A technique for synthesizing biocompatible hydrogels by cross-linking calcium-form poly(
Tissue engineering aims to create wound-covering biomaterials for skin-related diseases and biological body parts as alternatives to transplanted harvested tissues and organs. One example, hydrogel, has been exploited for medical and pharmaceutical applications for scaffolding or covering for the last three decades [
Alginate is used extensively as a hydrogel component because of its many advantages, such as hydrophilicity, high swellability, nontoxicity, and easy preparation [
Polyglutamic acid (PGA) is a natural linear polymer which is copolymerized from the amino acid glutamic acid, which is formed by peptide bonds between the amino group and the carboxyl group at the end of the glutamic acid side chain. It can be synthesized by bacilli such as
Pluronic F-127 is a triblock copolymer of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO), which has a nominal molecular weight of 12 500 and an average formula of EO99PO67EO99 [
We aimed to study biocompatibility and cytocompatibility of different proportions of F-127 with alginate-calcium-PGA (described as A-CP-F) in hydrogels prepared via a casting method. There are two advantages to using this blend as a bone tissue engineering material.
The blended hydrogel can provide mechanical strength, so A-CP-F gels would be more suitable for clinical application than a simple hydrogel. In this study, these A-CP-F gels were subject to hydrophilic tests, tensile tests, protein adsorption, and blood coagulation tests to demonstrate the clinical applicability of the A-CP-F matrix.
Sodium alginate (Acros, USA) with molecular weight about 22 kD was used without further purification. Sodium-form and calcium-form
A 1.5 wt% homogeneous alginate solution was prepared by dissolving sodium alginate powder in deionized water at room temperature for 24 h under stirring. Calcium-PGA powder was dissolved in deionized water at room temperature under stirring for 2 h to form a homogeneous solution of 3 wt%. Then F-127 solution was mixed with Ca-PGA solution at seven ratios, and 20 mL of the alginate solution was cast onto a glass plate. Finally, 20 mL of the Ca-PGA-F-127 solution was poured over the alginate to form a hydrogel. For comparison, sodium alginate hydrogel was soaked in either 10 wt% CaCl2, 3 wt% Na-PGA, or 3 wt% Ca-PGA aqueous solution. The resulting hydrogels were labeled as in Table
Preparation and composition of hydrogel blends.
Ingredient | Designation |
---|---|
Alginate | |
CaCl2 | A-C |
Na-PGA (with CaCl2) | A-NP |
Ca-PGA | |
0% F-127 | A-CP |
5% F-127 | A-CP-5F |
10% F-127 | A-CP-10F |
15% F-127 | A-CP-15F |
20% F-127 | A-CP-20F |
25% F-127 | A-CP-25F |
30% F-127 | A-CP-30F |
We measured the swelling behavior of the gels under different pH with normal saline solution and different temperatures of deionized water, and samples of 1 × 1 cm2 were dried in an oven for 2 h at 105°C [
A piece of swollen hydrogel was weighed
The tensile strength and breaking elongation of the blended hydrogels were measured with a tensile tester (MTS 810; Material Test System, USA) according to ASTM D882-02. The dry samples were prepared by vacuum drying at 40°C overnight. The specimens were cut into a specific dog-bone shape (11.5 cm long, 2.5 cm wide at the ends, and 0.6 cm wide in the middle). The thickness of each specimen was measured. The measurement was conducted at a crosshead speed of 10 mm/min under a tensile preload of 10 kg.
The adsorption of HAS and HPF was measured. A piece of hydrogel of 1 × 1 cm2 was immersed in 5 mL of pH 7.4 PBS containing 2 mg/dL HSA or HPF at 37°C for 24 h under 100 rpm shaking. Afterwards, the samples were gently taken out and rinsed five times with PBS, followed by placing them in 1 wt% aqueous solution of sodium dodecyl sulfate (SDS), and shaken for 60 min at room temperature to remove the protein adsorbed on the surface. The protein content of each sample was measured using the BCA reagents (Pierce). The absorbance at 562 nm was measured using a spectrometer to calculate the concentration of protein [
The determination of platelet adhesion and thrombus formation followed published procedures [
The
MG-63 osteosarcoma cells were seeded at 3000 cells/cm2 in 6-well plates under differentiating conditions until the assay endpoint. For dose-response assays, cells were seeded in 96-well plates at 30 000 cells/cm2 for 24 h and then dosed with glycosaminoglycans, alternative agents, or controls in 10-fold dilutions from 100 mg/mL to 100 pg/mL. Adenylate kinase activity (for membrane integrity) was measured using the ToxiLight assay kit (Cambrex Corporation) [
MTT assay followed the procedures given in the literature [
Microsoft Excel was used to calculate standard deviations and statistically significant differences between samples using two-tailed Student’s
The F-127 hydrogel had pH-sensitive swelling behavior. The swelling ratios for A-CP were 1.49 and 0.71 under pH 4 and pH 10, respectively, without F-127, while the average ratios for F-127 hydrogel were 1.21 and 0.52, respectively (Figure
Swelling ratios of A-C, A-NP, A-CP, and A-CP-F series hydrogels with normal saline at different pH.
Swelling ratios of A-C, A-NP, A-CP, and A-CP-F series hydrogels in deionized water at different temperature.
The water retention capacity of A-CP was higher than that of A-C and A-NP (Figure
Water retention of A-C, A-NP, A-CP, and A-CP-F series hydrogels with increase in centrifugal force.
With progressive 5% increases in F-127 content, the tensile strength was 1.56, 1.18, 1.37, 1.07, 1.41, and 1.22 times that of A-CP hydrogels, while the breaking point was 2.56, 3.56, 3.44, 2.72, 2.94, and 3.11 times that of A-CP hydrogels (Figure
Tensile strength and breaking elongation of A-CP-F series hydrogels.
The surface densities of adsorbed HSA and HPF on the A-CP surface were lower than those of A-CP-F and A-C (Figure
Dependence of adsorption of human serum albumin and human plasma fibrinogen on A-C, A-NP, A-CP, and A-CP-F series hydrogels.
After 90 min incubation, platelet adhesion for the A-CP-F series was much higher than that of the A-CP, A-NP, and A-C hydrogels (Figure
Platelet adhesion of A-C, A-NP, A-CP, and A-CP-F series hydrogels.
The APTT and PT of A-CP-F series hydrogel were about 62% and 84% of that of the blank plasma control (Figure
Anticoagulation times of A-C, A-NP, A-CP, and A-CP-F series hydrogels.
As shown in Figure
MTT assays of A-C, A-NP, A-CP, and A-CP-F series hydrogels with MG-63 cells.
Four kinds of alginate hydrogels (A-C, A-NP, A-CP, and A-CP-F series) were compared for mechanical properties, biocompatibility, and cytocompatibility. The swelling ratios decreased as F-127 increased because F-127 strongly interacts with the other compositions and wraps around the polymer chains due to their tight structure. Also, the linear chain of F-127 causes these blends to be more hydrophobic than the A-CP blends [
It is known that Ca2+ acts as a bridge that triggers the cross-linking and formation of hydrogels, but the higher the Ca2+ concentration, the worse the hydrogel. Alginate has hydrophilicity and high swellability originally but only forms a hydrogel with the aid of Ca2+, but Ca2+ might eliminate useful properties of alginate during the gelation process. Therefore we investigated the exceptional hydrogel formed by mixing
Traditionally, alginate gel beads are prepared from sodium or potassium alginate solution in an aqueous solution of calcium ions, typically from calcium chloride (CaCl2), to make alginate-calcium chloride hydrogel. The gelation of CaCl2 in varying cross-linking densities and a polymer concentration gradient within the gel influences the properties of alginate hydrogels, and the amount and nature of retained liquid substantially affect the porosity and mechanical strength of gel networks as well. Calcium alginate has recently been used as a cell delivery vehicle for
Hydrogel is used in various ways in medication because, in the swollen condition, its three-dimensional structure is formed by polymer chains with soft and flexible characteristics similar to natural tissue. Good hydrogels have several critical abilities that maintain three-dimensional networks and water retentiveness in use. However, the tighter the three-dimensional networks, the lesser the swellability of hydrogel, but the looser the structure, the poorer the maintenance.
Increasing the calcium content promoted the cross-linking density and reduced the molecular weight between cross-links in the alginate hydrogels. The increased cross-linking density reduces swellability, flexibility, and retentiveness of hydrogels resulting from the more compact calcium linkage network. The copolymerized A-Na-PGA hydrogels and A-Ca-PGA hydrogels overcame the defect because PGA has more hydrogen bonds in the carboxyl group replacing calcium-formed ionic networks. The carboxyl groups on the polymer chain residues of PGA are highly sensitive to pH, which creates flexible, highly absorbing, and smoother hydrogels. All the investigations of alginate hydrogels suggest that these hydrogels, especially A-Ca-PGA hydrogels, can lead to successful application for medical and pharmaceutical utilization.
As calcium plays a major role in bone metabolism, Ca-PGA has a positive effect on the reconstruction of bone. Therefore these A-CP-F series gels are applicable as injectable bone repair material. The molecular weight between cross-links and the cross-linking density of the hydrogels were characterized from the equilibrium swelling theory. Increasing the calcium content increased the cross-linking density and reduced the molecular weight between cross-links in the alginate hydrogels. Therefore, the A-CP-F composite could serve as a useful bone substitute for repairing bone defects.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors wish to thank Wan Fang Hospital and Taipei Medical University (Joint Research Grant 100TMU-WFH-08) for supporting this project.