Chuntao Liua,
Xin Liuab,
Changyun Quanab,
Xiaoqiong Lia,
Chaozhu Chena,
Hua Kanga,
Weikang Hua,
Qing Jiangab and
Chao Zhang*ab
aSchool of Engineering, Sun Yat-sen University, Guangzhou, Guangdong 510006, China. E-mail: zhchao9@mail.sysu.edu.cn; Fax: +86-20-39332152; Tel: +86-20-39332145
bGuangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instruments, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
First published on 9th February 2015
Methacrylated poly(γ-glutamic acid) (mPGA) was introduced to the spongy poly(ethylene glycol)-co-2-hydroxyethyl methacrylate (PEG-HEMA) cryogels. A suitable amount (<0.1% w/v) of poly(γ-glutamic acid) (γ-PGA) in cryogels was found to promote homogeneous mineralization, while higher concentration of γ-PGA inhibited the mineralization. The deposited minerals mainly consisted of hydroxyapatite, carbonated apatite, and calcium phosphate, and were independent on the concentration of γ-PGA. The mineralized cryogels can support adhesion, viability, and migration of rat bone marrow stromal cells (rMSCs). The γ-PGA/HEMA-PEGDA cryogels showed their great potential as promising scaffolding materials in bone tissue engineering.
Recently, cryogel1–3 has received increasing attention in tissue engineering due to its high porosity and interconnected macroporous structure. Varghese4 investigated poly(ethylene glycol) cryogels as scaffolds for cartilage tissue engineering. Their study indicated that the cryogels can support adhesion, viability, proliferation, and biosynthetic activity of seeded chondrocytes. The microarchitecture of PEG cryogel can also affect the osteogenic differentiation of human mesenchymal stem cells (hMSCs).5 Spongy cryogels can promote osteogenic differentiation to a greater extent than their columnar counterparts. Kumar6 prepared alginate/gelatin cryogels using glutaraldehyde or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide as crosslinkers, and found the type of crosslinker may affect the physiochemical properties of matrices and lead to different cell behaviors.
Stemming from the fact that anionic proteins can regulate the mineral phase deposition in natural bone,30 the incorporation of anionic groups, such as carboxyl group, onto polymeric substrate has become a common approach to inducing mineralization on the surface. Chappard7 discovered that modification of poly(2-hydroxyethyl methacrylate) (pHEMA) with carboxymethyl groups could significantly increase the deposition of calcium. Bertozzi8 studied the effect of carboxyl groups on the mineralization of pHEMA hydrogels. Carboxyl groups may act as calcium binding-sites to initiate the heterogeneous nucleation and high-affinity growth of calcium apatite on the gel surface and extensive calcification inside the gel. A later study on substrates containing biomimetic mineral-nucleating amino acid ligands revealed that the structure and density of the ligands could effectively control the morphology and crystallinity of the mineral.9
Glutamic acid and aspartic acid residues are widely found with extraordinary abundance in non-collagenous proteins of bone tissue and take part in the bio-function of such proteins. For example, consecutive glutamyl residues play a major role in bone sialoprotein as a mineral nucleating agent in vivo.10 Similarly, crosslinked poly(γ-glutamic acid) (γ-PGA) has proved to induce the heterogeneous nucleation of hydroxyapatite in a simulated body environment.11
In this work, the role of γ-PGA in mineralization of the spongy pHEMA cryogel scaffold was investigated; and the effect of concentration of γ-PGA on mineralization, as well as the adhesion, viability, and migration of rat bone marrow stromal cells (rMSCs) in the scaffolds were evaluated in detail.
Growth medium containing high-glucose Dulbecco's modified Eagle medium (DMEM) (Hyclone, USA), 10% of fetal bovine serum (FBS) (PAA, Germany), and 1% of penicillin–streptomycin (Tianjin Haoyang Biological Manufacture Co., LTD, China) was used in the cell culture. rMSCs were isolated from the femurs and tibias of four week-old male Sprague Dawley rats as described elsewhere.15 Cells were cultured in growth medium. Cells at forth passage were used for experiments.
Cryogels or mineralized cryogels were thoroughly rinsed with running DI water, and lyophilized before further characterization. The mineralized cryogels were presented as M-Px, where x stands for the percentage content of mPGA in the polymerization formulation; and the non-mineralized cryogels were termed Non-M-Px in the following context.
EWC% = [(mw − md)/mw] × 100 | (1) |
Cell-laden constructs (n = 3) were harvested at 0, 7, and 21 days for determination of the content of DNA.5 Constructs were frozen at −80 °C, lyophilized, weighed, and homogenized. The homogenized constructs were digested in papain solution (2.375 U mL−1 of papain, 10 mM of cysteine in PBE buffer) for 16 h at 60 °C. DNA content was then measured using Quant-iT Picogreen dsDNA Assay kit according to manufacturer's guidance.
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Fig. 1 ATR-FTIR spectra (a) and XRD spectra (b) of mPGA and cryogels before and after mineralization. |
The interconnectivity of the porous structure of the cryogels were examined under SEM and optical microscope (Fig. 2). Cryogels with different concentrations of γ-PGA (0, 0.01, 0.1, 0.25, 0.5% w/v) have similar average pore sizes with diameters about 50 μm (Table S1 and Fig. S1†). The thickness of the wall of the pores was found to be in the range of 11.9–19.3 μm, while no statistical difference was observed at different γ-PGA concentrations. Porosity of the cryogels with different concentration of γ-PGA is similarly above 80% (Fig. S2†). These findings suggested that the concentration of γ-PGA did not significantly affect the pore parameters of cryogel.
After mineralization, the interconnectivity of cryogels was maintained (Fig. 2) with very little variation in the structure of the pores as compared with that of non-mineralized cryogel (Table S1, Fig. S1 and S2†). ATR-FTIR analysis was performed on the surface of the mineralized cryogels (Fig. 1a). After mineralization, the intensity of peaks for the backbone of the polymeric matrix scaffold (P0 and P0.5) decreased greatly, while the bands of PO43− groups (ν1 ∼ 963 cm−1, ν3 ∼ 1036 cm−1) appeared. Additionally, bands at ∼870 cm−1 (ν2) due to CO32− can be found,23,24 suggesting that CO32− groups have been incorporated into the minerals. In the XRD patterns, significant intensity of peaks at (002) and peaks at (211), (112), (300) in the mineralized cryogels indicated that the minerals in the cryogels have similar crystal lattice to HA standard (ICDD 9-432).25
Due to its strong hydrophilicity, incorporating γ-PGA into the cryogel matrix could lead to higher EWC. The EWCs of the cryogels were similarly higher than 93% at concentration of γ-PGA lower than 0.25%; however, the EWC decreased greatly when the concentration of mPGA reached 0.25% w/v, this could probably be attributed to the increased crosslink density due to the increased amount of methacryloyl groups brought by γ-PGA. During the mineralization, carboxyl groups were consumed in chelation with calcium ion and induction of minerals, and the minerals formed in this process could in turn reduce the accessibility of carboxyl groups to water molecules, i.e. displaying a decrease in EWC.
The mineral deposited in the γ-PGA-modified cryogel was stained using Alizarin red S (Fig. 3). With 0.1% (w/v) of γ-PGA, the whole section was stained red, indicating the minerals were deposited homogenously in the cryogel; however, cryogels with γ-PGA concentration of 0, 0.01, and 0.5% showed relative lighter staining in the center of the sections. Minerals on the surface or inside the cryogels may be originated from either the precipitation from the metastable solution or the growth of the crystal nucleus induced by glutamyl residues on the backbone of cryogel. The staining on the surface of cryogel matrix with 0 or 0.01% (w/v) of γ-PGA probably mainly came from the precipitation of the metastable solution and the growth of such precipitation; once the surface is covered by these precipitates and nucleated minerals, less minerals formed inside cryogels, which was later confirmed by the SEM observation (Fig. 5). Meanwhile, the minerals which covered the surface of cryogels can hinder the mineral deposition in the interior space of the cryogels. As a result, less minerals were found inside the cryogels. However, as the concentration of γ-PGA increased, the mineral crystal nucleation and the growth of the crystal were promoted. Mineralization not only took place on the surface of the cryogel but also in the interior space of the matrix. Here, carboxyl groups on the cryogel matrix acted as Ca2+ binding-site and induced the heterogeneous nucleation of hydroxyapatite.7–9,11 The amount and adhesion strength of minerals in the cryogels were governed by the density of carboxyl groups on the surface of the matrix. Kawashita26 has demonstrated the catalytic effect of carboxyl groups on apatite nucleation, and acceleration of apatite nucleation from released Ca2+ ions. In this work, the apatite nucleation may also be catalyzed and accelerated by the carboxyl groups in the cryogel matrix following the similar process. After being treated with 40 mM Ca2+/24 mM HPO42− solution, a large number of the Ca2+ ions was combined with the carboxyl groups, which may develop high local concentration of Ca2+ ions and facilitate the nucleation of apatite in situ. With increasing concentration of γ-PGA, the amount of minerals firstly increased slightly and then decreased, which was in accordance with the quantification of Ca and P (Fig. 4a and b). Comparable amount of mineral was found in each group with mPGA concentration ranging from 0–0.1%. This could probably be attributed to the large amount of minerals from the precipitation of the metastable immersion solution onto the superficial area of the cryogel.
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Fig. 4 Ca & P content in the cryogels after mineralization. (a) Ca content & (b) P content from UV measurement; (c) SEM-EDS images. |
It has been reported that excessive carboxyl groups may inhibit the nucleation of mineral in the case of polyaspartic acid.27 In this study, maximum Ca and P contents were obtained at concentration of γ-PGA of 0.1% w/v, and were significantly higher than those in cryogels with 0.5% w/v of γ-PGA (P < 0.05). In other words, mineralization was inhibited at higher concentration of γ-PGA than 0.1% w/v, and the minerals were found mainly located on the surface of the cryogels with 0.1% or higher concentration of γ-PGA due to the growth of precipitation.
The Ca/P ratios of the mineral in the cryogels were quantified to be 1.63, 1.66, and 1.64 for P0, P0.1, and P0.5, respectively, which were close to that of hydroxyapatite (1.67).28 However, the Ca/P ratios on the surface of the mineralized cryogels as determined via SEM-EDS (Fig. 4c) were 1.48, 1.96, and 1.57 for P0, P0.1, and P0.5 cryogels, respectively. The discrepancy may come from the fact that EDS only detects the local composition of mineral, which may be greatly influenced by the presence of calcium carbonate.
More minerals were found on the surface than in the interior space of the cryogels (Fig. 5). Different morphologies of the minerals could be observed in different regions of the same cryogel; this could be attributed to the different stages of the mineral growth, which has little correlation with the amount of carboxyl groups but highly depends on the local concentration and composition of the SBF. Similar phenomenon had been reported by Mooney,29 who discovered that the Ca/P ratio and crystal morphology on the surface of poly(α-hydroxy ester) were dependent not on the polymer surface characteristics but on the composition of the SBF. In this case, the cryogels were soaked in 40 mM Ca2+/24 mM HPO42− solution prior to immersion in SBF to increase to the regional concentration of calcium and phosphate ions inside the cryogels, and consequently to accelerate the growth of the minerals. This process may change the formulation of SBF in the mineralization, and then influenced the chemical composition and morphology of the resultant mineral phase.30 With the aid of 40 mM Ca2+/24 mM HPO42− solution, the crystals growth not only originated from the amorphous minerals which was induced by the carboxyl groups on the surfaces of the scaffold, but also from the precipitate from the metastable solution.
In general, we confirm that the minerals in the cryogels have similar composition, mainly consisting of hydroxyapatite, carbonated apatite, and calcium phosphate, and were independent on the concentration of γ-PGA. However, the amount of minerals were closely related to the concentration of γ-PGA. The amount of minerals increased with the concentration of γ-PGA (<0.1% w/v) initially, and decreased when the concentration of mPGA was above 0.1% w/v.
In order to further confirm the role of γ-PGA in mineralization, cryogels with different concentrations of γ-PGA were immersed in SBF for four weeks without pre-soaking in the 40 mM Ca2+/24 mM HPO42− solution, the SBF was changed with every other day to ensure sufficient supply of ions. Under this slow process of mineralization, tiny amount of Ca and P were detected in the mineralized cryogels via colorimetric assay (Fig. S3a and b†), even though no P was detected by SEM-EDS (data not shown). The amount of Ca increased slightly with the increasing concentration of γ-PGA. There was no significant difference in P content between each group. However, more mineral nucleation was found on the pore wall of cryogel with 0.1% w/v of γ-PGA as evidenced by SEM (Fig. S3c†) and OM observations (Fig. S3d†) as compared with those with 0% w/v and 0.5% w/v of γ-PGA. This phenomenon further reveals that, at the early stage of mineralization, suitable amount (<0.1% w/v) of γ-PGA promotes the mineral nucleation, while larger amount (>0.1% w/v) of γ-PGA binds to and stabilizes the pre-nucleation clusters, forming loosely packed, diffusive structures that slowly aggregate and densify, and finally inhibits mineral nucleation, as described by Sommerdijk.27
Cytoskeleton staining was performed to evaluate the migration of cells from the surface to the interior space of the cryogels (Fig. 6d). After 21 days' culture, more cells in the M-P0.1 cryogel were observed as compared with those in other groups. The staining of F-actin (stained green) also indicated that cells preferentially adhered to the cryogel where minerals were fully developed. After 21 days' culture, a larger amount of cells migrated into and were distributed uniformly inside the M-P0.1 cryogels. However, cells were mainly distributed on the top of “M-P0” and “Non-M-P0.1” cryogels. This phenomenon indicated that minerals can not only support the adhesion but also the migration of cells. Take account of the Alizarin red S staining of M-P0.1 cryogel (Fig. 3), it seems that the uniform distribution of minerals may have positive consequences on biological response of cells in terms of better viability and benefit the migration of cells to bulk of the matrix.
Footnote |
† Electronic supplementary information (ESI) available: Porosity, pore size and distribution, pore morphology. See DOI: 10.1039/c4ra15893h |
This journal is © The Royal Society of Chemistry 2015 |