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 one of hydroxyapatite, calcite and silicate on at least some of said cells within said network.

2. A structure as set forth in claim 1 wherein said predetermined form is a flat panel shape with a thickness of 2.5 cm.

3. A process of making a mineralized mycelium scaffolding comprising the steps of obtaining a scaffold of fungal biopolymer having a network of interconnected mycelia cells; and thereafter mineralizing said scaffold.

4. A process as set forth in claim 3 wherein said fungal biopolymer of said scaffold has a chitin/chitosan backbone and said process further comprises a step of functionalizing said scaffold by removing a OH moiety therefrom and replacing said moiety with a phosphate group to increase an affinity for cations.

5. A process as set forth in claim 4 wherein said step of mineralizing said scaffold with at least one of silicates, apatites, and carbonates.

6. A process as set forth in claim 4 further comprising the steps of placing said functionalized scaffold in a saturated calcium hydroxide solution for a period of time sufficient to create calcium phosphate precursor sites on said mycelia cells of said scaffold; thereafter suspending said scaffold in a solution with ion concentrations 1.5 times that of the human body and a pH of 7.25 for a period of time sufficient to precipitate a hydroxyapatite coating on said cells of said scaffold; and thereafter removing said scaffold from said solution and drying said scaffold.

7. A process as set forth in claim 4 further comprising the steps of preparing a slurry of milled calcium carbonate and dicalcium phosphate in water; drying said slurry to remove residual water therefrom and to form a desiccated powder; calcinating said powder at temperature and time sufficient to create a hydroxyapatite powder; creating a ceramic slurry with said hydroxyapatite powder, water, a binder, a plasticizer and a dispersant imbibing said functionalized scaffold with said ceramic slurry; lyophilizing said imbibed scaffold to remove moisture therefrom; and thereafter sintering said imbibed scaffold at a temperature and time sufficient to create a ceramic with said predetermined form.

8. A process as set forth in claim 3 wherein said fungal biopolymer of said scaffold consists of aligned bundles of fungal biopolymer assembled into a predetermined microstructure.

9. A process as set forth in claim 8 further comprising the steps of functionalizing said scaffold by removing a OH moiety therefrom and replacing said moiety with a phosphate group to increase an affinity for cations; placing said functionalized scaffold in a saturated calcium hydroxide solution for a period of time sufficient to create calcium phosphate precursor sites on said mycelia cells of said scaffold; thereafter suspending said scaffold in a solution with ion concentrations 1.5 times that of the human body and a pH of 7.25 for a period of time sufficient to precipitate a hydroxyapatite coating on said cells of said scaffold; and thereafter removing said scaffold from said solution and drying said scaffold.

10. A process as set forth in claim 3 further comprising the steps of preparing a supersaturated solution of calcium nitrate and sodium bicarbonate of a pH of 8.5; suspending said scaffold in said solution for a period of time sufficient to mineralize said scaffold with calcite; and removing said scaffold from said solution and lyophilizing said scaffold to remove residual moisture therefrom.

11. A process as set forth in claim 3 further comprising the steps of deactyling said scaffold; preparing a calcium silicate solution is of finely ground quartz and calcium oxide with a pH of 12; autoclaving said calcium silicate solution at a temperature and time sufficient to create calcium silicate, drying said calcium silicate to a powder; thereafter mixing said dried silicate powder with water to from a slurry; and infusing said slurry into said deactylated scaffold to form a scaffold/slurry matrix.

12. A process as set forth in claim 11 wherein said slurry is infused into said deactylated scaffold at a slurry: scaffold ratio of 20:1

13. A process as set forth in claim 11 further comprising the steps of compressing said scaffold/slurry matrix; heating the compressed scaffold/slurry matrix at 150 C. for up to 2 hours; and then drying the heated compressed scaffold/slurry matrix.

14. A process as set forth in claim 3 further comprising the steps of deactyling said scaffold; creating a solution of catalytic agent, fluoride salts, water, and one of amino acid and an amine; imbibing said deactylated scaffold with said solution to form a scaffold/slurry matrix; Infusing said matrix with tetraethylorthosilicate for a period of time sufficient to condense said tetraethylorthosilicate onto said matrix; and thereafter drying said matrix.

Description

EXAMPLES

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

[0021] When low-energy hydroxyapatite [Ca.sub.5(PO.sub.4).sub.3(OH)] mineralization is desired .sub.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.

[0022] 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.

[0023] 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.

[0024] 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.+5.0mM; 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.21.0 mM; trishydroxymethyl aminomethane50 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.

[0025] 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.

[0026] 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

[0027] When hydroxyapatite [Ca.sub.5(PO.sub.4).sub.3(OH)] mineralization is quickly desired .sub.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.

[0028] A hydroxyapatite slurry is prepared by milling calcium carbonate and dicalcium phosphate in deionized water (DI) until most agglomerated particles are destroyed.

[0029] This slurry is then dried until most residual moisture has been removed to form a desiccated powder.

[0030] 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.

[0031] 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).

[0032] The ceramic slurry should be created according to the following percentages: hydroxyapatite54 wt %, DI water33.8 wt %, plasticizer6.2 wt %, binder4.4 wt %, and dispersant1.6 wt %. This is milled for 24 hours to destroy agglomerated particles.

[0033] 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.

[0034] 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

[0035] When low-energy hydroxyapatite [Ca.sub.5(PO.sub.4).sub.3(OH)] mineralization is desired .sub.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.

[0036] 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.

[0037] The mineralized scaffolds can be as small as 1 mm1 mm1 mm, and the largest piece that has been created is 15 cm5 cm2.5 cm.

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

[0038] 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.

[0039] 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 2.61610.sup.3 M. 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.

[0040] 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.

[0041] 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

[0042] 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 deactylated to prepare it for mineralization.

[0043] 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].

[0044] 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.

[0045] 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

[0046] 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.

[0047] This prepared scaffold is then deactylated to prepare the scaffold for functionalization.

[0048] 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.

[0049] 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.

[0050] The deactylated 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]).

[0051] 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.

[0052] 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.