Biomimetic Alginate/Gelatin Cross-Linked Hydrogels Supplemented with Polyphosphate for Wound Healing Applications

Abstract

In the present study, the fabrication of a biomimetic wound dressing that mimics the extracellular matrix, consisting of a hydrogel matrix composed of non-oxidized and periodate-oxidized marine alginate, was prepared to which gelatin was bound via Schiff base formation. Into this alginate/oxidized-alginate-gelatin hydrogel, polyP was stably but reversibly integrated by ionic cross-linking with Zn²⁺ ions. Thereby, a soft hybrid material is obtained, consisting of a more rigid alginate scaffold and porous structures formed by the oxidized-alginate-gelatin hydrogel with ionically cross-linked polyP. Two forms of the Zn-polyP-containing matrices were obtained based on the property of polyP to form, at neutral pH, a coacervate-the physiologically active form of the polymer. At alkaline conditions (pH 10), it will form nanoparticles, acting as a depot that is converted at pH 7 into the coacervate phase. Both polyP-containing hydrogels were biologically active and significantly enhanced cell growth/viability and attachment/spreading of human epidermal keratinocytes compared to control hydrogels without any adverse effect on reconstructed human epidermis samples in an in vitro skin irritation test system. From these data, we conclude that polyP-containing alginate/oxidized-alginate-gelatin hydrogels may provide a suitable regeneratively active matrix for wound healing for potential in vivo applications.

Keywords: alginate; cell migration; coacervate; gelatin; human epidermal keratinocytes; inorganic polyphosphate; ionic cross-linking; nanoparticles; periodate oxidation; zinc ions.

Figure 1

Oxidation of sodium alginate by periodate (NaIO₄) and cross-linking with gelatine.

Figure 2

Formation of Zn-polyP coacervate and preparation of alginate-oxidized alginate hydrogel. (I.) Fabrication of Zn-polyP coacervate and Zn-polyP-NP; SEM. (A,B) Addition of ZnCl₂ at super-stoichiometric concentration ratio to Na-polyP at pH 7 results in the formation of the aqueous coacervate phase which, after drying, appears as jagged material. (C,D) If the reaction is run at pH 10 nanoparticles (NP) are formed. (II.) Preparation of the alginate-oxidized alginate hydrogel. (A) Alginate was added dropwise to oxidized alginate (OA-50). (B) After gel formation (C) gelatin was added, resulting in an increased viscosity of the hydrogel. (D) Finally, the material was ionically cross-linked with ZnCl₂ and freeze-dried; ESEM.

Figure 3

“ALG/OA-HG” and the characterization. (I.) Ionic cross-linking within the “ALG/OA-HG”. (A) Dry alginate-OA-50 material. (B) After submersion in saline the hybrid material dissolves almost completely after 12 h. (C) Solid alginate-OA-50 material obtained after treatment with ZnCl₂ and drying. (D) After exposure to saline the material stays stable. (II.) Characterization of alginate and oxidized alginate (OA-50) by FTIR. Alginate shows the characteristic carboxylate (COO⁻) vibrational mode signals at 1593 cm⁻¹ and 1403 cm⁻¹. Then C-O-C stretching signals of the acetal group are recorded at 1083 cm⁻¹ and at 1015 cm⁻¹. The comparative FTIR spectrum to OA-50 shows that the signals for the latter material are less intensive.

Figure 4

Morphology and characterization of the hydrogels. (I.) Surface texture of the different hydrogels; ESEM. (A,B) The polyP lacking hydrogel, “ALG/OA-HG”. This hydrogel was supplemented with Na-polyP and then treated with ZnCl₂. (C,D) At pH 7 polyP forms a coacervate, “ALG/OA-polyP-Coa-HG”, while (E,F) the surface of the material, composed of alginate/OA-50/gelatin and polyP, and obtained at pH 10, is covered with nanoparticles (NP), “ALG/OA-polyP-NP-HG”. (II.) EDX spectra, taken from (A) Ca-polyP-NP and (B) “ALG/OA-polyP-NP-HG”.

Figure 5

Viability of cells on the hydrogels. (I.) MTT analysis to assess the growth/viability of the keratinocytes. The cells were cultivated onto “ALG/OA-HG”, “ALG/OA-polyP-Coa-HG” or “ALG/OA-polyP-NP-HG” matrices. The viability/cell number was determined after an incubation period of 2 d or 4 d. Then the cells were reacted with MTT and the intensity of the formed formazan dye was measured with a microplate reader at 550 nm. Ten parallel assays per point were performed. The data represent means ± SD. The significances within an individual group (*; p < 0.01) were calculated. The significances with respect to the assays without polyP are likewise marked (#; p < 0.01). (II.) Microscopic analysis of the cells onto the respective gels, “ALG/OA-HG”, “ALG/OA-polyP-Coa-HG” or “ALG/OA-polyP-NP-HG”, after an incubation period for 4 d. In A to C the cells were vitally stained with calcein and inspected with a fluorescence microscope. In D to F the cells were visualized by ESEM.

Figure 6

Determination of the migration activity of keratinocytes in the Boyden chamber. (A) Image of the Boyden chamber. The cells were placed into the upper reservoir, a transwell insert (TW), which is connected with the lower reservoir by a porous membrane (M). On the bottom of the lower reservoir, the hydrogel (G) is introduced. Attracted by the polyP released from the gel the cells migrate through the membrane. (B) Increase of the migration activity of keratinocytes in those assays into which the polyP-containing hydrogels, either “ALG/OA-polyP-Coa-HG” or “ALG/OA-polyP-NP-HG” had been added to the lower chamber. The migration activity is correlated with the one measured in the controls (without any hydrogel; set to 100%). Addition of the polyP-lacking “ALG/OA-HG” hydrogel to the system did not influence the activity (*; p < 0.01). (C) Light micrographs of the cells on the bottom side of the membrane (invasive cells) prior to fixation.

Figure 7

Determination of the viability of the hydrogels by using the in vitro reconstructed 3D human skin model. (A) the skin sample in the inserts of the well plates (a) prior and (b) after the addition of the hydrogel sample (200 µL). (c) Histological cross-section through the skin with the stratum corneum and the epidermis; staining with hematoxylin and eosin. (B) Short term skin irritation test by application of the MTT assay (n = 5).

Figure 8

Schematic outline of alginate spheres (left) and alginate/oxidized-alginate-gelatin hybrid hydrogel (right). (Left) The natural alginate comprises a compact hydrogel that is ionically linked to a resistant protection of cells against external attacks. (Right) The alginate/oxidized-alginate-collagen hybrid material is a relatively soft hydrogel into which cells can migrate and proliferate. This material is designed as wound dressing.