# Patent

## Title
Oxygenated three-dimensional matrix for bone growth

## Abstract

An encapsulated three-dimensional (3-D) bone matrix composition for inducing bone growth includes a 3-D bone matrix, an oxygen carrier incorporated into the 3-D bone matrix, and an encapsulation material which encapsulates the 3-D bone matrix incorporated with the oxygen carrier. Methods are disclosed for preparing an encapsulated 3-D bone matrix that can be maintained in packaging.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a National Phase Patent Application and claims the priority of International Application Number PCT/US2014/011359, filed on Jan. 13, 2014, which claims priority of U.S. Provisional Application No. 61/751,926, filed on Jan. 13, 2013. The disclosures of which are hereby incorporated by reference.


Improving the processing and administration of an oxygenated bone matrix composition will improve the osteogenesis, osteoconduction, and osteoinduction capabilities in bone grafting to advance the state of the art in bone repair. For three-dimensional (3-D) demineralized bone matrix (DBM), preserving the oxygenated matrix is challenging given the hydrophobic nature of the oxygen carrier material as well as the 3-D structure of the matrix. For example, perfluorocarbons (PFCs) are oxygen carriers, and are extremely hydrophobic viscous liquids which render making a formulation suitable for implantation into patients using PFCs very challenging. Accordingly, a need exists for an oxygenated 3-D bone matrix for bone growth that can be maintained in packaging from the time of manufacturing until its use.
SUMMARY
In some embodiments, an encapsulated three-dimensional (3-D) matrix composition for inducing bone growth includes a 3-D matrix, an oxygen carrier incorporated into the 3-D bone matrix, and an encapsulation material that encapsulates the 3-D bone matrix incorporated with the oxygen carrier.
In some embodiments, a method of making an encapsulated oxygenated three-dimensional (3-D) matrix includes incorporating an oxygen carrier into a 3-D bone matrix to form an oxygenated 3-D bone matrix, and incorporating an encapsulation material into the oxygenated 3-D bone matrix to form an encapsulated oxygenated 3-D bone matrix.


DETAILED DESCRIPTION
The present disclosure describes an oxygenated 3-D bone matrix for inducing bone growth that retains the incorporated oxygen carrier and is capable of being maintained in a package. Maintaining and preserving the incorporation of an oxygen carrier in a 3-D bone matrix is difficult because of the matrix structure that is not conducive to mixing and the hydrophobicity of oxygen carriers. For example, in a demineralized bone matrix (DBM) that is incorporated with a perfluorocarbon (PFC), the PFC is hydrophobic and the DBM is hydrophilic. As disclosed herein, a 3-D bone matrix is incorporated with an oxygen carrier and the oxygenated 3-D bone matrix is then encapsulated for packaging and later use.
It has previously been demonstrated that PFC's can be applied to various matrices including DBM and enhance cellular activity, as described in U.S. patent application Ser. No. 13/740,244, the entire contents of which are herein incorporated by reference.
As used herein the term demineralizing refers to demineralization of bone. The decrease in the amount of calcium in the bone determines the amount of demineralization. That is, completely demineralized bone has no calcium, or only trace amounts of calcium. In some embodiments completely demineralized bone has no more than 10% calcium.
In some embodiments, a 3-D bone matrix includes an oxygen carrier incorporated within a stable formulation. In some embodiments, the oxygen carrier is a PFC. A stable formation includes encapsulation which retains the incorporated PFC and decreases elution of the PFC from the matrix. In some embodiments, the oxygenated 3D matrix is stable while packaged.
Non-limiting examples of 3-D matrices include: a demineralized cancellous DBM sponge, a DBM fiber matrix, and non-demineralized cancellous strips. Non-limiting examples of demineralized cancellous DBM sponge are disclosed in U.S. Provisional Application No. 61/877,825, Appendix I, the entire contents of which are herein incorporated by reference. Non-limiting examples of a DBM fiber matrix include Grafton® Flex, Grafton® Matrix, Grafton® Matrix Strips, and those disclosed in U.S. Provisional Application No. 61/864,499, the entire contents of which are herein incorporated by reference. In some embodiments, the 3-D bone matrix is a demineralized cancellous DBM sponge.
In some embodiments, the oxygen carrier is a perfluorocarbon (PFC). Non-limiting examples of PFCs include perfluorodecalin, perfluorohexane, perfluoroperhydrophenanthrene, perfluorobutylamine (PFTBA or PFTBM), perfluorooctylbromide (PFOB), perfluoro-n-octane, octafluoropropane, perfluorodichlorooctane, perfluorodecane, perfluorotripropylamine, perfluorotrimethylcyclohexane, perfluoromethyladamantane, perfluorodimethyladamantane, perfluoromethyldecaline, perfluorofluorene, diphenyldimethylsiloxane, hydrogen-rich monohydroperfluorooctane, alumina-treated perfluorooctane, mixtures thereof, or any suitable oxygen carrier.
The choice of material used for encapsulation influences the performance of the 3-D bone matrix. Encapsulation material is characterized by the following: i) it retains the oxygen carrier within the 3-D bone matrix, ii) it does not interfere with the rehydration of the 3-D bone matrix, and/or iii) it is capable of being mixed with bone marrow aspirate. As such, encapsulation materials are hygroscopic, rehydrate rapidly, and rapidly dissolve or remodel.
In some embodiments, a porous 3-D bone matrix is soaked in an oxygen carrier (e.g., a PFC), and the oxygen-soaked 3-D bone matrix is then immersed in a collagen-derived material (e.g., gelatin) to form an encapsulated oxygenated 3-D bone matrix. Additional encapsulation materials include glycerols, lecithins, polyoxamers, phospholipids, and other suitable materials having the identified characteristics.
In some embodiments, the oxygen carrier is combined with other dispersing agents to form an emulsion, gel or admixture, and this mixture is then incorporated into the 3-D bone matrix. Non-limiting examples of dispersing agents include glycerols, phospholipids, lecithins, surfactants, and polyoxymers.
In some embodiments, DBM powder is combined with an oxygen carrier to form an oxygenated DBM slurry mixture as described in U.S. application Ser. No. 13/740,244, the entire contents of which are herein incorporated by reference. Specifically a PFC as disclosed herein is mixed with DBM (powder or chips) to form a DBM/PFC mixture. In some embodiments, the amount of PFC in the PFC/DBM mixture is in the range of about 32-48 weight %. Sterile saline is then added to provide a slurry with the handling characteristics of wet sand. This oxygenated DBM slurry mixture is then incorporated into the 3-D bone matrix. The addition of DBM powder enhances the osteoinductive potential of the 3-D bone matrix.
In some embodiments, the process for oxygenating and encapsulating the 3-D bone matrix includes soaking the 3-D bone matrix in a plasticizing agent (also known as a swelling agent) until the matrix is fully saturated. The saturated 3-D bone matrix it is then lyophilized (e.g., dehydration) to remove any water, leaving behind the plasticizing agent that keeps the matrix pliable. In some embodiments the plasticizing agent is mixed with water to help facilitate incorporation of the plasticizer into the matrix. Non-limiting examples of plasticizing agents include phospholipids, liquid polyhydroxyl compounds and liquid polyhydroxyl compound derivatives. The polyhydroxyl compounds and derivatives of this type include those which in the pure or concentrated state and at ambient temperature are normally solid or semi-solid but are soluble in a suitable solvent, e.g., water, physiological saline, ethanol, glycerol, glucose, propylene glycol, polyethylene glycol of from 200-1000 molecular weight, etc., or mixtures thereof, to provide a liquid composition.
In particular, useful polyhydroxyl swelling agents possess from 2 to about 18 carbons and include such classes of compounds as the acyclic polyhydric alcohols, non-reducing sugars, sugar alcohols, sugar acids, monosaccharides, disaccharides, water-soluble or water dispersible oligosaccharides, polysaccharides and known derivatives thereof. Specific polyhydroxyl compounds include ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol, trimethylolethane, trimethylopropane, erythritol, pentaerythritol, polyalkylene glycols such as the polyethylene glycols, xylitol, sorbitol, mannitol, dulcitol, arabinose, xylose, ribose, adonitol, arabitol, rhamose, inositol, fructose, galactose, glucose, mannose, sorbose, sucrose, maltose, lactose, maltitol, lactitol, stachyose, maltopentaose, cyclomaltohexaose, trehalose, carrageenan, agar, alginic acid, guar gum, gum tragacanth, locust bean gum, gum arabic, xanthan gum, amylose, mixtures and copolymers thereof.
After saturation and lyophilization, an oxygen carrier is then added to the saturated and lyophilized matrix. The oxygenated bone matrix is then placed into a collagen solution for encapsulation of the oxygenated matrix. This encapsulated and oxygenated 3-D bone matrix is placed into standard packaging formats without the need for special handling.
The following Examples are presented for illustrative purposes only, and do not limit the scope or content of the present application.
Example 1. Incorporation of PFTBM into a 3-D DBM Matrix
A piece of demineralized allograft cancellous bone sponge (DCS) (OsteoSponge®, Bacterin) weighing 0.3 grams was placed into a vial containing PFTBM (Sigma Aldrich), and agitated for 5 minutes. The DCS was removed from the vial and the free PFTBM allowed to drain. The weight was 1.36 grams. The sample was then blotted using non-linting paper and a final weight of 0.85 grams recorded. From the dimensions of the sponge it could be calculated that the PFTBM concentration was 0.92 g/cm3. The impregnated DCS was then dipped into a second solution of Pluronic F-68 in N-methyl-2-pyrrolidone (NMP) to encapsulate the construct, trapping the PFC inside.
Example 2. Incorporation of PFTBM into a 3-D DBM Matrix
A piece of demineralized allograft cancellous bone sponge (DCS) (OsteoSponge®, Bacterin) weighing 0.26 grams was placed into a vial containing PFTBM and agitated for 5 minutes. The DCS was removed from the vial and the free PFTBM allowed to drain. The weight was 1.05 grams. The sample was then blotted using non-linting paper and a final weight of 0.87 grams recorded. From the dimensions of the sponge it could be calculated that the PFTBM concentration was 1.04 g/cm3. The impregnated DCS was then dipped into a solution of lecithin in water to encapsulate the construct, trapping the PFC inside. The sample was then dried in a vacuum oven.
Example 3. Incorporation of PFTBM into a 3-D DBM Matrix
A piece of demineralized allograft cancellous bone sponge (DCS) (OsteoSponge®, Bacterin) weighing 0.14 grams was placed into a vial containing PFTBM and agitated for 5 minutes. The DCS was removed from the vial and the free PFTBM allowed to drain. The weight was 0.57 grams. The sample was then blotted using non-linting paper and a final weight of 0.37 grams recorded. From the dimensions of the sponge it could be calculated that the PFTBM concentration was 0.5 g/cm3. The impregnated DCS was then dipped into a second solution of gelatin to encapsulate the construct, trapping the PFC inside. 





















While the present invention has been illustrated and described with reference to certain exemplary embodiments, those of ordinary skill in the art will understand that various modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present invention, as defined in the following claims.

## Claims

1. An encapsulated 3-dimensional (3-D) bone matrix composition for inducing bone growth, comprising:
a 3-D bone matrix selected from a demineralized cancellous sponge or a non-demineralized cancellous bone strip;
an oxygen carrier incorporated into the 3-D bone matrix; and
an encapsulation material that encapsulates the 3-D bone matrix.

2. The encapsulated 3-D bone matrix composition of claim 1, wherein the oxygen carrier is selected from the group consisting of perfluorodecalin, perfluorohexane, perfluoroperhydrophenanthrene, perfluorobutylamine (PFTBA or PFTBM), perfluorooctylbromide (PFOB), perfluoro-n-octane, octafluoropropane, perfluorodichlorooctane, perfluorodecane, perfluorotripropylamine, perfluorotrimethylcyclohexane, perfluoromethyladamantane, perfluorodimethyladamantane, perfluoromethyldecaline, perfluorofluorene, diphenyldimethylsiloxane, monohydroperfluorooctane, alumina-treated perfluorooctane, and mixtures thereof.

3. The encapsulated 3-D bone matrix composition of claim 1, wherein the encapsulation material is a collagen-derived material.

4. The encapsulated 3-D bone matrix composition of claim 1, further comprising a demineralized bone matrix powder mixed with the oxygen carrier.

5. The encapsulated 3-D bone matrix composition of claim 1, further comprising a dispersing agent mixed with the oxygen carrier.

6. The encapsulated 3-D bone matrix composition of claim 5, wherein the dispersing agent is selected from the group consisting of glycerols, lecithins, polyoxamers, surfactants, and phospholipids.