Compositions for preparing bone graft

Abstract

Provided herein are formulations for treating bone graft materials prior to implantation. Treating the bone graft materials with the formulations disclosed herein can restore the physiological and biochemical characteristics of bone. The bone graft can be a bone allograft. Also provided herein are methods for preparing the formulations and methods for preparing a bone graft for transplantation.

Description

COMPOSITIONS FOR PREPARING BONE GRAFTCROSS-REFERENCE

[0001] This PCT application claims the benefit to U.S. Provisional Application No. 63 / 710,802, filed October 23, 2024, the content of which is incorporated herein in its entirety by reference.FIELD

[0002] This disclosure relates to compositions and methods for preparing grafts in general, and more particularly to, bone grafts.BACKGROUND

[0003] There remains a need for improved compositions formulated for restoring the physiological and biochemical characteristics of bone grafts prior to implantation.SUMMARY

[0004] In one aspect, provided herein is a formulation for rehydrating a bone graft material, comprising: about 5 mM of P-glycerophosphate; about 70 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and about 2.5 mM calcium lactate, in a buffered aqueous solution.

[0005] In another aspect, provided herein is a formulation for rehydrating a bone graft material, comprising: 1-25 mM of P-glycerophosphate; 1-100 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and 1-20 mM calcium lactate, in a buffered aqueous solution.

[0006] In another aspect, provided herein is a formulation for rehydrating a bone graft material, comprising: an organic phosphate, L-ascorbic acid or a derivative thereof, and an organic calcium salt.

[0007] In another aspect, provided herein is a method of preparing a formulation for hydrating a bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate an ascorbic acid-organic phosphate-organic calcium salt solution, thereby generating the formulation.

[0008] In another aspect, provided herein is a method of preparing a bone graft, comprising: (1) preparing a formulation for rehydrating a bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii)dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation; (2) providing the bone graft material; and (3) rehydrating the bone graft material in the formulation for a period of time, thereby generating the bone graft.

[0009] In another aspect, provided herein is a method of treating a bone condition or defect in a subject in need thereof, comprising:(l) providing a bone graft material; (2) preparing a formulation for rehydrating the bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation; (3) rehydrating the bone graft material in the formulation for a period of time, generating a bone graft; (4) implanting the bone graft to a bone site of the bone condition or defect in the subject.

[0010] In yet another aspect, provided herein is a kit for preparing a bone graft, comprising: (1) P-glycerophosphate; (2) L-ascorbic acid; (3) calcium lactate; (4) a solvent; and (5) a bone graft material.

[0011] In yet another aspect, provided herein is a kit for preparing a bone graft, comprising: (1) the formulation of any one of claims 1-34; (2) a bone graft material.

[0012] In yet another aspect, provided herein is a kit for preparing a bone graft, comprising: (1) an organic phosphate; (2) L-ascorbic acid, or a derivative thereof; (3) an organic calcium salt; (4) a solvent; and (5) a bone graft material.

[0013] Other features and advantages of the present disclosure will become apparent from the following detailed description. The detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only. These detailed descriptions and specific examples are provided for the purposes of explanation, and not limitation, of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will beobtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0015] FIG. 1 depicts an example of a sterile package including a bone allograft, a partially prepared buffer solution, and ascorbic acid, according to some embodiments.

[0016] FIG.2 depicts an example of a sterile package including a bone allograft, a partially prepared buffer solution, and ascorbic acid, according to some embodiments.

[0017] FIG.3 depicts an example of a sterile package including a bone allograft and a buffer solution, according to some embodiments.

[0018] FIG.4 depicts an example of a sterile package including a bone allograft and a buffer solution, according to some embodiments.

[0019] FIG.5 depicts an example of a sterile package including a bone allograft and a buffer solution, according to some embodiments.

[0020] FIG.6 depicts an example of a sterile package including a bone allograft and a buffer solution, according to some embodiments.

[0021] FIG.7 depicts a flowchart of an exemplary method for preparing the bone allograft illustrated in FIGS. 1 and 2 for implantation, according to some embodiments.

[0022] FIG.8 depicts a flowchart of an exemplary method for preparing the bone allograft illustrated in FIGS. 3-6 for implantation, according to some embodiments.

[0023] FIG.9 is a graph showing increase in OPN expression in bone grafts rehydrated with AlloWide Buffer according to some embodiments.

[0024] FIG. 10 is a graph showing increase in OCN expression in bone grafts rehydrated with AlloWide Buffer according to some embodiments.

[0025] FIG. 11 is a graph showing increase in ALP activity in bone grafts rehydrated with AlloWide Buffer according to some embodiments.

[0026] FIG. 12 is a graph showing increase in calcium deposition in bone grafts rehydrated with AlloWide Buffer according to some embodiments.

[0027] FIG. 13 is a graph showing increase in cell seeding and viability in bone grafts rehydrated with AlloWide Buffer according to some embodiments.DETAILED DESCRIPTION

[0028] In the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.I. Formulation

[0029] In one aspect, provided herein is a formulation. The formulation can be a formulation for rehydrating a graft material. The graft material can be a bone graft material. The formulation may include P-glycerophosphate. The P-glycerophosphate may have a concentration of l-25mM. The P-glycerophosphate may have a concentration of about 5 mM. The formulation may include L-ascorbic acid. The formulation may include L-ascorbic acid 2-phosphate. The formulation may include L-ascorbic acid and L-ascorbic acid 2-phosphate. The L-ascorbic acid and / or the L-ascorbic acid 2-phosphate may have a concentration of 1-100 pg / mL. The L-ascorbic acid and / or the L-ascorbic acid 2-phosphate may have a concentration of about 70 pg / mL. The formulation may include calcium lactate. The calcium lactate may have a concentration of l-20mM. The calcium lactate may have a concentration of about 2.5 mM. The formulation may be a buffered aqueous solution.

[0030] In one aspect, provided herein is a formulation for rehydrating a bone graft material, comprising: about 5 mM of P-glycerophosphate; about 70 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and about 2.5 mM calcium lactate, in a buffered aqueous solution.

[0031] In one aspect, provided herein is a formulation for rehydrating a bone graft material, comprising: 1-25 mM of P-glycerophosphate; 1-100 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and 1-20 mM calcium lactate, in a buffered aqueous solution.

[0032] In another aspect, provided herein is a formulation. The formulation can be a formulation for rehydrating a graft material. The graft material can be a bone graft material. The formulation may include an organic phosphate. The formulation may include L-ascorbic acid. The formulation may include a derivative of L-ascorbic acid. The formulation may include an organic calcium salt.

[0033] In another aspect, provided herein is a formulation for rehydrating a bone graft material, comprising: an organic phosphate, L-ascorbic acid or a derivative thereof, and an organic calcium salt.

[0034] As used herein, the term “rehydrating”, “rehydrate”, or the like, in the context of a bone graft material, refers to treating the bone graft material in an aqueous solution, for example, to prepare the bone graft for implantation. Rehydrating the bone graft material may or may notresult in an increase in water content of the bone graft material. The bone graft material may be in a dehydrated state, a partially dehydrated state, or hydrated state.

[0035] As used herein, the term “bone graft” refers to a bone tissue suitable for implantation. A bone graft may be prepared from a bone graft material. Any suitable bone graft material can be used. The bone graft material may be derived from a donor, which may be a human donor or non-human donor. The bone graft material may be derived from the same individual who will receive the bone graft. The bone graft material may be derived from a living organism or a deceased organism. The bone graft material may be partially or fully synthetic.

[0036] Organic phosphate

[0037] The formulation of the present disclosure can include an organic phosphate.

[0038] The organic phosphate of the formulation may act as an organic phosphate donor in mineralization processes. When hydrolyzed by alkaline phosphatase (an enzyme highly expressed by osteoblasts and mesenchymal stem cells (MSCs), the organic phosphate can release inorganic phosphate (Pi), which can combine with calcium ions to form hydroxyapatite crystals, the primary mineral component of bone. Beyond a role in biomineralization, the organic phosphate can actively drive osteogenic differentiation of mesenchymal stem cells (BM-MSCs) by, for example, upregulating key osteogenic markers such as Runx2, osteocalcin, and bone sialoprotein.

[0039] The organic phosphate can be P-glycerophosphate (C3H7Na2O6P). Other suitable organic phosphates may also be used, such as sugar phosphates, nucleotide phosphates, phosphorylated vitamins and cofactors, and phospholipid components.

[0040] In some embodiments, the organic phosphate comprises a substrate of alkaline phosphatase. In some embodiments, the organic phosphate comprises a phosphate donor. In some embodiments, the organic phosphate comprises a phosphate donor that releases inorganic phosphate upon hydrolysis by alkaline phosphatase. In some embodiments, the organic phosphate is capable of driving osteogenic differentiation of mesenchymal stem cells (BM-MSCs). In some embodiments, the organic phosphate is capable of upregulating osteogenic markers such as Runx2, osteocalcin, and bone sialoprotein. In some embodiments, the organic phosphate comprises P-glycerophosphate.

[0041] In some embodiments, the organic phosphate has a concentration of between about 1 mM and about 50 mM, between about 1 mM and about 40 mM, between about 1 mM and about 30 mM, between about 1 mM and about 25 mM, between about 1 mM and about 20 mM, between about 1 mM and about 15 mM, between about 1 mM and about 10 mM, between about 1 mMand about 9 mM, between about 1 mM and about 8 mM, between about 1 mM and about 7 mM, between about 1 mM and about 6 mM, between about 1 mM and about 5 mM, between about 1 mM and about 4 mM, between about 1 mM and about 3 mM, between about 1 mM and about 2 mM, between about 2 mM and about 50 mM, between about 2 mM and about 40 mM, between about 2 mM and about 30 mM, between about 2 mM and about 25 mM, between about 2 mM and about 20 mM, between about 2 mM and about 15 mM, between about 2 mM and about 10 mM, between about 2 mM and about 9 mM, between about 2 mM and about 8 mM, between about 2 mM and about 7 mM, between about 2 mM and about 6 mM, between about 2 mM and about 5 mM, between about 2 mM and about 4 mM, between about 2 mM and about 3 mM, between about 3 mM and about 50 mM, between about 3 mM and about 40 mM, between about 3 mM and about 30 mM, between about 3 mM and about 25 mM, between about 3 mM and about 20 mM, between about 3 mM and about 15 mM, between about 3 mM and about 10 mM, between about 3 mM and about 9 mM, between about 3 mM and about 8 mM, between about 3 mM and about 7 mM, between about 3 mM and about 6 mM, between about 3 mM and about 5 mM, between about 3 mM and about 4 mM, between about 4 mM and about 50 mM, between about 4 mM and about 40 mM, between about 4 mM and about 30 mM, between about 4 mM and about 25 mM, between about 4 mM and about 20 mM, between about 4 mM and about 15 mM, between about 4 mM and about 10 mM, between about 4 mM and about 9 mM, between about 4 mM and about 8 mM, between about 4 mM and about 7 mM, between about 4 mM and about 6 mM, between about 4 mM and about 5 mM, between about 5 mM and about 50 mM, between about 5 mM and about 40 mM, between about 5 mM and about 30 mM, between about 5 mM and about 25 mM, between about 5 mM and about 20 mM, between about 5 mM and about 15 mM, between about 5 mM and about 10 mM, between about 5 mM and about 9 mM, between about 5 mM and about 8 mM, between about 5 mM and about 7 mM, between about 5 mM and about 6 mM, between about 6 mM and about 50 mM, between about 6 mM and about 40 mM, between about 6 mM and about 30 mM, between about 6 mM and about 25 mM, between about 6 mM and about 20 mM, between about 6 mM and about 15 mM, between about 6 mM and about 14 mM, between about 6 mM and about 13 mM, between about 6 mM and about 12 mM, between about 6 mM and about 11 mM, between about 6 mM and about 10 mM, between about 6 mM and about 9 mM, between about 6 mM and about 8 mM, between about 6 mM and about 7 mM, between about 7 mM and about 50 mM, between about 7 mM and about 40 mM, between about 7 mM and about 30 mM, between about 7 mM and about 25 mM, between about 7 mM and about 20 mM, between about 7 mM and about 15 mM, between about7 mM and about 14 mM, between about 7 mM and about 13 mM, between about 7 mM and about 12 mM, between about 7 mM and about 11 mM, between about 7 mM and about 10 mM, between about 7 mM and about 9 mM, between about 7 mM and about 8 mM, between about 8 mM and about 50 mM, between about 8 mM and about 40 mM, between about 8 mM and about 30 mM, between about 8 mM and about 25 mM, between about 8 mM and about 20 mM, between about 8 mM and about 15 mM, between about 8 mM and about 14 mM, between about 8 mM and about 13 mM, between about 8 mM and about 12 mM, between about 8 mM and about 11 mM, between about 8 mM and about 10 mM, between about 8 mM and about 9 mM, between about 9 mM and about 50 mM, between about 9 mM and about 40 mM, between about 9 mM and about 30 mM, between about 9 mM and about 25 mM, between about 9 mM and about 20 mM, between about 9 mM and about 15 mM, between about 9 mM and about 14 mM, between about 9 mM and about 13 mM, between about 9 mM and about 12 mM, between about 9 mM and about 11 mM, between about 9 mM and about 10 mM, between about 10 mM and about 50 mM, between about 10 mM and about 40 mM, between about 10 mM and about 35 mM, between about 10 mM and about 30 mM, between about 10 mM and about 25 mM, between about 10 mM and about 20 mM, between about 10 mM and about 15 mM, between about 15 mM and about 50 mM, between about 15 mM and about 40 mM, between about 15 mM and about 35 mM, between about 15 mM and about 30 mM, between about 15 mM and about 25 mM, between about 15 mM and about 20 mM, between about 20 mM and about 50 mM, between about 20 mM and about 45 mM, between about 20 mM and about 40 mM, between about 20 mM and about 35 mM, between about 20 mM and about 30 mM, between about 20 mM and about 25 mM, between about 25 mM and about 50 mM, between about 25 mM and about 45 mM, between about 25 mM and about 40 mM, between about 25 mM and about 35 mM, between about 25 mM and about 30 mM, between about 30 mM and about 50 mM, between about 30 mM and about 45 mM, between about 30 mM and about 40 mM, between about 30 mM and about 35 mM, between about 35 mM and about 50 mM, between about 35 mM and about 45 mM, between about 35 mM and about 40 mM, between about 40 mM and about 50 mM, between about 40 mM and about 45 mM, or between about 45 mM and about 50 mM.

[0042] In some embodiments, the organic phosphate comprises P-glycerophosphate.

[0043] In some embodiments, the organic phosphate has a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, or more than 50 mM.

[0044] In some embodiments, the organic phosphate comprises P-glycerophosphate.

[0045] L-ascorbic acid or a derivative thereof

[0046] The formulation of the present disclosure can include an ascorbic acid, such as L-ascorbic acid, or a derivative thereof.

[0047] Ascorbic acid (CeHsOe) can play a multifaceted and essential role in connective tissue biology and stem cell fate determination. It can act as a cofactor for prolyl and lysyl hydroxylases, enzymes required for the post-translational modification and stabilization of collagen triple-helix formation — a foundational step in extracellular matrix (ECM) assembly. Without adequate ascorbic acid, collagen may remain structurally weak, impairing tissue integrity and delaying wound healing or graft incorporation. Moreover, ascorbic acid can function as a potent epigenetic modulator and stem cell differentiator, promoting the commitment of MSCs toward osteogenic and chondrogenic lineages while suppressing adipogenesis. Its antioxidant properties may also protect cells from oxidative stress during the critical early phases of graft revascularization and host integration.

[0048] In some embodiments, the ascorbic acid comprises L-ascorbic acid. In some embodiments, the ascorbic acid comprises L-ascorbic acid 2-phosphate.

[0049] In some embodiments, the L-ascorbic acid or derivative thereof has a concentration of between about 1 pg / mL and 200 pg / mL, between about 1 pg / mL and 190 pg / mL, between about 1 pg / mL and 180 pg / mL, between about 1 pg / mL and 170 pg / mL, between about 1 pg / mL and 160 pg / mL, between about 1 pg / mL and 150 pg / mL, between about 1 pg / mL and 140 pg / mL, between about 1 pg / mL and 130 pg / mL, between about 1 pg / mL and 120 pg / mL, between about 1 pg / mL and 110 pg / mL, between about 1 pg / mL and 100 pg / mL, between about 1 pg / mL and 90 pg / mL, between about 1 pg / mL and 80 pg / mL, between about 1 pg / mL and 70 pg / mL, between about 1 pg / mL and 60 pg / mL, between about 1 pg / mL and 50 pg / mL, between about 1 pg / mL and 40 pg / mL, between about 1 pg / mL and 30 pg / mL, between about 1 pg / mL and 20 pg / mL, between about 1 pg / mL and 10 pg / mL, between about 5 pg / mL and 200 pg / mL, between about 5 pg / mL and 190 pg / mL, between about 5 pg / mL and 180 pg / mL, between about 5 pg / mL and 170 pg / mL, between about 5 pg / mL and 160 pg / mL, between about 5 pg / mL and 150gg / mL, between about 5 gg / mL and 140 gg / mL, between about 5 gg / mL and 130 gg / mL, between about 5 gg / mL and 120 gg / mL, between about 5 gg / mL and 110 gg / mL, between about 5 gg / mL and 100 gg / mL, between about 5 gg / mL and 90 gg / mL, between about 5 gg / mL and 80 gg / mL, between about 5 gg / mL and 70 gg / mL, between about 5 gg / mL and 60 gg / mL, between about 5 gg / mL and 50 gg / mL, between about 5 gg / mL and 40 gg / mL, between about 5 gg / mL and 30 gg / mL, between about 5 gg / mL and 20 gg / mL, between about 5 gg / mL and 10 gg / mL, between about 10 gg / mL and 200 gg / mL, between about 10 gg / mL and 190 gg / mL, between about 10 gg / mL and 180 gg / mL, between about 10 gg / mL and 170 gg / mL, between about 10 gg / mL and 160 gg / mL, between about 10 gg / mL and 150 gg / mL, between about 10 gg / mL and 140 gg / mL, between about 10 gg / mL and 130 gg / mL, between about 10 gg / mL and 120 gg / mL, between about 10 gg / mL and 110 gg / mL, between about 10 gg / mL and 100 gg / mL, between about 10 gg / mL and 90 gg / mL, between about 10 gg / mL and 80 gg / mL, between about 10 gg / mL and 70 gg / mL, between about 10 gg / mL and 60 gg / mL, between about 10 gg / mL and 50 gg / mL, between about 10 gg / mL and 40 gg / mL, between about 10 gg / mL and 30 gg / mL, between about 10 gg / mL and 20 gg / mL, between about 15 gg / mL and 200 gg / mL, between about 15 gg / mL and 190 gg / mL, between about 15 gg / mL and 180 gg / mL, between about 15 gg / mL and 170 gg / mL, between about 15 gg / mL and 160 gg / mL, between about 15 gg / mL and 150 gg / mL, between about 15 gg / mL and 140 gg / mL, between about 15 gg / mL and 130 gg / mL, between about 15 gg / mL and 120 gg / mL, between about 15 gg / mL and 110 gg / mL, between about 15 gg / mL and 100 gg / mL, between about 15 gg / mL and 90 gg / mL, between about 15 gg / mL and 80 gg / mL, between about 15 gg / mL and 70 gg / mL, between about 15 gg / mL and 60 gg / mL, between about 15 gg / mL and 50 gg / mL, between about 15 gg / mL and 40 gg / mL, between about 15 gg / mL and 30 gg / mL, between about 15 gg / mL and 20 gg / mL, between about 20 gg / mL and 200 gg / mL, between about 20 gg / mL and 190 gg / mL, between about 20 gg / mL and 180 gg / mL, between about 20 gg / mL and 170 gg / mL, between about 20 gg / mL and 160 gg / mL, between about 20 gg / mL and 150 gg / mL, between about 20 gg / mL and 140 gg / mL, between about 20 gg / mL and 130 gg / mL, between about 20 gg / mL and 120 gg / mL, between about 20 gg / mL and 110 gg / mL, between about 20 gg / mL and 100 gg / mL, between about 20 gg / mL and 90 gg / mL, between about 20 gg / mL and 80 gg / mL, between about 20 gg / mL and 70 gg / mL, between about 20 gg / mL and 60 gg / mL, between about 20 gg / mL and 50 gg / mL, between about 20 gg / mL and 40 gg / mL, between about 20 gg / mL and 30 gg / mL, between about 25 gg / mL and 200 gg / mL, between about 25 gg / mL and 190 gg / mL, between about 25 gg / mL and 180 gg / mL, between about 25 gg / mL and 170 gg / mL, between about 25 gg / mL and 160gg / mL, between about 25 gg / mL and 150 gg / mL, between about 25 gg / mL and 140 gg / mL, between about 25 gg / mL and 130 gg / mL, between about 25 gg / mL and 120 gg / mL, between about 25 gg / mL and 110 gg / mL, between about 25 gg / mL and 100 gg / mL, between about 25 gg / mL and 90 gg / mL, between about 25 gg / mL and 80 gg / mL, between about 25 gg / mL and 70 gg / mL, between about 25 gg / mL and 60 gg / mL, between about 25 gg / mL and 50 gg / mL, between about 25 gg / mL and 40 gg / mL, between about 25 gg / mL and 30 gg / mL, between about 30 gg / mL and 200 gg / mL, between about 30 gg / mL and 190 gg / mL, between about 30 gg / mL and 180 gg / mL, between about 30 gg / mL and 170 gg / mL, between about 30 gg / mL and 160 gg / mL, between about 30 gg / mL and 150 gg / mL, between about 30 gg / mL and 140 gg / mL, between about 30 gg / mL and 130 gg / mL, between about 30 gg / mL and 120 gg / mL, between about 30 gg / mL and 110 gg / mL, between about 30 gg / mL and 100 gg / mL, between about 30 gg / mL and 90 gg / mL, between about 30 gg / mL and 80 gg / mL, between about 30 gg / mL and 70 gg / mL, between about 30 gg / mL and 60 gg / mL, between about 30 gg / mL and 50 gg / mL, between about 30 gg / mL and 40 gg / mL, between about 35 gg / mL and 200 gg / mL, between about 35 gg / mL and 190 gg / mL, between about 35 gg / mL and 180 gg / mL, between about 35 gg / mL and 170 gg / mL, between about 35 gg / mL and 160 gg / mL, between about 35 gg / mL and 150 gg / mL, between about 35 gg / mL and 140 gg / mL, between about 35 gg / mL and 130 gg / mL, between about 35 gg / mL and 120 gg / mL, between about 35 gg / mL and 110 gg / mL, between about 35 gg / mL and 100 gg / mL, between about 35 gg / mL and 90 gg / mL, between about 35 gg / mL and 80 gg / mL, between about 35 gg / mL and 70 gg / mL, between about 35 gg / mL and 60 gg / mL, between about 35 gg / mL and 50 gg / mL, between about 35 gg / mL and 45 gg / mL, between about 35 gg / mL and 40 gg / mL, between about 40 gg / mL and 200 gg / mL, between about 40 gg / mL and 190 gg / mL, between about 40 gg / mL and 180 gg / mL, between about 40 gg / mL and 170 gg / mL, between about 40 gg / mL and 160 gg / mL, between about 40 gg / mL and 150 gg / mL, between about 40 gg / mL and 140 gg / mL, between about 40 gg / mL and 130 gg / mL, between about 40 gg / mL and 120 gg / mL, between about 40 gg / mL and 110 gg / mL, between about 40 gg / mL and 100 gg / mL, between about 40 gg / mL and 90 gg / mL, between about 40 gg / mL and 80 gg / mL, between about 40 gg / mL and 70 gg / mL, between about 40 gg / mL and 60 gg / mL, between about 40 gg / mL and 50 gg / mL, between about 50 gg / mL and 200 gg / mL, between about 50 gg / mL and 190 gg / mL, between about 50 gg / mL and 180 gg / mL, between about 50 gg / mL and 170 gg / mL, between about 50 gg / mL and 160 gg / mL, between about 50 gg / mL and 150 gg / mL, between about 50 gg / mL and 140 gg / mL, between about 50 gg / mL and 130 gg / mL, between about 50 gg / mL and 120 gg / mL, between about 50 gg / mL and 110 gg / mL,between about 50 |ag / mL and 100 jag / mL, between about 50 gg / mL and 90 gg / mL, between about 50 |ag / mL and 80 gg / mL, between about 50 gg / mL and 70 gg / mL, between about 50 gg / mL and 60 gg / mL, between about 60 gg / mL and 200 gg / mL, between about 60 gg / mL and 190 gg / mL, between about 60 gg / mL and 180 gg / mL, between about 60 gg / mL and 170 gg / mL, between about 60 |ag / mL and 160 gg / mL, between about 60 gg / mL and 150 gg / mL, between about 60 gg / mL and 140 gg / mL, between about 60 gg / mL and 130 gg / mL, between about 60 gg / mL and 120 gg / mL, between about 60 gg / mL and 110 gg / mL, between about 60 gg / mL and 100 gg / mL, between about 60 gg / mL and 90 gg / mL, between about 60 gg / mL and 80 gg / mL, between about 60 |ag / mL and 70 gg / mL, between about 70 gg / mL and 200 gg / mL, between about 70 gg / mL and 190 gg / mL, between about 70 gg / mL and 180 gg / mL, between about 70 gg / mL and 170 gg / mL, between about 70 jag / mL and 160 gg / mL, between about 70 gg / mL and 150 gg / mL, between about 70 gg / mL and 140 gg / mL, between about 70 gg / mL and 130 gg / mL, between about 70 gg / mL and 120 gg / mL, between about 70 gg / mL and 110 gg / mL, between about 70 gg / mL and 100 gg / mL, between about 70 gg / mL and 90 gg / mL, between about 70 gg / mL and 80 gg / mL, between about 80 gg / mL and 200 gg / mL, between about 80 gg / mL and 180 gg / mL, between about 80 |ag / mL and 160 gg / mL, between about 80 gg / mL and 140 gg / mL, between about 80 |ag / mL and 120 gg / mL, between about 80 gg / mL and 100 gg / mL, between about 100 gg / mL and 200 |ag / mL, between about 100 gg / mL and 180 gg / mL, between about 100 gg / mL and 160 |ag / mL, between about 100 gg / mL and 140 gg / mL, between about 100 gg / mL and 120 gg / mL, between about 120 gg / mL and 200 gg / mL, between about 120 gg / mL and 180 gg / mL, between about 120 |ag / mL and 160 gg / mL, between about 120 gg / mL and 140 gg / mL, between about 140 |ag / mL and 200 gg / mL, between about 140 gg / mL and 180 gg / mL, between about 140 gg / mL and 160 gg / mL, between about 160 gg / mL and 200 gg / mL, between about 160 gg / mL and 180 |ag / mL, or between about 180 gg / mL and 200 gg / mL.

[0050] In some embodiments, the L-ascorbic acid or derivative thereof has a concentration of about 1 |ag / mL, about 2 gg / mL, about 3 gg / mL, about 4 gg / mL, about 5 gg / mL, about 6 gg / mL, about 7 |ag / mL, about 8 gg / mL, about 9 gg / mL, about 10 gg / mL, about 11 gg / mL, about 12 jag / mL, about 13 gg / mL, about 14 gg / mL, about 15 gg / mL, about 16 gg / mL, about 17 gg / mL, about 18 |ag / mL, about 19 gg / mL, about 20 gg / mL, about 25 gg / mL, about 30 gg / mL, about 35 jag / mL, about 40 gg / mL, about 45 gg / mL, about 50 gg / mL, about 55 gg / mL, about 60 gg / mL, about 65 |ag / mL, about 70 gg / mL, about 75 gg / mL, about 80 gg / mL, about 85 gg / mL, about 90 jag / mL, about 95 gg / mL, about 100 gg / mL, about 110 gg / mL, about 120 gg / mL, about 130gg / mL, about 140 gg / mL, about 150 gg / mL, about 160 gg / mL, about 170 gg / mL, about 190 gg / mL, about 200 gg / mL, or more than about 200 gg / mL.

[0051] In some embodiments, the L-ascorbic acid or derivative thereof is L-ascorbic acid. In some embodiments, the L-ascorbic acid or derivative thereof is L-ascorbic acid 2-phosphate. In some embodiments, the L-ascorbic acid or derivative thereof is L-ascorbic acid and L-ascorbic acid 2-phosphate.

[0052] Organic calcium salt

[0053] The formulation of the present disclosure can include an organic calcium salt.

[0054] The organic calcium salt of the formulation may contribute not only as a bioavailable source of calcium ions — an essential element for hydroxyapatite deposition and bone mineralization — but may also act as a signaling molecule that influences stem cell behavior. Extracellular calcium can activate the calcium-sensing receptor on MSCs, triggering intracellular pathways (e.g., MAPK / ERK, Wnt / p-catenin) that promote osteoblast proliferation, differentiation, and survival. In this context, the organic calcium salt can act as a stem cell differentiator, and may synergize with the organic phosphate and ascorbic acid to create a pro-osteogenic microenvironment. Additionally, the 1 organic calcium salt may support cellular metabolism under hypoxic conditions often encountered in the early post-implantation niche, further enhancing graft viability.

[0055] In some embodiments, the organic calcium salt is capable of promoting osteoblast proliferation, differentiation, and / or survival. In some embodiments, the organic calcium salt is capable of supporting cellular metabolism under hypoxic conditions.

[0056] In some embodiments, the organic calcium salt has a concentration of between about 1 mM and about 50 mM, between about 1 mM and about 40 mM, between about 1 mM and about 30 mM, between about 1 mM and about 25 mM, between about 1 mM and about 20 mM, between about 1 mM and about 15 mM, between about 1 mM and about 10 mM, between about 1 mM and about 9 mM, between about 1 mM and about 8 mM, between about 1 mM and about 7 mM, between about 1 mM and about 6 mM, between about 1 mM and about 5 mM, between about 1 mM and about 4 mM, between about 1 mM and about 3 mM, between about 1 mM and about 2 mM, between about 2 mM and about 50 mM, between about 2 mM and about 40 mM, between about 2 mM and about 30 mM, between about 2 mM and about 25 mM, between about 2 mM and about 20 mM, between about 2 mM and about 15 mM, between about 2 mM and about 10 mM, between about 2 mM and about 9 mM, between about 2 mM and about 8 mM, between about 2 mM and about 7 mM, between about 2 mM and about 6 mM, between about 2mM and about 5 mM, between about 2 mM and about 4 mM, between about 2 mM and about 3 mM, between about 3 mM and about 50 mM, between about 3 mM and about 40 mM, between about 3 mM and about 30 mM, between about 3 mM and about 25 mM, between about 3 mM and about 20 mM, between about 3 mM and about 15 mM, between about 3 mM and about 10 mM, between about 3 mM and about 9 mM, between about 3 mM and about 8 mM, between about 3 mM and about 7 mM, between about 3 mM and about 6 mM, between about 3 mM and about 5 mM, between about 3 mM and about 4 mM, between about 4 mM and about 50 mM, between about 4 mM and about 40 mM, between about 4 mM and about 30 mM, between about 4 mM and about 25 mM, between about 4 mM and about 20 mM, between about 4 mM and about 15 mM, between about 4 mM and about 10 mM, between about 4 mM and about 9 mM, between about 4 mM and about 8 mM, between about 4 mM and about 7 mM, between about 4 mM and about 6 mM, between about 4 mM and about 5 mM, between about 5 mM and about 50 mM, between about 5 mM and about 40 mM, between about 5 mM and about 30 mM, between about 5 mM and about 25 mM, between about 5 mM and about 20 mM, between about 5 mM and about 15 mM, between about 5 mM and about 10 mM, between about 5 mM and about 9 mM, between about 5 mM and about 8 mM, between about 5 mM and about 7 mM, between about 5 mM and about 6 mM, between about 6 mM and about 50 mM, between about 6 mM and about 40 mM, between about 6 mM and about 30 mM, between about 6 mM and about 25 mM, between about 6 mM and about 20 mM, between about 6 mM and about 15 mM, between about 6 mM and about 14 mM, between about 6 mM and about 13 mM, between about 6 mM and about 12 mM, between about 6 mM and about 11 mM, between about 6 mM and about 10 mM, between about 6 mM and about 9 mM, between about 6 mM and about 8 mM, between about 6 mM and about 7 mM, between about 7 mM and about 50 mM, between about 7 mM and about 40 mM, between about 7 mM and about 30 mM, between about 7 mM and about 25 mM, between about 7 mM and about 20 mM, between about 7 mM and about 15 mM, between about 7 mM and about 14 mM, between about 7 mM and about 13 mM, between about 7 mM and about 12 mM, between about 7 mM and about 11 mM, between about 7 mM and about 10 mM, between about 7 mM and about 9 mM, between about 7 mM and about 8 mM, between about 8 mM and about 50 mM, between about 8 mM and about 40 mM, between about 8 mM and about 30 mM, between about 8 mM and about 25 mM, between about 8 mM and about 20 mM, between about 8 mM and about 15 mM, between about 8 mM and about 14 mM, between about 8 mM and about 13 mM, between about 8 mM and about 12 mM, between about 8 mM and about 11 mM, between about 8 mM and about 10 mM, between about 8 mM and about 9 mM,between about 9 mM and about 50 mM, between about 9 mM and about 40 mM, between about 9 mM and about 30 mM, between about 9 mM and about 25 mM, between about 9 mM and about 20 mM, between about 9 mM and about 15 mM, between about 9 mM and about 14 mM, between about 9 mM and about 13 mM, between about 9 mM and about 12 mM, between about 9 mM and about 11 mM, between about 9 mM and about 10 mM, between about 10 mM and about 50 mM, between about 10 mM and about 40 mM, between about 10 mM and about 35 mM, between about 10 mM and about 30 mM, between about 10 mM and about 25 mM, between about 10 mM and about 20 mM, between about 10 mM and about 15 mM, between about 15 mM and about 50 mM, between about 15 mM and about 40 mM, between about 15 mM and about 35 mM, between about 15 mM and about 30 mM, between about 15 mM and about 25 mM, between about 15 mM and about 20 mM, between about 20 mM and about 50 mM, between about 20 mM and about 45 mM, between about 20 mM and about 40 mM, between about 20 mM and about 35 mM, between about 20 mM and about 30 mM, between about 20 mM and about 25 mM, between about 25 mM and about 50 mM, between about 25 mM and about 45 mM, between about 25 mM and about 40 mM, between about 25 mM and about 35 mM, between about 25 mM and about 30 mM, between about 30 mM and about 50 mM, between about 30 mM and about 45 mM, between about 30 mM and about 40 mM, between about 30 mM and about 35 mM, between about 35 mM and about 50 mM, between about 35 mM and about 45 mM, between about 35 mM and about 40 mM, between about 40 mM and about 50 mM, between about 40 mM and about 45 mM, or between about 45 mM and about 50 mM.

[0057] In some embodiments, the organic calcium salt comprises calcium lactate. In some embodiments, the calcium salt comprises calcium ascorbate.

[0058] In some embodiments, the organic calcium salt has a concentration of about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, or more than 50 mM.

[0059] Any suitable organic calcium salt can be used, such as calcium lactate, calcium ascorbate, calcium citrate, calcium gluconate, calcium carbonate, calcium glycerophosphate, calcium phosphate (tribasic), calcium stearate, calcium citrate malate, calcium acetate, calcium propionate, calcium sorbate, calcium glycinate.

[0060] In some embodiments, the organic calcium salt comprises calcium lactate. In some embodiments, the calcium salt comprises calcium ascorbate.

[0061] Buffered solution

[0062] The formulation can be an aqueous solution, optionally a buffered aqueous solution.

[0063] In some embodiments, the aqueous solution comprises saline. In some embodiments, the aqueous solution comprises water for injection. In some embodiments, the aqueous solution comprises a buffered isotonic solution. In some embodiments, the aqueous solution comprises HEPES.

[0064] In some embodiments, the aqueous solution comprises Normosol. In some embodiments, the aqueous solution comprises Plasma-Lyte.

[0065] In some embodiments, the formulation has a pH of between about 7.2 and 7.4.

[0066] Bone graft material

[0067] The formulation disclosed herein comprises physiological solutions intended for the rehydration and preparation of bone grafts, such as cancellous and cortical bone in various shapes such as shafts, chips, powder, and geometrical shapes, in mineral or demineralized bone matrix. Bone graft materials can be freeze-dried, frozen, or in fresh format.

[0068] In some embodiments, the bone graft material comprises one or more of: bone powder; crushed bone; bone chips; cortical bone; cancellous bone; bone shaft; and bone in the form of a plurality of cubes, matchsticks, wedges, or other geometric shape.

[0069] In some embodiments, the bone graft material comprises a bone material in a dry state. In some embodiments, the bone graft material comprises a bone material that has been freeze-dried. In some embodiments, the bone graft material comprises a bone material in a wet state. In some embodiments, the bone graft material comprises a bone material in a frozen state.

[0070] It is demonstrated herein that when a bone graft material is rehydrated in the formulation of the present disclosure, the resulting bone graft has an increased expression of osteopontin (OPN), osteocalcin (OCN), increased alkaline phosphatase (ALP) activity, increased calcium deposition, and provides increased cell seeding and cell viability, when compared to a bone graft rehydrated in saline.

[0071] In some embodiments, rehydrating the bone graft material with the formulation disclosed herein results in a bone graft that has an increased expression in osteopontin (OPN) compared to a control bone graft.

[0072] In some embodiments, the increase in OPN expression when compared to a control bone graft is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold, at about 21 days after being rehydrated.

[0073] In some embodiments, the control is a bone graft rehydrated with normal saline.

[0074] In some embodiments, rehydrating the bone graft material with the formulation results in a bone graft that has an increased expression in osteocalcin (OCN) compared to a control bone graft.

[0075] In some embodiments, the increase in OCN expression when compared to a control bone graft is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold, at about 21 days after being rehydrated.

[0076] In some embodiments, the control is a bone graft rehydrated with normal saline

[0077] In some embodiments, rehydrating the bone graft material with the formulation results in a bone graft that has an increased alkaline phosphatase (ALP) activity compared to a control bone graft.

[0078] In some embodiments, the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, at about 7 days after being rehydrated with the solution.

[0079] In some embodiments, the control is a bone graft rehydrated with normal saline.

[0080] In some embodiments, the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, at about 14 days after being rehydrated.

[0081] In some embodiments, the control is a bone graft rehydrated with normal saline.

[0082] In some embodiments, the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, at about 21 days after being rehydrated.

[0083] In some embodiments, the control is a bone graft rehydrated with normal saline.

[0084] In some embodiments, rehydrating the bone graft material with the formulation results in a bone graft that has an increased level of calcium deposition compared to a control bone graft.

[0085] In some embodiments, the increase in calcium deposition level when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at about 14 days after being rehydrated.

[0086] In some embodiments, the control is a bone graft rehydrated with normal saline.

[0087] In some embodiments, rehydrating the bone graft material with the formulation results in a bone graft that allows an increased level of seeding of a primary cell compared to a control bone graft.

[0088] In some embodiments, the increase in the level of seeding of the primary cell when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 140%, at least about 160%, at least about 180%, or at least about 200%.

[0089] In some embodiments, the control is a bone graft rehydrated with normal saline.

[0090] In some embodiments, rehydrating the bone graft material with the formulation results in a bone graft that allows an increased viability of a primary cell compared to a control bone graft.

[0091] In some embodiments, the increase in viability of the primary cell when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 140%, at least about 160%, at least about 180%, or at least about 200%.

[0092] The primary cell may be any type of cells normally associated with bone, such as mesenchymal stem cells, osteoprogenitor cells, osteoclast precursors, and endothelial cells.

[0093] In some embodiments, the primary cell comprises a mesenchymal stem cell (MSC).

[0094] In some embodiments, the control is a bone graft rehydrated with normal saline.II. Methods

[0095] In another aspect, provided herein is a method of preparing a formulation for hydrating a bone graft material. The method can comprise dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution. The method can comprise dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution. The method can comprise dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate an ascorbic acid-organic phosphate-organic calcium salt solution, thereby generating the formulation.

[0096] In another aspect, provided herein is a method of preparing a formulation for hydrating a bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate an ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation.

[0097] One or more embodiments provide a method for preparing the buffer solution, including dissolving a suitable concentration of P-glycerophosphate (C3H7Na2O6P), ascorbic acid (C6H8O6), and calcium lactate (C6H10CaO6) in sterile normal saline, sterile distilled water or water suitable for injection(WFI) or HEPES-buffered sterile saline (10-25 mM HEPES in isotonic saline) at 37°C. For example, use either water, normal saline, or HEPES 37°C at from the first step, or the solution can be warmed to 37°C once the salts are added to form the final buffer solution.

[0098] For example, 1-25 mM of the P-glycerophosphate (C3H7Na2O6P), 1-100 pg / mL of the ascorbic acid (C6H8O6), and 1-20 mM of the calcium lactate (C6H10CaO6) may be dissolved within 100 mL of normal saline, distilled sterile water or HEPES. The amount of the buffer solution depending on form, a shape, and a size of the bone allograft, as the bone allograft is to be fully immersed in the buffer solution prior to implantation.

[0099] In some embodiments, the formulation for rehydrating a bone graft material is prepared by sequentially dissolving specific bioactive components in sterile Water for Injection (WFI) under controlled conditions to ensure stability, solubility, and biological activity. For example, first, ascorbic acid (CeHsOe) is dissolved in a portion of the WFI at 37°C temperature with gentle stirring; this initial step can minimize oxidative degradation by avoiding exposure to high ionic strength or alkaline conditions early in the process. Next, P-glycerophosphate (C3H?Na2O6P) is slowly added to the ascorbic acid solution under continuous mixing; this order can prevent premature precipitation and allows gradual pH adjustment, as P-glycerophosphate can elevate thesolution pH, which, if introduced too early, could accelerate ascorbic acid degradation. Finally, calcium lactate (C<,H loCaCX.) is carefully dissolved into the mixture. The use of calcium lactate can provide bioavailable calcium without forming insoluble calcium-phosphate complexes during preparation. The final solution can be adjusted to pH 7.2-7.4 if necessary, filtered through a 0.22 pm sterilizing-grade membrane, and aseptically filled into de-pyrogenated Type I glass vials (for example 10 mL fill volume). This dissolution sequence — ascorbic acid — > 0-glycerophosphate — > calcium lactate, can preserve the chemical integrity of each component, can prevent cross-precipitation, and can yield a clear, stable, and osteoinductive buffer that can actively support stem cell differentiation, collagen synthesis, and mineralized matrix formation upon allograft rehydration and implantation. The rehydration buffer can be prepared in any suitable dissolution. For example, the solution can be prepared first by dissolving the other salts, and the solution may be shaken well so that a white precipitate does not form and finally a suitable and clear solution is obtained. Beta-glycerophosphate or calcium lactate can be dissolved first, and then other salts are added.

[0100] Different variations of at least one of 0-glycerophosphate, ascorbic acid, and the calcium lactate may be used instead of the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), and the calcium lactate (C6H10CaO6), respectively, in different embodiments, whereby the specific form, the molecular structure, the stereochemistry, and / or the chemical composition may vary. For instance, in some embodiments, the ascorbic acid comprises the stereochemistry and the structure denoted by (2R)-2-[(lS)-l,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one. In different embodiments, at least one of ^-glycerophosphate, ascorbic acid, and calcium lactate dissolved in the normal saline or WFI may exist in different forms, such as solid or liquid. For instance, each compound may exist in solid form as, for example, a powder or crystal, or in liquid form, wherein the compound may be dissolved in a solvent (e.g., sterile distilled water or water for injection). Further, in some embodiments, a substance or compound including at least one of 0-glycerophosphate, ascorbic acid, and calcium lactate may be added into the WFI or normal saline or HEPES instead of the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), and the calcium lactate (C6H10CaO6). For example, calcium ascorbate (C12H14CaO12), a calcium salt of ascorbic acid, may be dissolved in the WFI instead of the ascorbic acid (C6H8O6). In some embodiments, the 0-glycerophosphate (C3H7Na2O6P) and the calcium lactate (C6H10CaO6) are first dissolved in the WFI and the ascorbic acid (C6H8O6) is dissolved in the WFI at a later time, closer to the time of use of the buffer solution, as the ascorbic acid (C6H8O6) or ascorbic acid 2-phosphate may become unstable and decomposewithin the buffer solution after a short period of time (e.g., a few weeks). In some embodiments, the buffer solution is completely stable at room temperature for a period of time (e.g., one month) and has a pH between 7.2 to 7.4.

[0101] In some embodiments, the method further comprises adjusting the pH. In some embodiments, the method further comprises adjusting the pH to between about 7.2 and 7.4.

[0102] In some embodiments, the method further comprises sterilizing the formulation.

[0103] In some embodiments, the L-ascorbic acid or derivative thereof comprises L-ascorbic acid or L-ascorbic acid 2-phosphate.

[0104] In some embodiments, the organic phosphate comprises P-glycerophosphate.

[0105] In some embodiments, the organic calcium salt comprises calcium ascorbate. In some embodiments, the organic calcium salt comprises calcium lactate.

[0106] In some embodiments, the formulation comprises between about 1 mM and about 25 mM of P-glycerophosphate.

[0107] In some embodiments, the formulation comprises between about 1 mM and about 20 mM of calcium lactate.

[0108] In some embodiments, the formulation comprises between about 1 pg / mL and 100 pg / mL of L-ascorbic acid or derivative thereof.

[0109] In some embodiments, the solvent comprises sterile water. In some embodiments, the solvent comprises water for injection. In some embodiments, the solvent comprises saline. In some embodiments, the solvent comprises HEPES.

[0110] In some embodiments, the method minimizes oxidative degradation of the L-ascorbic acid or the derivative thereof. In some embodiments, the method minimizes precipitation of the L-ascorbic acid or the derivative thereof, and / or the P-glycerophosphate.

[0111] In some embodiments, the formulation is the formulation disclosed herein.

[0112] In another aspect, provided herein is a method of preparing a bone graft. The method can include preparing a formulation for rehydrating a bone graft material. Preparing the formulation can include dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution. Preparing the formulation can include dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution. Preparing the formulation can include dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate an ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation. The method can include providing the bone graft material. The method can include rehydrating the bone graft material in the formulation for a period of time.

[0113] In another aspect, provided herein is a method of preparing a bone graft, comprising: (1) preparing a formulation for rehydrating a bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation; (2) providing a bone graft material; and (3) rehydrating the bone graft material in the formulation for a period of time, thereby generating the bone graft.

[0114] One or more embodiments provide a method for preparing a bone allograft for implantation. In some embodiments, the method includes preparing the buffer solution as described above. In some embodiments, the method further includes submerging or rehydration of the bone allograft within and / or mixing the bone allograft with the buffer solution and waiting for a period of time (e.g., 20 minutes, 30 minutes, etc.), after which the bone allograft is ready for implantation within a human body. In some embodiments, the period of time the bone allograft is submerged is 20 minutes and in other embodiments 30 minutes. In other embodiments the submersion time may be between 20 and 30 minutes. In yet other embodiments the submersion time is between 20 and 30 minutes. The purpose of submerging the bone allograft within and / or mixing the bone allograft with the buffer solution is to reintroduce water into the bone allograft that was previously removed (e.g., during freeze drying) prior to implantation of the bone allograft. This is similar to other buffer solutions currently used. However, the buffer solution described herein can also provide another purpose, namely, improving the quantity and quality of bone formation as well as speed of ossification post-implantation of the bone allograft facilitated by the absorption of P-glycerophosphate (C3H7Na2O6P), ascorbic acid (C6H8O6) or ascorbic acid 2-phosphate, and calcium lactate (C6H10CaO6) during immersion of the bone allograft in the buffer solution. In some embodiments, air bubbles may be removed from porous bone allograft using an instrument and then rehydrated with a buffer solution.

[0115] One or more embodiments provide a bone allograft. A form, a shape, and a size of the bone allograft may vary in different embodiments. Examples of bone allografts with different forms and shapes include, but are not limited to, bone powder; a bone shaft; bone in the form of a plurality of cubes, matchsticks, wedges, or other geometric shape; crushed bone; bone chips; cortical or cancellous. In some embodiments, more than one form, shape, and / or size of bone allograft are provided. The size of each different form and shape of the bone allograft may vary depending on the application. Further, a state of the bone allograft may vary in differentembodiments. Examples of different bone allograft states include, but are not limited to, a dry state (i.e. , after removal of water from the bone allograft via freeze drying or other drying methods), a wet state (i.e., without the removal of water from the bone allograft), a frozen state (i.e., below zero degrees Celsius), and an unfrozen state (e.g., at room temperature).

[0116] In another aspect, provided herein is a method of treating a bone condition or defect in a subject in need thereof. The method can include providing a bone graft material. The method can include preparing a formulation for rehydrating a bone graft material. Preparing the formulation can include dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution. Preparing the formulation can include dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution. Preparing the formulation can include dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate an ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation. The method can include rehydrating the bone graft material in the formulation for a period of time, generating a bone graft. The method can include implanting the bone graft to a bone site of the bone condition or defect in the subject.

[0117] In another aspect, provided herein is a method of treating a bone condition or defect in a subject in need thereof, comprising: (1) providing a bone graft material; (2) preparing a formulation for rehydrating the bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphate-organic calcium salt solution, thereby forming the formulation; (3) rehydrating the bone graft material in the formulation for a period of time, generating a bone graft; (4) implanting the bone graft to a bone site of the bone condition or defect in the subject.

[0118] In some embodiments, the bone condition or defect is resulted from spine surgery, orthopedic trauma surgery, joint reconstruction / arthroplasty surgery, orthopedic surgery for tumor, foot and ankle Surgery, sports medicine surgery, or oral and maxillofacial surgery.

[0119] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0120] In some embodiments, the L-ascorbic acid or derivative thereof comprises L-ascorbic acid or L-ascorbic acid 2-phosphate. In some embodiments, the organic phosphate comprises [3-glycerophosphate. In some embodiments, the organic calcium salt comprises calcium ascorbate. In some embodiments, the organic calcium salt comprises calcium lactate.

[0121] In some embodiments, the bone graft material comprises one or more of: bone powder; crushed bone; bone chips; cortical bone; cancellous bone; bone shaft; and bone in the form of a plurality of cubes, matchsticks, wedges, or other geometric shape.

[0122] In some embodiments, the bone graft material comprises a bone material in a dry state. In some embodiments, the bone graft material comprises a bone material that has been freeze-dried. In some embodiments, the bone graft material comprises a bone material in a wet state. In some embodiments, the bone graft material comprises a bone material in a frozen state.

[0123] In some embodiments, the bone graft is an allograft. In some embodiments, the bone graft is an autograft. In some embodiments, the bone graft is a xenograft.

[0124] In some embodiments, the period of time is between about 20 minutes and about 30 minutes.

[0125] In some embodiments, the formulation is prepared according to the method disclosed herein.

[0126] In some embodiments, the formulation is the formulation disclosed herein.III. Kits

[0127] In a further aspect, provided herein is a kit for preparing a bone graft, comprising: (1) [:1-glycerophosphate; (2) L-ascorbic acid; (3) calcium lactate; (4) a solvent; and (5) a bone graft material.

[0128] In a further aspect, provided herein is a kit for preparing a bone graft, comprising: (1) the formulation of any one of claims 1-34; (2) a bone graft material.

[0129] In a further aspect, provided herein is a kit for preparing a bone graft, comprising: (1) an organic phosphate; (2) L-ascorbic acid, or a derivative thereof; (3) an organic calcium salt; (4) a solvent; and (5) a bone graft material.

[0130] In some embodiments, the kit further comprises an applicator.

[0131] In some embodiments, the kit further comprises a try comprising at least two compartments.

[0132] In some embodiments, the organic phosphate, the L-ascorbic acid or derivative thereof, and the organic calcium salt are stored within a first compartment, the solvent is stored within a second compartment, and the bone graft material is stored within a third compartment.

[0133] In some embodiments, the solvent is contained within a container in the second compartment.

[0134] In some embodiments, the organic phosphate, the L-ascorbic acid or derivative thereof, and the organic calcium salt are dissolved in the solvent and provided as a rehydration buffer.

[0135] In some embodiments, the rehydration buffer is stored in a first compartment, and the bone graft material is stored in a second compartment.

[0136] In some embodiments, the rehydration buffer is contained within a container stored in the first compartment.

[0137] In some embodiments, the organic phosphate and the organic calcium salt are dissolved in the solvent and provided as a partially prepared rehydration buffer

[0138] In some embodiments, the L-ascorbic acid or derivative thereof is stored within a first compartment, the partially prepared rehydration buffer is stored within a second compartment, and the bone graft material is stored within a third compartment.

[0139] In some embodiments, the partially prepared rehydration buffer is contained within a container stored in the second compartment.

[0140] In some embodiments, at least one of the compartments is configured to contain a volume of the rehydration buffer and the bone graft material in a period of time, thereby allowing rehydration of the bone graft material in the at least one compartment.

[0141] One or more embodiments provide a sterile package. In some embodiments, the bone allograft is stored in a first cavity of the sterile package, a partially prepared buffer solution, whereby only the normal saline or WFI, is stored within a second cavity of the sterile package, and the salts separately are stored within a third cavity of the sterile package. In some embodiments, more than one form, shape, and / or size of bone allograft is included in the sterile package in a same cavity or in separate cavities. For example, bone powder may be stored in a first cavity, bone in the form of a plurality of cubes may be stored in a second cavity, the partially prepared solution may be stored in a third cavity, and the salts may be stored in a fourth cavity of the sterile package. A quantity of the partially prepared buffer solution (e.g., Icc, 75cc, 200cc, etc.) and the salts (e.g., 0.2, 0.5, 1, 2, 4, or 5grams, etc.) provided within the sterile package depends on at least one of a form, a shape, a size, and a volume of the bone allograft provided within the sterile package. In some embodiments, the salts mixed with the partially prepared buffer solution within the second cavity or another cavity of the sterile package to produce the buffer solution and the bone allograft is submerged within and / or mixed with the buffer solution within the second cavity or another cavity of the sterile package. In some embodiments, theascorbic acid (C6H8O6) ascorbic acid 2-phosphate is mixed with the partially prepared buffer solution within a container included in or separate from the sterile package (e.g., a bottle, a pouch, a bag, or a vial within which the partially prepared buffer solution may or may not be initially stored within the sterile package) to produce the buffer solution and the bone allograft is submerged within and / or mixed with the buffer solution within the container or a cavity of the sterile package. In some embodiments, the sterile package further includes an applicator (e.g., stirring rod) stored in its own separate cavity or in one of the other cavities of the sterile package for mixing the salts with the partially prepared buffer solution and / or the bone allograft with the buffer solution. Packaging the bone allograft, the solution or the partially prepared buffer solution, and the salts in a single sterile package omits the need for sourcing a sterile container and a sterile buffer solution in an operating room, facilitating the process and reducing the time of preparing the bone allograft for implantation. Moisture control (<1% RH in packaging) is essential. Further, the use of the sterile package and preparing the buffer solution within a cavity of the sterile package reduces the risk of contamination. Surgeons can select the optimal graft morphology intraoperatively based on defect geometry without opening multiple packages or risking cross-contamination. Dosing of buffer volume and salt mass scaled to graft volume ensures consistent rehydration kinetics and biological activity across use cases.

[0142] In some embodiments, the sterile package includes only two cavities, a first for storing the bone allograft and a second for storing the buffer solution. In such embodiments, the bone allograft is submerged within and / or mixed with the buffer solution within the first or second cavity of the sterile package. In some embodiments, the bone allograft is submerged within and / or mixed with the buffer solution within a container included in or separate from the sterile package (e.g., a bottle, a pouch, a bag, or a vial within which the partially prepared buffer solution may or may not be initially stored within the sterile package). In some embodiments, the sterile package further includes an applicator (e.g., stirring rod) stored in its own separate cavity or in one of the other cavities of the sterile package for mixing the bone allograft with the buffer solution. An expiration date of items included within the sterile package may vary in different embodiments, depending on planned use of the items. For instance, given that the bone allograft is needed within a short period of time, there is minimal concern of the buffer solution expiring due to the instability and decomposition of the ascorbic acid (C6H8O6) or ascorbic acid 2-phosphate within the buffer solution. As such, the buffer solution may be included in the sterile package rather than the partially prepared buffer solution and the ascorbic acid (C6H8O6). In instances wherein the bone allograft may not be used for a longer period of time, the partiallyprepared buffer solution (including the 0-glycerophosphate (C3H7Na2O6P) or the calcium lactate (C6H10CaO6) or both of them dissolved in the normal saline or WFI) and the ascorbic acid (C6H8O6) or ascorbic acid 2-phosphate are provided separately within the sterile package and the ascorbic acid (C6H8O6) or ascorbic acid 2-phosphate is dissolved within the partially prepared buffer solution closer to the time of use of the buffer solution.

[0143] In some embodiments, the sterile package includes a plurality of cavities to store the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), the calcium lactate (C6H10CaO6), the normal saline or WFI, and the bone allograft. In such embodiments, the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), and the calcium lactate (C6H10CaO6) are mixed with the normal saline or WFI within a cavity of the sterile package to produce the buffer solution and the bone allograft is submerged within and / or mixed with the buffer solution within the same cavity or another cavity of the sterile package. In some embodiments, the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), and the calcium lactate (C6H10CaO6) are mixed with the normal saline or WFI within a container included in or separate from the sterile package (e.g., a bottle, a pouch, a bag, or a vial within which the partially prepared buffer solution may or may not be initially stored within the sterile package) and the bone allograft is submerged within and / or mixed with the buffer solution within the container or a cavity of the sterile package. In some embodiments, the sterile package further includes an applicator (e.g., stirring rod) stored in its own separate cavity or in one of the other cavities of the sterile package for mixing the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), and the calcium lactate (C6H10CaO6) with the PBS and / or the bone allograft with the buffer solution. In different embodiments, at least one of the 0-glycerophosphate (C3H7Na2O6P), the ascorbic acid (C6H8O6), and the calcium lactate (C6H10CaO6) is dissolved in the normal saline or WFI prior to packaging or is included in the sterile package as a raw compound (e.g., a powder) for dissolution at the time of use.

[0144] A size (e.g., 5 cm3, 10 cm3, etc.), a color (e.g., black, brown, etc.), an opacity (e.g., partially transparent, opaque, etc.), and a shape (e.g., cubic, cylindrical, etc.) of each cavity of the sterile package may vary, depending on the item being stored within the respective cavity.Further, the size, the color, the opacity, and the shape of each cavity may vary in different embodiments. In some embodiments, each cavity is separately sealed and unsealed, each cavity being accessible independent of the other cavities. Alternatively, all cavities are accessed by removing a single seal. In some embodiments, at least one cavity is resealable after unsealing of the at least one cavity. For instance, the cavity within which the bone allograft is submerged inthe buffer solution may include a resealable seal such that the cavity may be resealed during submersion of the bone allograft in the buffer solution and unsealed at the time of use of the bone allograft. This may reduce the risk of contamination during submersion of the bone allograft in the buffer solution.

[0145] In some embodiments, the bone allograft, the partially prepared buffer solution, and / or the ascorbic acid (C6H8O6) are positioned directly within their respective cavity of the sterile package. In some embodiments, the bone allograft, the partially or completely prepared buffer solution, and / or the salts are stored in a respective container (e.g., a vial, plastic or nylon paper pouch or bag, a bottle, etc.) positioned within their respective cavity of the sterile package. A size (e.g., 5 cm3, 10 cm3, etc.), a color (e.g., black, white, brown etc.), an opacity (e.g., partially transparent, opaque, etc.), a material (e.g., glass, plastic, etc.), and a shape (e.g., cubic, cylindrical, etc.) of each container may vary, depending on the item being stored within the container. Further, the size, the color, the opacity, and the shape of each container may vary in different embodiments. Further, the order in which the bone allograft, the partially or completely prepared buffer solution, and the salts are packaged within the sterile package may vary in different embodiments.

[0146] A size, a color, an opacity, and a shape (of the sterile package as well as one or more materials from which the sterile package is fabricated may vary in different embodiments. In some embodiments, at least a portion of the sterile package is dark in color and / or opaque to prevent light from coming into contact with one or more of the bone allografts, the partially or fully prepared buffer solution, and the salts. In particular, preventing contact between the light and the partially or fully prepared buffer solution is important as light reduces the stability of the partially prepared buffer solution. In embodiments, the sterile package including the bone allograft, the partially prepared buffer solution, and the salts (C6H8O6) may be shipped and stored at room temperature or above freezing temperatures. In one or more embodiments, the sterile package within which the bone allograft, the partially or fully prepared buffer solution, and the salts are included is stored in a refrigerator to increase the shelf life of the partially or fully prepared buffer solution.

[0147] In some embodiments, the bone allograft is sterilized (e.g., by gamma rays) prior to packaging the bone allograft. In embodiments, the partially or fully prepared buffer solution is sterilized (e.g., by filtration process) prior to packaging the partially prepared buffer solution. In embodiments, the salts are prepared sterilely. In one or more embodiments, a package is sterilized aseptically to obtain the sterile package and the sterilized allograft, the sterilized buffersolution, and the sterile salts are stored and sealed in the sterile package under aseptic conditions. In one or more embodiments, a package is sterilized after storing and sealing the sterilized allograft, the sterilized partially or fully prepared buffer solution, and the sterile salts within the package.

[0148] Referring to FIGS. 1 and 2. A sterile package 100 includes a first cavity 101 storing a bone allograft 102, a second cavity 103 storing a glass vial 104 containing a partially prepared buffer solution 105, a third cavity 106 storing a plastic bag 107 containing salts 108, and an applicator 109. In FIG. 2, the applicator 109 is stored in a fourth cavity 110.

[0149] Referring to FIGS.3-6. A sterile package 300 includes a first cavity 301 storing a bone allograft 302 and a second cavity 303 storing a glass vial 304 containing a buffer solution 305 or the buffer solution 305 directly. In FIGS. 5 and 6, an applicator 306 is included.

[0150] Referring to FIG. 7, which depicts a method for preparing the bone allograft 102 illustrated in FIGS. 1 and 2 for implantation. The method includes opening the sterile package 100, pouring the partially prepared buffer solution 105 in the second cavity 103, adding the ascorbic acid (C6H8O6) 108 to the partially prepared buffer solution 105 in the second cavity 103 and mixing the two together using the applicator 109 to produce a buffer solution, submerging the bone allograft 102 within and / or mixing the bone allograft 102 with the buffer solution using the applicator 109, waiting for a period of time (e.g., 30 minutes, etc.), and a surgeon obtaining the bone allograft 102 for implantation within a human body. FIG. 8 illustrates a flowchart describing a method for preparing the bone allograft 302 illustrated in FIGS. 3-6 for implantation. The method includes opening the sterile package 300, pouring the buffer solution 305 in the second cavity 303, submerging the bone allograft 302 within and / or mixing the bone allograft 302 with the buffer solution 305, waiting for a period of time (e.g., 30 minutes, etc.), and a surgeon obtaining the bone allograft 302 for implantation within a human body.

[0151] The buffer solution described herein can improve the quality of bone regeneration as well as speed of ossification post-implantation of the bone allograft, as compared to the current use of a neutral buffer solution. The chemical compounds forming the buffer solution described herein can cause the osteogenesis and bone regeneration processes to occur faster and improved quality of bone as the chemical compounds promote better and more seeding of stem cells into the bone allograft and increase stem cell differentiation into bone-forming cells. Further, the buffer solution does not contain growth factors that may cause side effects, but rather naturally increases the ossification properties of the bone allograft.

[0152] The sterile package described herein can provide a means for preparing a bone allograft for implantation that can reduce an overall procedure time and reduces a risk of contamination. Current practices require surgical staff to source a sterile buffer solution and a sterile container for submerging the bone allograft in or mixing the bone allograft with the buffer solution. These steps add time to the overall bone allograft implantation procedure. Further, these steps introduce a risk of contamination, such as by accidental use of a contaminated container. Since the bone allograft and the buffer solution are provided in the sterile package and the sterile package includes a cavity for submerging the bone allograft in or mixing the bone allograft with the buffer solution, both procedure time and risk of contamination may be reduced.IV. Definitions

[0153] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure pertains.

[0154] As used herein, the singular forms “a,” “an,” and “the” include plural references, unless indicated otherwise. For example, a reference to “a ligand” can be a reference to more than one ligand.

[0155] The term “or” is used to mean “and / or”, unless it is indicated explicitly to refer to alternatives only. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the expression “A, B, and / or C” can mean A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.

[0156] The term “comprise”, “comprising”, or the like means additional elements or components other than those recited may be present. Other terms such as “include”, “contain”, “have” and the like have similar meaning.

[0157] The term “consist of’, “consisting of’ or the like means no additional component is present.

[0158] The term “about” or “approximately” when used in reference to a particular recited value, means the value may vary from the recited value by no more than 10%, 5%, 2% or 1%. Where a particular value is recited, it can be understood that the value is modified by the term “about” or “approximately”, unless indicated otherwise.

[0159] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0160] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0161] Although any compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of compositions and methods are now described.EXAMPLESExample 1

[0162] Allografts, such as bone, skin, tendon, and heart valve, are tissues donated from deceased donors. Allograft tissues are utilized in a wide variety of surgical specialties including ophthalmology, orthopedics, neurology, podiatry, oncology, pediatrics, burn care, emergency medicine, cardiology, urology, dental surgery, and general surgery. These donated tissues are delivered to tissue engineering facilities under established rules and regulations for processing prior to their surgical use. Processing of an allograft tissue may include harvesting the particular tissue of interest, shaping the allograft tissue as required, and decellularization to remove specific donor cells and immunogenic proteins from the allograft tissue. Processed allograft tissues may be available in different forms and states. For bone allografts in particular, they are most commonly available in a dried state, whereby the bone allografts are subjected to a freeze-drying process to remove intracellular water from the bone allograft. Freeze-dried bone allografts provide suitable expiration dates and may be transported at room temperature. Typically, at the time of use, a dried bone allograft is submerged in or rehydrated with a sterile solution (i.e., normal serum) for a short period of time until the water removed from the bone allograft during the drying process has returned to some extent, upon which the bone allograft is ready for implantation in a human body. Therefore, rehydration of lyophilized (freeze-dried) bone allografts is a critical pre-implantation step that can restore the graft’s structural integrity, porosity, and biocompatibility by reintroducing moisture in a controlled, sterile manner. Proper rehydration ensures the removal of residual processing agents, facilitates cellular infiltration postimplantation, and minimizes the risk of inflammatory responses. The rehydration medium mustbe endotoxin- free, sterile, and matched to physiological pH and osmolality to preserve graft property and support optimal regeneration within the host tissue. Post-implantation of a bone allograft, proper ossification is desired as soon as possible, whereby a suitable amount of stem cells are absorbed in the bone allograft, remain attached, and become suitable bone-forming cells. The more attractive the bone allograft is to stem cells, the better the processes of bone ossification perform. In traditional procedures, the buffer solution only moistens and removes the bone allograft from the dry state and does not help with facilitating quicker bone formation or improved quantity and quality of bone formation. Historically, the rehydration of lyophilized bone allografts has relied almost exclusively on 0.9% sodium chloride (normal saline) due to its widespread availability, sterility, and isotonic nature. In some specialized settings, lactated Ringer’s solution or antibiotic-containing saline has been used, though these remain the exceptions rather than the standard. These traditional solutions are chosen primarily for their compatibility with intravenous use and regulatory familiarity, rather than their biological suitability for tissue rehydration. Despite decades of clinical use, none of these conventional fluids are specifically designed to support the biochemical and cellular demands of bone graft integration, highlighting a significant gap between clinical convenience and biological optimization.

[0163] Although normal saline is isotonic, it lacks physiological buffering capacity, resulting in a suboptimal pH (~4.5-5.5) that can induce local acidosis upon implantation, potentially triggering inflammation and impairing osteoblast function. Its non-physiological ionic composition containing only Na+and Cl" without essential ions like calcium, phosphate, or bicarbonate — fails to support early cellular adhesion, mineralization, or extracellular matrix remodeling.Furthermore, normal saline provides no antioxidant or osmo-protective agents, leaving the graft vulnerable to oxidative stress during rehydration. These limitations may compromise graft resorption kinetics, delay vascularization, and ultimately reduce the clinical performance of the allograft, underscoring the need for a purpose-designed, biocompatible rehydration buffer.

[0164] Currently, the standard intraoperative protocol for preparing lyophilized bone allografts for implantation involves multiple manual steps: surgical personnel must first retrieve a sterile container (e.g., a stainless-steel basin or disposable fray), then draw or pour a sterile buffer solution — typically 0.9% normal saline — into that container and finally submerge the allograft for a prescribed rehydration period. This multi-step process is not only time-consuming but also highly dependent on the availability of ancillary sterile supplies in the operating room. Delays may occur if the appropriate container or sufficient volume of sterile solution is not immediatelyat hand, potentially disrupting surgical flow, especially in high-acuity or time-sensitive procedures such as trauma reconstructions or spinal fusions. In busy operating room environments, even minor procedural inefficiencies can cumulatively impact case turnover, anesthesia duration, and overall resource utilization. Because the current rehydration system is open — requiring transfer of fluid from vials or bags into an exposed basin — it creates a direct pathway for airborne or contact-based microbial ingress during the critical moments before implantation. This open configuration significantly elevates the risk of introducing low-level contaminants that may not cause immediate infection but could impair graft integration or trigger subclinical inflammation. Given that the operating room prioritizes both maximal sterility assurance and procedural efficiency, there is a clinical and operational need for an integrated, ready-to-use rehydration system that eliminates manual transfer steps, reduces procedural time, and minimizes human error, thereby enhancing both patient safety and surgical workflow.Example 2Preparation of buffer solution

[0165] Sterile, isotonic, and physiologically optimized rehydration buffer for bone allografts is prepared by sequentially dissolving the components in sterile Water for Injection (WFI) under controlled conditions to ensure maximal stability, solubility, and biological activity. By way of example, 1-25 mM of the P-glycerophosphate (C3H7Na2O6P), 1-100 pg / mL of the ascorbic acid (C6H8O6), and 1-20 mM of the calcium lactate (C6H10CaO6) may be dissolved within 100 mL of normal saline, distilled sterile water or HEPES. Regarding the process steps, first, ascorbic acid (CeHsOe) is dissolved in a portion of the WFI at 37°C temperature with gentle stirring; this initial step can minimize oxidative degradation by avoiding exposure to high ionic strength or alkaline conditions early in the process. Next, P-glycerophosphate (C3H7Na2O6P) is slowly added to the ascorbic acid solution under continuous mixing; this order may prevent premature precipitation and allow gradual pH adjustment, as P-glycerophosphate can elevate the solution pH, which, if introduced too early, could accelerate ascorbic acid degradation. Finally, calcium lactate (CeHioCaOe) is slowly added and dissolved into the mixture. The use of calcium lactate provides bioavailable calcium without forming insoluble calcium-phosphate complexes during preparation. The final solution is adjusted to pH 7.2-7.4 if necessary, filtered through a 0.22 pm sterilizing-grade membrane, and aseptically filled into de-pyrogenated Type I glass vials (for example 10 mL fill volume). This precise dissolution sequence — ascorbic acid — > P-glycerophosphate — > calcium lactate, can preserve the chemical integrity of each component, prevent cross-precipitation, and yield a clear, stable buffer solution.Example 3

[0166] Methods and materials for examining buffer efficacy in an ex vivo setting with bone allograft

[0167] A rehydration buffer was prepared with the following components: L-ascorbic acid 2-phosphate (AsA2P) :70 pg-mL1, 0-glycerophosphate (0-GP) : 5 mM, Calcium lactate (as calcium source) : 2.5 mM total extracellular Ca2+. Dissolution sequence: ascorbic acid — > 0-glycerophosphate — > calcium lactate, pH 7.4 adjusted, filtered through a 0.22 pm, Osmolarity 290 mOsm / kg, temperature 37°C. In rehydrating the bone graft, a buffer volume of at least 10x the graft volume (by liquid volume) was used, with gentle rocking or slow rotation, and a rehydration time of 30 minutes. Treated bone graft was used immediately after rehydration.

[0168] Cell Seeding-. hBM-MSCs (Mesenchymal Stem Cells) (passage 1-3), Seeding Density: 1.0 x 106cells per 100 mg of bone allograft, seeding method: static seeding in 24-well plates; cells suspended in 100 pL DMEM + 10% FBS, pipetted directly onto grafts, incubated 2 h (37°C, 5% CO2), then 1 mL osteogenic medium added. Group A: allografts rehydrated 30 min in the buffer, Group B: allografts rehydrated 30 min in 0.9% normal saline (control).

[0169] Rehydration & Culture Conditions'. Rehydration: 30 min at 37°C with gentle rocking (30 rpm) in 10x graft volume of buffer (e.g., 10 mL buffer per 100 mg graft), Post-rehydration: Grafts transferred to 24-well plates, seeded immediately, Culture Medium: DMEM + 10% FBS + 1% penicillin / streptomycin.

[0170] Cell Viability Assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at Days 7, 14, 21. MTT assay was conducted using a standard procedure.

[0171] Alkaline Phosphatase (ALP) activity and Alizarin Red S. assays'. Alkaline Phosphatase (ALP) Activity was measured using an ALP ELISA kit at day 7, 14 and 21, and Alizarin Red S (ARS) Staining for Calcium Deposition were measured at day 21. Comparisons were made between buffer (Group A) and Saline (control) (Group B).Example 4

[0172] In this example, buffer efficacy was demonstrated in an ex vivo setting with a bone allograft. The bone allograft was prepared according to the method described in Example 3.

[0173] Osteopontin (OPN) is a phosphorylated glycoprotein expressed during the late stages of osteogenic differentiation, playing critical roles in cell adhesion, mineralization, and matrix maturation. In this study, bone allografts pre-treated for 30 minutes with AlloWide buffer exhibited a 4.4-fold upregulation of OPN gene expression relative to normal saline controls at Day 21, normalized to housekeeping genes TBP and ACTB (FIG. 9). This significant increase may reflect enhanced functional maturation of seeded mesenchymal stem cells (MSCs), which may be driven by the synergistic action of calcium lactate and P-glycerophosphate in the buffer. Without wishing to be bound by a theory, calcium ions activate integrin-mediated signaling pathways that promote OPN transcription, while phosphate released from 0-GP supports hydroxyapatite nucleation — a key trigger for OPN expression as part of the mineralization feedback loop.

[0174] The robust upregulation of OPN in the allograft pretreated with the rehydration buffer shows successful osteogenic commitment and also suggests improved graft integration potential. OPN serves as both a structural component of the mineralized matrix and a signaling molecule that regulates osteoclast recruitment and bone remodeling — making it a valuable biomarker for predicting long-term graft performance. Importantly, this effect was achieved without modifying the culture medium itself, demonstrating that brief rehydration with the rehydration buffer primes the graft surface to actively direct cellular behavior. Clinically, elevated OPN correlates with accelerated bone healing and reduced resorption rates, supporting the translational value of this approach.Example 5

[0175] In this example, buffer efficacy was demonstrated in an ex vivo setting with a bone allograft. The bone allograft was prepared according to the method described in Example 3.

[0176] Osteocalcin (OCN) is one of the most specific markers of terminal osteoblastic differentiation, synthesized exclusively by mature osteoblasts and directly involved in regulating hydroxyapatite crystal growth. The 3.1 -fold increase in OCN gene expression observed in MSCs cultured on AlloWide-primed allografts compared to saline controls demonstrates a profound enhancement in late-stage osteogenesis (FIG. 10). Without wishing to be bound by a theory, this effect can be attributed to the combined influence of ascorbic acid 2-phosphate, which promotes collagen I deposition — providing the necessary scaffold for mineralization — and calcium lactate, which activates the calcium-sensing receptor (CaSR) to initiate downstream osteogenic transcriptional programs including OCN. The timing of peak OCN expression at Day 21 isconsistent with known kinetics of osteoblast maturation, indicating that the AlloWide buffer does not merely accelerate differentiation but supports its full progression to a functionally mature phenotype.

[0177] Clinically, OCN expression is strongly associated with active bone formation and mineralization capacity — making it a key predictor of graft success in vivo. The fact that OCN levels rose significantly despite identical culture conditions post-seeding underscores the importance of initial graft conditioning: the 30-minute rehydration step effectively “preprogrammed” the microenvironment to support advanced osteoblast function. This finding has important implications for clinical practice, where rapid and reliable bone regeneration is paramount. Furthermore, the use of dual housekeeping genes (TBP and ACTB) ensures normalization accuracy, eliminating concerns about variable gene stability during differentiation. Together, these data provide compelling molecular evidence that AlloWide enhances the biological competence of bone allografts beyond passive rehydration.Example 6

[0178] Alkaline phosphatase (ALP) activity serves as an early and sensitive indicator of osteogenic commitment, peaking around Days 10-17 before declining as cells transition into the mineralization phase. FIG. 11 shows that ALP activity was consistently higher in MSCs cultured on AlloWide-primed grafts across all timepoints — reaching 111 U / L at Day 21 versus 81 U / L in saline controls. Without wishing to be bound by a theory, this progressive elevation may reflect sustained osteogenic stimulation provided by the buffer’s components: P-glycerophosphate supplies inorganic phosphate essential for ALP substrate availability, while ascorbic acid 2-phosphate supports ECM synthesis, facilitating cell-matrix interactions that amplify ALP expression. The non-linear trajectory — steeper rise in the AlloWide group — suggests accelerated differentiation kinetics. From a clinical standpoint, elevated ALP activity correlates with faster bone repair and improved graft incorporation, particularly in challenging defects where early osteoinduction is critical. The fact that ALP levels remained significantly elevated even at Day 21 indicates prolonged osteogenic signaling — possibly due to slow release of bioactive ions from the graft surface, maintaining a pro-differentiation milieu. The combined data suggests that AlloWide initiates osteogenesis earlier, sustains it longer, and culminates in greater mineral deposition — all without altering the standard culture protocol.Example 7

[0179] Alizarin Red S staining quantifies calcium deposition, which may serve as a definitive endpoint for evaluating mineralization capacity in vitro. FIG. 12 shows that the optical density (OD) at 550 nm was 1.1 for AlloWide -primed grafts versus 0.5 for saline controls, representing a 120% increase in mineral content. Without wishing to be bound by a theory, this dramatic enhancement can be explainable by the cumulative effects of the buffer’s components: calcium lactate provides the cationic source, P-glycerophosphate generates phosphate via hydrolysis, and ascorbic acid 2-phosphate enables collagen I fibrillogenesis — creating a template for hydroxyapatite nucleation. The fact that mineralization occurs despite identical culture media shows that the 30-minute rehydration step conferred lasting biochemical modifications to the graft surface, enabling it to act as a reservoir of osteoinductive cues over the 21 -day period. In clinical translation, the degree of mineralization directly impacts graft stability, load-bearing capacity, and integration with host bone. An OD value of 1.1 indicates substantial mineral deposition — approaching the threshold typically seen in mature bone tissue — whereas 0.5 suggests minimal calcification, characteristic of poorly differentiated or inactive cultures. This level of mineralization is clinically relevant and exceeds what is typically achieved with saline-rehydrated grafts, highlighting the therapeutic advantage of AlloWide. As a final validation point, the ALZ data corroborates the protein (ALP) and genomic (OPN / OCN) results, forming a triad of evidence that AlloWide transforms inert bone allografts into bioactive, regenerative implants.Example 8

[0180] The cellular evaluation demonstrates that 30-minute rehydration of decellularized bone allografts with AlloWide buffer significantly enhances both initial cell attachment (“Primary Cell Seeding”) and short-term survival (“Cell Alive after 24h”) compared to normal saline controls (FIG. 13). Specifically, 79% of seeded MSCs adhered to AlloWide -primed grafts, versus only 52% on saline-treated grafts — representing a 52% improvement in seeding efficiency. Without wishing to be bound by a theory, this dramatic increase may be attributable to the buffer’s calcium lactate component, which provides bioavailable Ca2+ions that bind to integrin receptors (e.g., a2pi) on MSC membranes, promoting focal adhesion formation and cytoskeletal organization. Additionally, ascorbic acid 2-phosphate supports collagen I synthesis even during early culture phases, enhancing ECM-mediated anchorage. The second metric — 69% cell viability at 24h for AlloWide vs. 44% for saline — reflects reduced apoptosis and improvedmetabolic adaptation, likely due to decreased oxidative stress (from ascorbate’s antioxidant properties) and better nutrient exchange facilitated by superior cell-scaffold integration.

[0181] From a clinical and translational standpoint, these results are profoundly significant. Efficient cell seeding is the critical first step in successful tissue regeneration — poor engraftment leads to graft failure, delayed healing, or non-union. The fact that AlloWide improves primary seeding by over 50% suggests it could substantially enhance the efficacy of bone allografts in vivo, particularly in challenging environments such as infected sites or elderly patients with compromised MSC function. Furthermore, the 24-hour viability data indicates that cells not only attach but also survive the initial post-seeding stress phase — a prerequisite for subsequent proliferation and differentiation. This early survival advantage may translate into faster functional recovery in surgical applications. When combined with downstream markers like ALP, OPN, OCN, and mineralization, these cellular-level outcomes show AlloWide actively primes bone allografts for enhanced regenerative performance — not merely rehydrating them.

[0182] ENUMERATED EMBODIMENTSEnumerated Embodiment 1. A formulation for rehydrating a bone graft material, comprising:a. 1-25 mM of P-glycerophosphate;b. 1-100 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and c. 1-20 mM calcium lactate,in a buffered aqueous solution.Enumerated Embodiment 2. A formulation for rehydrating a bone graft material, comprising:a. about 5 mM of P-glycerophosphate;b. about 70 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and c. about 2.5 mM calcium lactate,in a buffered aqueous solution.Enumerated Embodiment 3. A formulation for rehydrating a bone graft material, comprising:a. an organic phosphate;b. L-ascorbic acid, or a derivative thereof; andc. an organic calcium salt.Enumerated Embodiment 4. The formulation of any of the enumerated embodiments, wherein the organic phosphate comprises a substrate of alkaline phosphatase.Enumerated Embodiment 5. The formulation of any of the enumerated embodiments, wherein the organic phosphate comprises a phosphate donor.Enumerated Embodiment 6. The formulation of any of the enumerated embodiments, wherein the organic phosphate comprises a phosphate donor that releases inorganic phosphate upon hydrolysis by alkaline phosphatase.Enumerated Embodiment 7. The formulation of any of the enumerated embodiments, wherein the organic phosphate is capable of driving osteogenic differentiation of mesenchymal stem cells (BM-MSCs).Enumerated Embodiment 8. The formulation of any of the enumerated embodiments, wherein the organic phosphate is capable of upregulating osteogenic markers such as Runx2, osteocalcin, and bone sialoprotein.Enumerated Embodiment 9. The formulation of any of the enumerated embodiments, wherein the organic phosphate comprises P-glycerophosphate.Enumerated Embodiment 10. The formulation of any of the enumerated embodiments, wherein the organic phosphate has a concentration of between about 1 mM and about 25 mM.Enumerated Embodiment 11. The formulation of any of the enumerated embodiments, wherein the P-glycerophosphate has a concentration of between about 1 mM and about 25 mM.Enumerated Embodiment 12. The formulation of any of the enumerated embodiments, wherein the P-glycerophosphate has a concentration of about 5mM.Enumerated Embodiment 13. The formulation of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof comprises L-ascorbic acid.Enumerated Embodiment 14. The formulation of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof comprises L-ascorbic acid 2-phosphate. Enumerated Embodiment 15. The formulation of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof has a concentration of between about 1 pg / mL and 100 pg / mL.Enumerated Embodiment 16. The formulation of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof has a concentration of about 70 pg / mL.Enumerated Embodiment 17. The formulation of any of the enumerated embodiments, wherein the organic calcium salt comprises a source of calcium ion for hydroxyapatite deposition and / or bone mineralization.Enumerated Embodiment 18. The formulation of any of the enumerated embodiments, wherein the organic calcium salt comprises calcium lactate.Enumerated Embodiment 19. The formulation of any of the enumerated embodiments, wherein the organic calcium salt comprises calcium ascorbate.Enumerated Embodiment 20. The formulation of any of the enumerated embodiments, wherein the organic calcium salt is capable of promoting osteoblast proliferation, differentiation, and / or survival.Enumerated Embodiment 21. The formulation of any of the enumerated embodiments, wherein the organic calcium salt is capable of supporting cellular metabolism under hypoxic conditions.Enumerated Embodiment 22. The formulation of any of the enumerated embodiments, wherein the organic calcium salt has a concentration of between about 1 mM and about 20 mM.Enumerated Embodiment 23. The formulation of any of the enumerated embodiments, wherein the calcium lactate has a concentration of between about 1 The formulation of any of the enumerated embodiments, wherein the calcium lactate has a concentration of about 2.5 mM.Enumerated Embodiment 24. The formulation of any of the enumerated embodiments, wherein the formulation is an aqueous solution.Enumerated Embodiment 25. The formulation of any of the enumerated embodiments, wherein the aqueous solution is a buffered aqueous solution.Enumerated Embodiment 26. The formulation of any of the enumerated embodiments, wherein the aqueous solution comprises saline.Enumerated Embodiment 27. The formulation of any of the enumerated embodiments, wherein the aqueous solution comprises water for injection.Enumerated Embodiment 28. The formulation of any of the enumerated embodiments, wherein the aqueous solution comprises a buffered isotonic solution.Enumerated Embodiment 29. The formulation of any of the enumerated embodiments, wherein the aqueous solution comprises Normosol.Enumerated Embodiment 30. The formulation of any of the enumerated embodiments, wherein the aqueous solution comprises Plasma-Lyte.Enumerated Embodiment 31. The formulation of any of the enumerated embodiments, wherein the formulation has a pH of between about 7.2 and 7.4.Enumerated Embodiment 32. The formulation of any of the enumerated embodiments, wherein the bone graft material comprises one or more of: bone powder; crushed bone; bone chips; cortical bone; cancellous bone; bone shaft; and bone in the form of a plurality of cubes, matchsticks, wedges, or other geometric shape.Enumerated Embodiment 33. The formulation of any of the enumerated embodiments, wherein the bone graft material comprises a bone material in a dry state.Enumerated Embodiment 34. The formulation of any of the enumerated embodiments, wherein the bone graft material comprises a bone material that has been freeze-dried. Enumerated Embodiment 35. The formulation of any of the enumerated embodiments, wherein the bone graft material comprises a bone material in a wet state.Enumerated Embodiment 36. The formulation of any of the enumerated embodiments, wherein the bone graft material comprises a bone material in a frozen state.Enumerated Embodiment 37. The formulation of any of the enumerated embodiments, wherein rehydrating the bone graft material results in a bone graft that has an increased expression in osteopontin (OPN) compared to a control bone graft.Enumerated Embodiment 38. The formulation of any of the enumerated embodiments, wherein the increase in OPN expression when compared to a control bone graft is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5 -fold, at about 21 days after being rehydrated.Enumerated Embodiment 39. The formulation of any of the enumerated embodiments, wherein rehydrating the bone graft material results in a bone graft that has an increased expression in osteocalcin (OCN) compared to a control bone graft.Enumerated Embodiment 40. The formulation of any of the enumerated embodiments, wherein the increase in OCN expression when compared to a control bone graft is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5 -fold, at about 21 days after being rehydrated.Enumerated Embodiment 41. The formulation of any of the enumerated embodiments, wherein rehydrating the bone graft material results in a bone graft that has an increased alkaline phosphatase (ALP) activity compared to a control bone graft.Enumerated Embodiment 42. The formulation of any of the enumerated embodiments, wherein the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 7 days after being rehydrated.Enumerated Embodiment 43. The formulation of any of the enumerated embodiments, wherein the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 14 days after being rehydrated.Enumerated Embodiment 44. The formulation of any of the enumerated embodiments, wherein the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 21 days after being rehydrated.Enumerated Embodiment 45. The formulation of any of the enumerated embodiments, wherein rehydrating the bone graft material results in a bone graft that has an increased level of calcium deposition compared to a control.Enumerated Embodiment 46. The formulation of any of the enumerated embodiments, wherein the increase in calcium deposition level when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, at about 14 days after being rehydrated.Enumerated Embodiment 47. The formulation of any of the enumerated embodiments, wherein rehydrating the bone graft material results in a bone graft that allows an increased level of seeding of a primary cell compared to a control bone graft.Enumerated Embodiment 48. The formulation of any of the enumerated embodiments, wherein the increase in the level of seeding of the primary cell when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.Enumerated Embodiment 49. The formulation of any of the enumerated embodiments, wherein rehydrating the bone graft material results in a bone graft that allows an increased viability of a primary cell compared to a control bone graft.Enumerated Embodiment 50. The formulation of any of the enumerated embodiments, wherein the increase in viability of the primary cell when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.Enumerated Embodiment 51. The formulation of any of the enumerated embodiments, wherein the primary cell comprises a mesenchymal stem cell (MSC).Enumerated Embodiment 52. The formulation of any of the enumerated embodiments, wherein the rehydrated bone graft material comprises a bone graft that has an increased expression of osteopontin (OPN) compared to a control bone graft, wherein the increase in OPN expression is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5-fold, at about 21 days after being rehydrated.Enumerated Embodiment 53. The formulation of any of the enumerated embodiments, wherein the rehydrated bone graft material comprises a bone graft that has an increased expression of osteocalcin (OCN) compared to a control bone graft, wherein the increase in OPN expression is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5-fold, at about 21 days after being rehydrated.Enumerated Embodiment 54. The formulation of any of the enumerated embodiments, wherein the rehydrated bone graft material comprises a bone graft that has an increased alkaline phosphatase (ALP) activity compared to a control bone graft, wherein the increase in ALP activity is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 7 days after being rehydrated. Enumerated Embodiment 55. The formulation of any of the enumerated embodiments, wherein the rehydrated bone graft material comprises a bone graft that has an increased level of calcium deposition compared to a control wherein the increase in calcium deposition level is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, at about 14 days after being rehydrated.Enumerated Embodiment 56. The formulation of any of the enumerated embodiments, wherein the rehydrated bone graft material comprises a bone graft that allows an increased level of seeding of a primary cell compared to a control bone graft, wherein the increase in the level of seeding of the primary cell is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.Enumerated Embodiment 57. The formulation of any of the enumerated embodiments, wherein the rehydrated bone graft material comprises a bone graft that allows an increased viability of a primary cell compared to a control bone graft, wherein the increase in viability of the primary cell is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.Enumerated Embodiment 58. The formulation of any of the enumerated embodiments, wherein the control is a bone graft rehydrated with normal saline.Enumerated Embodiment 59. A method of preparing a formulation for hydrating a bone graft material, comprising:(i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution;(ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution;(iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate an ascorbic acid-organic phosphate-organic calcium salt solution,thereby forming the formulation.Enumerated Embodiment 60. The method of any of the enumerated embodiments, further comprising adjusting the pH to between about 7.2 and 7.4.Enumerated Embodiment 61. The method of any of the enumerated embodiments, further comprising sterilizing the formulation.Enumerated Embodiment 62. The method of any of the enumerated embodiments, wherein the method minimizes oxidative degradation of the L-ascorbic acid or the derivative thereof.Enumerated Embodiment 63. The method of any of the enumerated embodiments, wherein the method minimizes precipitation of the L-ascorbic acid or the derivative thereof, and / or the P-glycerophosphate.Enumerated Embodiment 64. The method of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof comprises L-ascorbic acid or L-ascorbic acid 2-phosphate.Enumerated Embodiment 65. The method of any of the enumerated embodiments, wherein the organic phosphate comprises P-glycerophosphate.Enumerated Embodiment 66. The method of any of the enumerated embodiments, wherein the organic calcium salt comprises calcium ascorbate.Enumerated Embodiment 67. The method of any of the enumerated embodiments, wherein the organic calcium salt comprises calcium lactate.Enumerated Embodiment 68. The method of any of the enumerated embodiments, wherein the formulation comprises between about 1 mM and about 25 mM of P- glycerophosphate.Enumerated Embodiment 69. The method of any of the enumerated embodiments, wherein the formulation comprises between about 1 mM and about 20 mM of calcium lactate.Enumerated Embodiment 70. The method of any of the enumerated embodiments, wherein the formulation comprises between about 1 pg / mL and 100 pg / mL of L-ascorbic acid or derivative thereof.Enumerated Embodiment 71. The method of any of the enumerated embodiments, wherein the solvent comprises sterile water.Enumerated Embodiment 72. The method of any of the enumerated embodiments, wherein the solvent comprises water for injection.Enumerated Embodiment 73. The method of any of the enumerated embodiments, wherein the solvent comprises saline.Enumerated Embodiment 74. The method of any of the enumerated embodiments, wherein the solvent comprises HEPES.Enumerated Embodiment 75. The method of any of the enumerated embodiments, wherein the formulation is the formulation of any of the enumerated embodiments.Enumerated Embodiment 76. A method of preparing a bone graft, comprising:(1) preparing a formulation for rehydrating a bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (hi) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphateorganic calcium salt solution, thereby forming the formulation;(2) providing the bone graft material; and(3) rehydrating the bone graft material in the formulation for a period of time, thereby generating the bone graft.Enumerated Embodiment 77. A method of treating a bone condition or defect in a subject in need thereof, comprising:(1) providing a bone graft material;(2) preparing a formulation for rehydrating the bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (hi) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphateorganic calcium salt solution, thereby forming the formulation;(3) rehydrating the bone graft material in the formulation for a period of time, generating a bone graft;(4) implanting the bone graft to a bone site of the bone condition or defect in the subject.Enumerated Embodiment 78. The method of any of the enumerated embodiments, wherein the bone condition or defect is resulted from spine surgery, orthopedic trauma surgery joint reconstruction / arthroplasty surgery, orthopedic surgery for tumor, foot and ankle Surgery, sports medicine surgery, or oral and maxillofacial surgery.Enumerated Embodiment 79. The method of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof comprises L-ascorbic acid or L-ascorbic acid 2-phosphate.Enumerated Embodiment 80. The method of any of the enumerated embodiments, wherein the organic phosphate comprises P-glycerophosphate.Enumerated Embodiment 81. The method of any of the enumerated embodiments, wherein the organic calcium salt comprises calcium ascorbate.Enumerated Embodiment 82. The method of any of the enumerated embodiments, wherein the organic calcium salt comprises calcium lactate.Enumerated Embodiment 83. The method of any of the enumerated embodiments, wherein the bone graft material comprises one or more of: bone powder; crushed bone; bone chips; cortical bone; cancellous bone; bone shaft; and bone in the form of a plurality of cubes, matchsticks, wedges, or other geometric shape.Enumerated Embodiment 84. The method of any of the enumerated embodiments, wherein the bone graft material comprises a bone material in a dry state.Enumerated Embodiment 85. The method of any of the enumerated embodiments, wherein the bone graft material comprises a bone material that has been freeze-dried. Enumerated Embodiment 86. The method of any of the enumerated embodiments, wherein the bone graft material comprises a bone material in a wet state.Enumerated Embodiment 87. The method of any of the enumerated embodiments, wherein the bone graft material comprises a bone material in a frozen state.Enumerated Embodiment 88. The method of any of the enumerated embodiments, wherein the bone graft is an allograft.Enumerated Embodiment 89. The method of any of the enumerated embodiments, wherein the bone graft is an autograft.Enumerated Embodiment 90. The method of any of the enumerated embodiments, wherein the bone graft is a xenograft.Enumerated Embodiment 91. The method of any of the enumerated embodiments, wherein the period of time is between about 20 minutes and about 30 minutes.Enumerated Embodiment 92. The method of any of the enumerated embodiments, wherein the formulation for rehydrating the bone graft material is the formulation of any of the enumerated embodiments.Enumerated Embodiment 93. The method of any of the enumerated embodiments, wherein the bone graft has an increased expression in osteopontin (OPN) compared to a control bone graft.Enumerated Embodiment 94. The method of any of the enumerated embodiments, wherein the increase in OPN expression when compared to a control bone graft is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5 -fold, at about 21 days after being rehydrated.Enumerated Embodiment 95. The method of any of the enumerated embodiments, wherein the bone graft has an increased expression in osteocalcin (OCN) compared to a control bone graft.Enumerated Embodiment 96. The method of any of the enumerated embodiments, wherein the increase in OCN expression when compared to a control bone graft is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5 -fold, at about 21 days after being rehydrated.Enumerated Embodiment 97. The method of any of the enumerated embodiments, wherein the bone graft has an increased alkaline phosphatase (ALP) activity compared to a control bone graft.Enumerated Embodiment 98. The method of any of the enumerated embodiments, wherein the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 7 days after being rehydrated.Enumerated Embodiment 99. The method of any of the enumerated embodiments, wherein the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 14 days after being rehydrated.Enumerated Embodiment 100. The method of any of the enumerated embodiments, wherein the increase in ALP activity when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 21 days after being rehydrated.Enumerated Embodiment 101. The method of any of the enumerated embodiments, wherein the bone graft has an increased level of calcium deposition compared to a control.Enumerated Embodiment 102. The method of any of the enumerated embodiments, wherein the increase in calcium deposition level when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, at about 14 days after being rehydrated.Enumerated Embodiment 103. The method of any of the enumerated embodiments, wherein the bone graft allows an increased level of seeding of a primary cell compared to a control.Enumerated Embodiment 104. The method of any of the enumerated embodiments, wherein the increase in the level of seeding of the primary cell when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.Enumerated Embodiment 105. The method of any of the enumerated embodiments, wherein the bone graft allows an increased viability of a primary cell compared to a control.Enumerated Embodiment 106. The method of any of the enumerated embodiments, wherein the increase in viability of the primary cell when compared to a control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.Enumerated Embodiment 107. The method of any of the enumerated embodiments, wherein the primary cell comprises a mesenchymal stem cell (MSC).Enumerated Embodiment 108. The method of any of the enumerated embodiments, wherein the control is a bone graft rehydrated with normal saline.Enumerated Embodiment 109. A kit for preparing a bone graft, comprising: (1) 0- glycerophosphate; (2) L-ascorbic acid; (3) calcium lactate; (4) a solvent; and (5) a bone graft material.Enumerated Embodiment 110. A kit for preparing a bone graft, comprising: (1) the formulation of any one of claims 1-34; (2) a bone graft material.Enumerated Embodiment 111. A kit for preparing a bone graft, comprising: (1) an organic phosphate; (2) L-ascorbic acid, or a derivative thereof; (3) an organic calcium salt; (4) a solvent; and (5) a bone graft material.Enumerated Embodiment 112. The of any of the enumerated embodiments, further comprising an applicator.Enumerated Embodiment 113. The kit of any of the enumerated embodiments, further comprising a tray comprising at least two compartments.Enumerated Embodiment 114. The kit of any of the enumerated embodiments, wherein the organic phosphate, the L-ascorbic acid or derivative thereof, and the organic calcium salt are stored within a first compartment, the solvent is stored within a second compartment, and the bone graft material is stored within a third compartment.Enumerated Embodiment 115. The kit of any of the enumerated embodiments, wherein the solvent is contained within a container in the second compartment.Enumerated Embodiment 116. The kit of any of the enumerated embodiments, wherein the organic phosphate, the L-ascorbic acid or derivative thereof, and the organic calcium salt are dissolved in the solvent and provided as a rehydration buffer.Enumerated Embodiment 117. The kit of any of the enumerated embodiments, wherein the rehydration buffer is stored in a first compartment, and the bone graft material is stored in a second compartment.Enumerated Embodiment 118. The kit of any of the enumerated embodiments, wherein the rehydration buffer is contained within a container stored in the first compartment.Enumerated Embodiment 119. The kit of any of the enumerated embodiments, wherein the organic phosphate and the organic calcium salt are dissolved in the solvent and provided as a partially prepared rehydration buffer.Enumerated Embodiment 120. The kit of any of the enumerated embodiments, wherein the L-ascorbic acid or derivative thereof is stored within a first compartment, the partially prepared rehydration buffer is stored within a second compartment, and the bone graft material is stored within a third compartment.Enumerated Embodiment 121. The kit of any of the enumerated embodiments, wherein the partially prepared rehydration buffer is contained within a container stored in the second compartment.Enumerated Embodiment 122. The kit of any of the enumerated embodiments, wherein at least one of the compartments is configured to contain a volume of the rehydration buffer and the bone graft material in a period of time, thereby allowing rehydration of the bone graft material in the at least one compartment.

[0183] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0184] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and devices within the scope of these claims and their equivalents be covered thereby.

[0185] The scope of the claims should not be limited by the embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMSWhat is claimed is:

1. A method of preparing a bone graft, comprising:(1) preparing a formulation for rehydrating a bone graft material, comprising: (i) dissolving L-ascorbic acid or a derivative thereof in a solvent to generate an ascorbic acid solution; (ii) dissolving an organic phosphate in the ascorbic acid solution to generate an ascorbic acid-organic phosphate solution; (iii) dissolving an organic calcium salt in the ascorbic acid-organic phosphate solution to generate a ascorbic acid-organic phosphateorganic calcium salt solution, thereby forming the formulation;(2) providing the bone graft material; and(3) rehydrating the bone graft material in the formulation for a period of time, thereby generating the bone graft.

2. The method of claim 1 , wherein the L-ascorbic acid or derivative thereof comprises L- ascorbic acid or L-ascorbic acid 2-phosphate.

3. The method of any one of claims 1-2, wherein the organic phosphate comprises 0- glycerophosphate.

4. The method of any one of claims 1-3, wherein the organic calcium salt comprises calcium lactate.

5. The method of any one of claims 1-4, wherein the formulation comprises 1-25 mM of 0- glycerophosphate; 1-100 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and 1- 20 mM calcium lactate.

6. The method of any one of claims 1-5, wherein the bone graft material comprises one or more of: bone powder; crushed bone; bone chips; cortical bone; cancellous bone; bone shaft; and bone in the form of a plurality of cubes, matchsticks, wedges, or other geometric shape.

7. The method of any one of claims 1-6, wherein the bone graft material comprises a bone material in a dry state or frozen state.

8. The method of any one of claims 1-6, wherein the bone graft material comprises a bone material that has been freeze-dried.

9. The method of any one of claims 1-8, wherein the bone graft is an allograft.

10. The method of any one of claims 1-9, wherein the period of time is between about 20 minutes and about 30 minutes.

11. The method of any one of claims 1-9, wherein the bone graft has an increased expression in osteopontin (OPN) compared to a control bone graft.

12. The method of any one of claims 1-9, wherein the increase in OPN expression is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5-fold, at about 21 days after being rehydrated.

13. The method of any one of claims 1-9, wherein the bone graft has an increased expression in osteocalcin (OCN) compared to a control bone graft.

14. The method of claim 13, wherein the increase in OCN expression is at least about 2 -fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5 -fold, at about 21 days after being rehydrated.

15. The method of any one of claims 1-14, wherein the bone graft has an increased alkaline phosphatase (ALP) activity compared to a control bone graft.

16. The method of claim 15, wherein the increase in ALP activity when compared to the control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 7 days after being rehydrated.

17. The method of any one of claims 1-16, wherein the bone graft has an increased level of calcium deposition compared to a control.

18. The method of claim 17, wherein the increase in calcium deposition level when compared to the control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, at about 14 days after being rehydrated.

19. The method any one of claims 1-18, wherein the bone graft allows an increased level of seeding of a primary cell compared to a control.

20. The method of claim 19, wherein the increase in the level of seeding of the primary cell when compared to the control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.

21. The method of any one of claims 19-20, wherein the bone graft allows an increased viability of a primary cell compared to a control.

22. The method of claim 20, wherein the increase in viability of the primary cell when compared to the control bone graft is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.

23. The method of any one of claims 21-22, wherein the primary cell comprises a mesenchymal stem cell (MSC).

24. The method of any one of claims 11-23, wherein the control is a bone graft rehydrated with normal saline.

25. A formulation for rehydrating a bone graft material, comprising:1-25 mM of P-glycerophosphate;1-100 pg / mL L-ascorbic acid and / or L-ascorbic acid 2-phosphate; and1-20 mM calcium lactate,in a buffered aqueous solution.

26. The formulation of claim 25, wherein the rehydrated bone graft material comprises a bone graft that has an increased expression of osteopontin (OPN) compared to a control bone graft, wherein the increase in OPN expression is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5-fold, at about 21 days after being rehydrated.

27. The formulation of claim 25 or 26, wherein the rehydrated bone graft material comprises a bone graft that has an increased expression of osteocalcin (OCN) compared to a control bone graft, wherein the increase in OPN expression is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 4.5-fold, at about 21 days after being rehydrated.

28. The formulation of any one of claims 25-27, wherein the rehydrated bone graft material comprises a bone graft that has an increased alkaline phosphatase (ALP) activity compared to a control bone graft, wherein the increase in ALP activity is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at about 7 days after being rehydrated.

29. The formulation of any one of claims 25-28, wherein the rehydrated bone graft material comprises a bone graft that has an increased level of calcium deposition compared to acontrol wherein the increase in calcium deposition level is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, at about 14 days after being rehydrated.

30. The formulation of any one of claims 25-29, wherein the rehydrated bone graft material comprises a bone graft that allows an increased level of seeding of a primary cell compared to a control bone graft, wherein the increase in the level of seeding of the primary cell is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.

31. The formulation of any one of claims 25-30, wherein the rehydrated bone graft material comprises a bone graft that allows an increased viability of a primary cell compared to a control bone graft, wherein the increase in viability of the primary cell is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.

32. The formulation of any one of claims 25-31, wherein the control is a bone graft rehydrated with normal saline.

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