower pH (5.5) than Tris-buffered saline. Approximately 70% of bone-like mineral was dissolved in Tris-buffered saline within 6 h relative to 48 h period, according to ICP results. SEM results showed that the most mineral in scaffolds was dissolved at 48 h for all three medias. The scattered tiny mineral particles were observed for mineralized scaffolds immersed in Tris-buffered saline and cell culture media. But very few mineral particles were observed for mineralized scaffolds in the MES due to the lower pH (acidic

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100 80 60 40 20 0 0

10

20

30

40

50

Time (hours) Fig. 4. Ca dissolution in Tris-buffered saline media. Relative dissolution was calculated from the ratio of mineral dissolution at each time to the final value (48 h); it does not count the complete scaffold degradation.

environment) compared with other two medias. The mineral dissolution rate can be adjusted by controlling the several parameters of biomimetic process such as pH, ionic constituents and temperature. 5. Conclusions The BLCA layer was coated in-vitro in the surfaces of 3D PLGA scaffolds using hydrolysis techniques and mSBF, within 5 days at normal pressure and temperature. The most BLCA in the scaffolds, which immersed in each physiological media and culture media, was dissolved within 48 h, except dispersed mineral particles. This study suggests that BLCA has a potential to use as a carrier for biological molecules for short-term localized release applications. In order to use BLCA layers as a therapeutic carrier for long-term release required for orthopaedic and dental implants, mineral dissolution rate should be adjusted by controlling the parameters of biomimetic procedure. References

Fig. 3. SEM images showing mineral dissolution at 48 h in (a) Tris-buffered saline, (b) MES and (c) culture media; arrows indicate the tiny dispersed mineral particles.

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