Article pubs.acs.org/Langmuir
Effect of Organic Acids on Calcium Phosphate Nucleation and Osteogenic Differentiation of Human Mesenchymal Stem Cells on Peptide Functionalized Nanofibers Danial Barati, Joshua D. Walters, Seyed Ramin Pajoum Shariati, Seyedsina Moeinzadeh, and Esmaiel Jabbari* Biomimetic Materials and Tissue Engineering Laboratory, Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, United States S Supporting Information *
ABSTRACT: Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(DL-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs. in CaP nanocrystals.8−10 Further, citrate ions inhibit the formation of large and stable CaP crystals in solution11 and catalyze CaP nucleation on surfaces.12 Furthermore, carboxylate-rich organic acids account for 5.5% of the organic matter in bone, and citric acid plays a significant role in controlling the extent of CaP nucleation and mineralization in the natural bone.8 We previously showed that functionalization of nanofibers with a sequence of glutamic acids (GLU) that acted as a calcium chelating agent increased the extent of CaP crystal formation on the nanofibers by as much as 200% based on the fiber weight.4 The CaP content achieved with the functionalized nanofiber microsheets was higher than cancellous bone at 190%13 but lower than compact bone with >230% CaP content.14 The objective of this work was to investigate the effect of supplementing the nucleation solution with organic acids on
1. INTRODUCTION Reconstruction of large incomplete bone segments remains a significant clinical problem.1 Frozen allogeneic bone graft is used, but its long-term failure rate in reconstruction of large defects is 25%.1 Demineralized bone provides a supportive matrix for differentiation and maturation of osteoprogenitor cells, but it fails to provide rigidity for segmental defects.2 Calcium phosphate (CaP) ceramics have attracted much attention as a bone substitute due to their osteoconductivity and osteointegrative properties, but they are brittle in tension and shear.3,4 Fiber-reinforced composites of calcium phosphates and degradable polymers have superior properties compared to their individual components, but they are limited by the amount of CaP crystals that can be incorporated in the composite matrix.5−7 Therefore, there is a need for composite biomaterials that can stabilize the regenerating volume, have tunable resorption, and support differentiation and maturation of osteoprogenitor cells. Recent reports indicate that the spacing between carboxylate groups in citric acid matches the spacing between calcium ions © XXXX American Chemical Society
Received: February 24, 2015 Revised: April 10, 2015
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DOI: 10.1021/acs.langmuir.5b00615 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. (a) Molecular structure of the organic acids. Effect of organic acid concentration added to mSBF on CaP content of the microsheets with (b) and without (c) GLU after 16 h incubation. (d) Effect of incubation time in mSBF supplemented with one of the organic acids (6 mM) on CaP content of the microsheets. (e) CaP content of the GLU microsheets plotted against the sum of acidic strength and H-bonding hydroxyl groups of the organic acids. None and Ctrl are the microsheets with and without GLU, respectively, incubated in mSBF without organic acids. There was a significant pairwise difference between CaP content of all groups. Error bars correspond to means ±1 SD for n = 3.
medium. The cell-seeded microsheets were characterized by cell number, alkaline phosphatase (ALP) activity, calcium content, and total collagen content with incubation time. Results indicate that the addition of HCA, CA, and TART to the nucleation medium significantly increased the CaP content of the microsheets, but osteogenic differentiation of hMSCs significantly increased only on those microsheets incubated in CA or AsA supplemented nucleation solution.
the extent of CaP crystal formation on the functionalized nanofiber microsheets and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaPnucleated microsheets. The CaP nucleating peptide Glu-GluGly-Gly-Cys (EEGGC or GLU peptide) was conjugated to a low molecular weight poly(L-lactide) to produce a GLULMPLA conjugate. The GLU-LMPLA conjugate was mixed with high molecular weight PLA, and the mixture was electrospun to generate aligned GLU-functionalized PLA nanofiber microsheets. The microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with one of the following organic acids to grow CaP crystals on the surface of nanofibers. The organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), salicylic acid (SalA), and ascorbic acid (AsA) with different acidic strength and number of hydroxyl groups. The selected organic acids naturally exist in fruits and vegetables. CA is the dominant organic acid in bone15 and citrus fruits16 while MA is found in apple and pear.16 Tomato contains 9% CA, 4% MA, and 0.5% AsA based on dry mass.17 SalA is found in rice and soybean18 while grapes and grapefruit are a major source of TART and HCA.19 The CaP nucleated microsheets were characterized with respect to particle size, crystallite size, percent crystallinity, calcium to phosphate (Ca/P) ratio, CaP content, and compressive modulus. Next, hMSCs were seeded on the CaP nucleated microsheets and cultivated in osteogenic
2. MATERIALS AND METHODS 2.1. Materials. Poly(DL-lactide) (DL-PLA) with intrinsic viscosity of 0.65 dL/g and weight-average molecular weight (M̅ w) of 90 kDa and poly(lactide-co-glycolide) (PLGA; 50/50 copolymer) with intrinsic viscosity of 1.1 dL/g and M̅ w of 105 kDa were received from Lactel (Cupertino, CA). L-Lactide (LA; >99.5% purity) was received from Ortec (Easly, SC). Diethylene glycol (DEG), sodium chloride (NaCl), potassium chloride (KCl), calcium chloride monohydrate (CaCl2 ·H 2 O), magnesium chloride hexahydrate (MgCl2·6H2O), sodium bicarbonate (NaHCO3), and monosodium phosphate (NaH2PO4) were from Fisher (Waltham, MA). Rink Amide NovaGel resin and Fmoc-protected amino acids were from EMD Biosciences (San Diego, CA). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), tartaric acid (TART), hydroxycitric acid (HCA), citric acid (CA), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA) were from VWR (West Chester, PA). Tin(II) 2-ethylhexanoate (TOC), acryloyl chloride (AC), and triethylamine (TEA) were from Sigma-Aldrich (St. Louis, MO). Dichloromethane (DCM, Acros Organics, Pittsburgh, PA) was dried by distillation over calcium B
DOI: 10.1021/acs.langmuir.5b00615 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir hydride. Diethyl ether and hexane were obtained from VWR (Bristol, CT) and used as received. Ethylenediaminetetraacetic acid disodium salt (EDTA), penicillin, streptomycin, paraformaldehyde, and Alizarin red stain were from Sigma-Aldrich (St. Louis, MO). Phosphatebuffered saline (PBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were from GIBCO BRL (Grand Island, NY). Trypsin and fetal bovine serum (FBS) were received from Invitrogen (Carlsbad, CA) and Atlas Biologicals (Fort Collins, CO), respectively. The QuantiChrom calcium and alkaline phosphatase (ALP) assays were received from Bioassay Systems (Hayward, CA). The Quant-it PicoGreen assay was received from Invitrogen (Carlsbad, CA). 2.2. Instrumentation. The chemical structure and degree of acrylation of the synthesized PLA were characterized by a Varian Mercury-300 1H NMR (Varian, Palo Alto, CA) in CDCl3 as described.20 The molecular weight distribution of the polymers was measured by gel permeation chromatography (GPC, Waters, Milford, MA) as described.20 The synthesized peptide was purified by preparative HPLC using a photodiode array refractive index detector, and the purified peptide was characterized with a Finnigan 4500 electrospray ionization (ESI) spectrometer (Thermo Electron, Waltham, MA) as described.21 2.3. Polymer Synthesis and Peptide Conjugation. Low molecular weight poly(L-lactide) (LMPLA) was synthesized by ringopening polymerization of LA monomer with DEG and TOC as initiator and catalyst, respectively, as we previously described.4 Acrylate-terminated LMPLA (Ac-LMPLA) was synthesized by reacting LMPLA with acryloyl chloride, as we previously described.22 The M̅ n and polydispersity index (PI) of Ac-LMPLA were 5.3 kDa and 1.2, respectively, as determined from the NMR spectrum.4 The amino acid sequence Glu-Glu-Gly-Gly-Cys, hereafter denoted by GLU peptide, was synthesized manually on Rink Amide NovaGel resin as we previously described.4 The GLU peptide was conjugated to AcLMPLA by Michael addition reaction to produce the GLU-LMPLA conjugate as described.4 The average number of peptides per GLULMPLA conjugate from the NMR spectrum was 1.3.4 2.4. Nanofiber Electrospinning. The electrospinning solution was prepared by dissolving 8 wt % PLA and 1.5 wt % GLU-LMPLA in HFIP.4 There was no GLU-LMPLA in the samples denoted by “without GLU”. The electrospinning solution was injected from a 1 mL syringe through a 21-gauge needle (Becton-Dickinson, Franklin, NJ) via a programmable syringe pump (KDS100, KD Scientific, Holliston, MA) as we previously described.4,23 The positive and ground electrodes of the high-voltage power source (ES40P-5W/ DAM, Gamma High Voltage Research, Ormond Beach, FL) were connected to the needle and a custom-built aluminum rotating wheel (20 cm diameter and 5 mm thickness), respectively.4,23 The aligned nanofibers were electrospun using a 0.8 mL/h injection rate, 20 kV electric potential, 7.5 cm needle-to-wheel distance, and wheel diameter and rotation speed of 20 cm and 1800 rpm, respectively, as previously optimized.23,24 The average thickness of the microsheets was 6 ± 1 μm, and the average fiber diameter was 200 ± 60 nm.4 The PLA/ GLU-LMPLA fibers are hereafter denoted by NF. 2.5. NF Mineralization. A stock solution of 10-fold concentrated SBF (10xSBF) was prepared by dissolving NaCl, KCl, CaCl2·H2O, MgCl2·6H2O, and NaH2PO4 in deionized (DI) water as described.4 It should be noted that a saturated solution of calcium and phosphate ions is normally used for nucleation and growth of CaP crystals on substrates25−27 as it takes many weeks to grow appreciable amount of CaP in 1xSBF.28 Next, one of the organic acids (Figure 1a) was added to the nucleation solution with a concentration of
