Process for making mineralized mycelium scaffolding and product made thereby

Abstract

The process of making a mineralized mycelium scaffolding requires obtaining a scaffold of fungal biopolymer having a network of interconnected mycelia cells, functionalizing the biopolymer to create precursor sites and thereafter mineralizing the scaffold with one of silicates, apatites and carbonates. The mineralized mycelium scaffolding may be used for medical applications in place of mineralized collagen membranes and collagen/hydroxyapatite composite scaffolds.

Claims

1. A structure comprising a scaffold of fungal biopolymer of predetermined form characterized in being formed of a network of interconnected mycelia cells; and a coating of at least one of an apatite, a carbonate, and a silicate on at least some of said cells within said network.

2. The structure of claim 1, wherein said predetermined form is a flat panel shape.

3. The structure of claim 2, wherein said flat panel shape has a thickness of 2.5 cm.

4. The structure of claim 1, wherein said coating is one of a hydroxyapatite, calcite, and silicate.

5. The structure of claim 1, wherein said coating is a ceramic made from hydroxyapatite powder, water, a binder, a plasticizer, and a dispersant.

6. The structure of claim 1, wherein said fungal biopolymer of said scaffold consists of aligned bundles of fungal biopolymer assembled into a predetermined microstructure.

7. The structure of claim 1, wherein said coating is made from dried calcium silicate powder and water.

8. The structure of claim 1, wherein said coating is made from a solution of catalytic agent, fluoride salts, water, and one of an amino acid and an amine.

9. The structure of claim 8, wherein said coating is infused with tetraethylorthosilicate.

10. The structure of claim 1, wherein said fungal biopolymer of said scaffold has a chitin/chitosan backbone.

11. The structure of claim 10, wherein an —OH moiety in said scaffold is replaced with a phosphate group.

12. The structure of claim 11, wherein said coating is a ceramic made from hydroxyapatite powder, water, a binder, a plasticizer, and a dispersant.

Description

EXAMPLES

1A Hydroxyapatite Mineralization of Mycelial Scaffold via a Solution-Based Reaction

(1) When low-energy hydroxyapatite [Ca.sub.5(PO.sub.4).sub.3(OH)] mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents (cleaning while preserving structure). This will serve as the raw scaffold, upon which mineralization will occur.

(2) The cleaned scaffold is then phosphorylated, functionalizing the chitin/chitosan backbone, replacing the —OH moiety with a phosphate group, thereby increasing the scaffold's affinity for cations to prepare the scaffold for mineralization.

(3) The scaffold should be thoroughly rinsed of any interfering residue and is then attached to a nonreactive clip to be freely suspended in and/or imbibed with in a saturated calcium hydroxide solution for 1-30 days, depending on desired degree of calcification, purity of starting material, degree of phosphorylation, or the like. In this step, calcium penetrates the scaffold which creates the calcium phosphate precursor sites necessary for hydroxyapatite formation.

(4) After rinsing off residual calcium hydroxide solution, the scaffold is suspended in a 36.5° C. solution with ion concentrations 1.5 times that of the human body (1×=Na.sup.+—142.0 mM; K.sup.+—5.0 mM; Mg.sup.2+—1.5 mM; Ca.sup.2+—2.5 mM; Cl.sup.−—148.8 mM; HCO.sub.3.sup.−—4.2 mM; HPO.sub.4.sup.2−—1.0 mM; trishydroxymethyl aminomethane—50 mM). The salts used to create this solution are: NaCl, NaHCO.sub.3, KCl, K.sub.2HPO.sub.4.3H.sub.2O, MgCl.sub.2.6H.sub.2O, CaCl, trishydroxymethyl aminomethane.

(5) Once prepared, the solution is buffered to pH 7.25 with concentrated HCl. To ensure process consistency, the ion concentration should be monitored and adjusted regularly either with a chemostat or manual inspection. This step requires upwards of 30 days of active soaking of the scaffold (with or without initial imbibing) to precipitate a hydroxyapatite coating of the desired thickness on the cells of the mycelial mass of the scaffold.

(6) The mineralized scaffold should then be dried, completing this mineralization process. The resultant scaffold of fungal biopolymer has a network of interconnected mycelia cells and a hydroxyapatite coating on the cells within the network.

1B Hydroxyapatite Mineralization of Mycelial Scaffold via a Solid-State Reaction

(7) When hydroxyapatite [Ca.sub.5(PO.sub.4).sub.3(OH)] mineralization is “quickly” desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur.

(8) A hydroxyapatite slurry is prepared by milling calcium carbonate and dicalcium phosphate in deionized water (DI) until most agglomerated particles are destroyed.

(9) This slurry is then dried until most residual moisture has been removed to form a desiccated powder.

(10) The desiccated powder is then calcinated at 900° C. for 1 hour at a heating rate of 5° C./min. This reaction creates a hydroxyapatite powder.

(11) The next step involves creating a ceramic slurry with the powder, DI water, a plasticizer (including, but not limited to, polyethylene glycol, glycerin, sorbitol, alkyl citrates, or acetylated monoglycerides), a binder (including, but not limited to: polyvinyl alcohol, lecithin, soy lecithin, or sodium stearoyl lactylate), and dispersant (including, but not limited to, polycarboxylate ether based superplasticizers, or Dispex polyacrylate dispersant).

(12) The ceramic slurry should be created according to the following percentages: hydroxyapatite—54 wt %, DI water—33.8 wt %, plasticizer—6.2 wt %, binder—4.4 wt %, and dispersant—1.6 wt %. This is milled for 24 hours to destroy agglomerated particles.

(13) The prepared scaffold is then imbibed with the milled ceramic slurry via vacuum infusion and lyophilized to remove moisture. The dry slurry/scaffold matrix is then sintered in a 1300° C. in a furnace for four hours at a heating rate of 5° C./min., creating a ceramic in the form of the original fungal scaffold.

(14) The resultant ceramic consists of a scaffold of fungal biopolymer with a network of interconnected mycelia cells and a hydroxyapatite coating on at least some of the cells within the network.

1C Hydroxyapatite Mineralization of Fungal Biopolymer Scaffold via a Solution-Based Reaction

(15) When low-energy hydroxyapatite [Ca.sub.5(PO.sub.4).sub.3(OH)] mineralization is desired upon a scaffold consisting of aligned bundles of fungal biopolymer assembled into a desired microstructure (e.g., helicodical to increase compressive strength by hindering crack propagation), one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur. From here, the procedure from 1A, beginning with functionalizing the chitin/chitosan backbone, followed through completion.

(16) The scaffolds mineralized with hydroxyapatite may be of a size and shape to be put to use as a biomedical material, for example, the scaffold may be of a flat panel shape with a thickness of 2.5 cm.

(17) The mineralized scaffolds can be as small as 1 mm×1 mm×1 mm, and the largest piece that has been created is 15 cm×5 cm×2.5 cm.

2A Calcite Mineralization of Mycelial Scaffold Via a Solution-Based Reaction

(18) When a low-energy calcite (CaCO.sub.3) mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur.

(19) A supersaturated solution is prepared from filtered and standardized stock calcium nitrate and sodium bicarbonate at 25° C. with a calcium and carbonate concentration of the working solution of 2.616×10.sup.−3M. The pH of this solution is then adjusted to 8.5 with standardized 0.1M potassium hydroxide solution and this is allowed to equilibrate in temperature and CO.sub.2 partial pressure.

(20) The cleaned scaffold is then suspended in this solution, making sure the solution is fully infused into the scaffold. Ion concentrations are constantly monitored and corrected by the addition of calcium nitrate, sodium carbonate, sodium bicarbonate, and potassium hydroxide, either manually or via a pH stat.

(21) Once the desired level of mineralization is achieved, the matrix is lyophilized to remove residual moisture and to complete the process.

3A Silication of Mycelial Scaffold Via Hydrothermal Hot Pressing

(22) When silicate mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur. This prepared scaffold is then deacetylated to prepare it for mineralization.

(23) A calcium silicate solution is created from finely ground quartz and calcium oxide, mixed well with a 1:1 Ca:Si ratio and a 20:1 water:powder ratio. The pH of this solution is adjusted to 12 with ammonium hydroxide and is transferred to a nonreactive vessel to be autoclaved at 150° C. for 24 hours to create calcium silicate, likely xonotlite [Ca.sub.6Si.sub.6O.sub.17(OH).sub.2].

(24) The silicate product is then collected, washed with DI water, and dried for 24 hours to remove residual water. The dry silicate powder is then mixed well into a 90 wt % slurry with DI water and is then infused into the prepared scaffold at a slurry:scaffold ratio of 20:1.

(25) The scaffold/slurry matrix is then moderately compressed (upwards of 50 MPa) and returned to the autoclave at 150° C. for upwards of 2 hours. The mineralized product is then dried to complete the process.

3B Silication of Mycelial Scaffold Via a Solution-based Reaction

(26) When silicate mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur.

(27) This prepared scaffold is then deacetylated to prepare the scaffold for functionalization.

(28) The deacetylation step removes the primary amine from chitin, which cannot be functionalized, and transitions the functional group to a hydroxyl, which is chitosan. This hydroxyl can then serve as the targeted site for phosphorylation or the like.

(29) The deacetylation process uses a 5 molar concentration of NaOH at 90 C for 30 to 120 minutes. The specimen is immersed in solution during this time.

(30) The deacetylated scaffold, under air or an inert atmosphere, is then imbibed with catalytic agent (including, but not limited to, an appropriate concentration and type of acid [e.g., acetic acid, hydrochloric acid, phosphoric acid, or the like], fluoride salts (e.g., potassium fluoride, sodium fluoride, tetra-n-butylammonium fluoride, or like like), water, an amino acid (e.g., cysteine or the like) or an amine [e.g., urea, imidazole, or the like]).

(31) The catalyst/scaffold matrix is then infused with tetraethylorthosilicate and silica allowed to condense onto the scaffold for upwards of 24 hours. The product is then dried to complete the process.

(32) The invention thus provides a relatively simple process for producing a mineralized biocompatible material as well as a unique biomedical material that may be used for medical applications in place of mineralized collagen membranes and collagen/hydroxyapatite composite scaffolds.