Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation

Abstract

The objective of this study is to design bioabsorbable injectable hydrogels based on the physico-chemical interaction between biocompatible polymers and ferric ions, and evaluate the survival, proliferation, and osteogenic differentiation of cells encapsulated in the hydrogels. The injectable hydrogels were prepared by simply mixing mixed alginate/gelatin solution at various ratios and FeCl₃ solution. The hydrogels prepared disappeared within a few days in the phosphate buffered-saline solution (PBS) with containing collagenase although the disappearance rate increased with an increase of the gelatin ratio in the hydrogel. For the hydrogel of alginate/gelatin low ratio, the survival and proliferation of cells in the hydrogel-encapsulated condition were significantly high compared with those of hydrogel at the higher ratios. The cells collected 3 days after cultured in the hydrogel also proliferated to a significantly higher extent than those collected from other hydrogels. The proliferation ability of cells was similar that of cells cultured on the standard tissue culture polystyrene (TCPS) dish. When evaluated to compare with cells cultured on the TCPS dish, the expression of runt-related transcription factor-2 (RUNX2) gene, the alkaline phosphatase (ALP) activity, and the calcium precipitation were significantly high. The cells were encapsulated by the mixed alginate/gelatin and FeCl₃ hydrogel and injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the injectable hydrogel prepared by simple mixing mixed alginate/gelatin solution and FeCl₃ solution is a promising material for the cell transplantation.

Statement of Significance

Injectable hydrogels prepared by simple mixing mixed alginate/gelatin solution at various ratios and FeCl₃ solution. For the hydrogel of alginate/gelatin low ratio, the survival, the proliferation, and the differentiate properties of cells in the hydrogel-encapsulated condition were similar those of cells cultured on the TCPS dish. When the cells encapsulated hydrogels were injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the present injectable hydrogel is a promising material for the cell transplantation.

Introduction

Cell transplantation is one of the promising therapies in regenerative medicine to induce the regeneration and repairing of damaged tissues and organs [1], [2], [3]. Especially, the cell transplantation via injection is attractive as a minimally invasive treatment since surgical operations are not required. Potential cell transplantation is most typically introduced into the tissues of interest directly via the injection of cells, suspended in an appropriate medium [4], [5], [6]. However, it is well recognized that the retention of cells transplanted at the injection site is extremely poor by the method [7]. Therefore, it is of prime importance to create a technology and methodology for an enhanced retention of cells transplanted at the injection site.

Under these circumstances, significant research efforts have been investigated to design injectable hydrogels that can be injected in a solution form into the body and then rapidly form the hydrogel at the injection site. Injectable hydrogels have many advantages for various biomedical applications, such as their ease of administration, the minimally invasive treatment, and simple cell encapsulation [8]. Various injectable hydrogels have been mainly prepared by chemically or covalently crosslinked of polymers [9], [10], [11]. However, the injectable hydrogels of chemical gelation require the introduction of chemically active residues or catalysts to initiate the in situ reaction [12]. Moreover, the cells transplanted with such injectable hydrogels do not always survive nor function efficiently in the body. This is mainly because the injectable hydrogels of chemical crosslinking are not degraded in the body synchronizing with the cell proliferation [13], [14], [15]. As one trial to tackle the issue, injectable hydrogels formed through a physico-chemical interaction force need to be designed. For the hydrogel formed based on the physico-chemical interaction, there are no needs to use the crosslinking agents for in situ reaction. In addition, the survival, proliferation, and biological functions are not suppressed by the in vivo remaining of hydrogels due to the fast degradation [12], [13], [14], [15].

Gelatin and alginate are both biocompatible polymers and have been widely used for food, pharmaceutical, and medical applications. The bio-safety also have been proven through their long-term these applications. In addition, ferric ions present in the body and do not show cytotoxicity at the low concentration. It is demonstrated that both the gelatin and alginate physically interact with ferric ions [16,17]. The properties of hydrogels crosslinked by the physical interaction can be controlled by changing the polymer concentration, molecular weight, and ion concentration [14].

In this study, injectable hydrogels based on the physico-chemical interaction among gelatin, alginate, and ferric ion are designed. The gelation behavior of hydrogels prepared by changing the mixing ratio of alginate and gelatin in the presence of ferric ions are evaluated. The viability and proliferation of cells encapsulated in the hydrogels were investigated. As one application of hydrogel, cells are transplanted with the injectable hydrogel for bone regeneration. Therefore, here, the osteogenic differentiation of cells was evaluated as one cell ability. We also evaluate the in vivo cell retention after injection with the hydrogel of alginate/gelatin and ferric ions.