Inorganic Polymeric Materials for Injured Tissue Repair: Biocatalytic Formation and Exploitation

Abstract

Two biocatalytically produced inorganic biomaterials show great potential for use in regenerative medicine but also other medical applications: bio-silica and bio-polyphosphate (bio-polyP or polyP). Biosilica is synthesized by a group of enzymes called silicateins, which mediate the formation of amorphous hydrated silica from monomeric precursors. The polymeric silicic acid formed by these enzymes, which have been cloned from various siliceous sponge species, then undergoes a maturation process to form a solid biosilica material. The second biomaterial, polyP, has the extraordinary property that it not only has morphogenetic activity similar to biosilica, i.e., can induce cell differentiation through specific gene expression, but also provides metabolic energy through enzymatic cleavage of its high-energy phosphoanhydride bonds. This reaction is catalyzed by alkaline phosphatase, a ubiquitous enzyme that, in combination with adenylate kinase, forms adenosine triphosphate (ATP) from polyP. This article attempts to highlight the biomedical importance of the inorganic polymeric materials biosilica and polyP as well as the enzymes silicatein and alkaline phosphatase, which are involved in their metabolism or mediate their biological activity.

Keywords: alkaline phosphatase; biomaterial; biosilica; coacervate; energy delivery; morphogenetic activity; nanoparticle; polyphosphate; silicatein; tissue regeneration.

Figure 1

Biocatalytic activities of silicatein and alkaline phosphatase (ALP), the principle enzymes involved silica and calcium phosphate/hydroxyapatite biomineral synthesis. (A) Silicatein exhibits both a hydrolase activity (hydrolytic cleavage of Si-O-C bonds, e.g., of tetraalkoxysilane compounds), and silica polymerase activity, mediating the formation of polymeric biosilica from natural monomeric silicic acid precursors. (B) ALP can act both as a hydrolase, which degrades polyphosphate (polyP) to monomeric orthophosphate forming calcium phosphate deposits, and as a transferase that transfers a metaphosphate intermediate formed by cleavage of the polymer to AMP. The ADP formed is then used as a substrate by adenylate kinase (ADK). (C–F) Reactions proceeding at the catalytic site of silicatein and (G–J) of ALP; the reactions numbered are described in the text. (C) Computer model of silicatein showing the catalytic triad amino acids His, Ser and Asn (in red). The propeptide sequence of the immature silicatein is given in green. (D,E) Proposed mechanism of biosilica formation. The reaction starts by nucleophilic attack of the OH group of a Ser residue at the silicic acid monomer, facilitated by a hydrogen bridge formation to the His imidazole group. (F) Subsequent steps of the proposed silicatein mechanism, leading to the formation of a reactive cyclic trisilicic acid species. (G–I) Involvement of the zinc ions bound to the His imidazole rings of the catalytic center of ALP (G,H) in binding of the metaphosphate intermediate (I) during the enzymatic reaction. (J) Transfer reaction of the enzyme-bound metaphosphate species to AMP catalyzed by ALP.

Figure 2

Different forms and phases of biosilica and bioinorganic polyP. (A–C) Enzymatically formed biosilica is obtained first as a water-rich gel-like product (A) that undergoes a hardening process by syneresis (B). In the presence of the silicatein, the formed amorphous spherical silica nanoparticles sinter together at ambient temperature under formation of solid biosilica blocks (C). (D–F) PolyP can be present either in a soluble form (D), e.g., as sodium salt (Na-polyP), or prepared in the form of nanoparticles (at alkaline pH in the presence of a stoichiometric surplus [based on phosphate] of divalent cations, e.g., Ca²⁺ (E) or as a water-rich coacervate (at neutral pH) (F).

Figure 3

Biosilica and polyP based materials; scanning electron microscopy (SEM) analysis. (A–C): Biosilica. (A) Fusion of silica particles under (B,C) formation of solid silica structures. In (B), a sterraster of the demosponge Geodia cydonium and in (C) spicules of the demosponge S. domuncula are shown. The sterraster in (B) has been broken to make the silicatein axial filaments visible in the centers of the holes (axial canals). (D–F): PolyP. (D,E) Amorphous Ca-polyP nanoparticles (different magnifications. Adapted with permission from ref. [87]. Copyright 2018, John Wiley and Sons. (F) Coacervate formed by Ca-polyP.

Figure 4

Delivery of metabolic energy during hydrolytic degradation of inorganic polyP. (A) Release of Gibb’s free energy (ΔG⁰) during stepwise hydrolysis of the energy-rich phosphoanhydride bonds of linear polyP molecules. During each hydrolytic step, a ΔG⁰ of approximately −30 kJ·mol⁻¹ is liberated. (B) Short-chain triphosphate (polyP₃) can exist either as a linear molecule or in a cyclic form. The cyclic polyP₃ has one phosphoanhydride bond more than the linear molecule and can deliver 50% more energy (ΔG⁰ = −90 kJ·mol⁻¹) compared to linear polyP₃ (ΔG⁰ = −60 kJ·mol⁻¹).

Figure 5

Different routes of administration of biosilica and polyP for tissue regeneration/repair. Both biosilica, either alone or together with silicatein, and polyP, as soluble polyP or polyP nanoparticles, can be applied by using (A) 3D printing techniques, (B) electrospinning, or (C) in a microparticular form, after encapsulation in poly(d,l-lactide-co-glycolide (PLGA). (D–F) Biosilica. (D) 3D printed grid of a biosilica-supplemented and SaOS-2 cells containing hydrogel. (E) Patches of biosilica (bs) deposits formed by silicatein on the surface of a TEOS-containing electrospun PCL nanofiber mat; SEM. Adapted with permission from ref. [113]. Copyright 2014, John Wiley and Sons. (F) Healing of a bone defect after implantation of PLGA-based microspheres, containing silica/silicatein. A tissue section from patella of rabbit in vivo labeled with oxytetracycline [115] is shown, allowing visualization of new bone formation; ms, microsphere. (G–I) PolyP. (G) 3D bio-printed disc prepared with a polyP-supplemented bio-ink solution, based on N,O-carboxymethyl chitosan; environmental scanning electron microscopy (ESEM). Adapted with permission from ref. [112]. Copyright 2022, IOP Publishing. (H) Electrospun fibrous mat fabricated with poly(lactic acid) (PLA) and amorphous Ca-polyP nanoparticles. After incubation with mouse calvaria MC3T3-E1 cells, the formation of cell (c) layers on the mats is visible. Adapted with permission from ref. [114]. Copyright 2015, Elsevier. (I) Discs prepared from amorphous polyP encapsulated into PLGA microspheres. Adapted with permission from ref. [116]. Copyright 2016, John Wiley and Sons.