Method of tuning mineralization of a bone matrix with protein modification

Abstract

A method of tuning mineralization of a bone matrix includes enhancing formation of carboxymethyl-lysine (CML) on the bone matrix. The demineralized bone matrix is positioned between a reservoir of a calcium solution and a reservoir of a phosphate solution. The demineralized bone matrix, the calcium solution and the phosphate solution is incubated for a period of time to enable calcium and phosphate ions to form mineral precursors and deposit on the bone matrix enhanced with CML.

Description

Attorney Docket No.: 105026-101 METHOD OF TUNING MINERALIZATION OF A BONE MATRIX WITH PROTEIN MODIFICATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 655,254,filed June 3, 2024, which is incorporated by reference as if disclosed herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with U.S. Government support under Grant Number AG075654awarded by the National Institutes of Health (NIH). The United States Government has certain rights in the invention. BACKGROUND

[0003] Medical implants formed at least partially from demineralized bone matrices (DBMs) area popular type of autograft for dental and orthopedic procedures due to their ease of use and high osteoinductivity (i.e., capacity to promote bone growth). Accordingly, much effort has been spent on improving the efficacy of select DBM products. The primary function of DBMs is to facilitate and accelerate bone growth within the cavity or void where it is placed in order to create a region of mineralized tissue that is of similar quality and integrity to the surrounding area. A vital aspect in the growth of mineralized tissue is the mineralization process itself, which can be categorized into two phases for bone. Primary mineralization is a short (e.g., 10 days) but rapid period of mineral precipitation that occurs within the collagenous matrix of bone, whereas secondary mineralization is a slow (e.g., 30 months) and gradual process that gives sufficient time for the previously deposited mineral to grow and mature. There are proteins and molecules in the bone matrix that can regulate this two-stage mineralization process, including type 1 collagen, which is the main component of DBMs.

[0004] Modification of these proteins in a bone matrix via glyoxation may act as a template formineral growth through the changing of lysine amino functional groups to carboxymethyl groups. This phenomenon may be attributed to the carboxyl groups formed on the lysine residues following glyoxation, a shared feature in other proteins and molecules known to regulate mineralization. The idea that proteins, such as collagen, may be modified to facilitate mineralization, a crucial process for bone growth, may lead to ways to improve the osteoconductivity of DBMs. 1 29252478.1Attorney Docket No.: 105026-101 SUMMARY

[0005] Aspects of the present disclosure are directed to a method of tuning mineralization of abone matrix. In some embodiments, the method includes enhancing formation of carboxymethyl- lysine (CML) on the bone matrix. In some embodiments, the bone matrix is positioned between a reservoir of a calcium solution and a reservoir of a phosphate solution. In some embodiments, the calcium solution is formed from 89 mM CaCl2 and the phosphate solution is formed from 55 mM NaH2PO4. In some embodiments, the bone matrix, the calcium solution and the phosphate solution are incubated for a period of time, to enable calcium and phosphate ions to form mineral precursors and precipitate onto the bone matrix enhanced with CML.

[0006] In some embodiments, the bone matrix is washed after the incubating. In someembodiments, the bone matrix includes collagen. In some embodiments, the pH of the calcium and phosphate solutions is adjusted to 7.4. In some embodiments, the formation of the CML includes synthesizing a glyoxating buffer, submerging the bone matrix in the glyoxating buffer for about 72 hours, and dialyzing the bone matrix in phosphate buffer saline after the about 72 hours. In some embodiments, the bone matrix, the calcium solution and the phosphate solution are incubated for about 7 days at 37ºC while agitating.

[0007] In some embodiments, the reservoir of calcium solution and the reservoir of phosphatesolution are separated from the bone matrix using at least one semi-permeable membrane. In some embodiments, the separation of the bone matrix from the calcium and phosphate solutions includes securing the at least one semi-permeable membrane to an opening of a vial containing the reservoir of calcium solution. In some embodiments, the vial is inverted and submerged into the reservoir of phosphate solution. In some embodiments, the at least one semi-permeable membrane at least partially surrounds the bone matrix.

[0008] Aspects of the present disclosure are directed to a method of making a medical implant. Insome embodiments, the medical implant comprises a body at least partially comprised of a bone matrix. In some embodiments, the method includes enhancing formation of carboxymethyl-lysine (CML) on the bone matrix of the body. In some embodiments, the method includes positioning the body between a reservoir of calcium solution and a reservoir of phosphate solution and incubating the body, the calcium solution and the phosphate solution for a period of time to enable calcium and phosphate ions to form mineral precursors that deposit on the bone matrix enhanced with CML. In some embodiments, the calcium solution is formed from 89 mM CaCl2and the phosphate solution is formed from 55 mM NaH2PO4. 2 29252478.1Attorney Docket No.: 105026-101

[0009] In some embodiments, the medical implant comprises an autograft. In some embodiments,the medical implant comprises an allograft. In some embodiments, the body is washed after incubation. In some embodiments, the bone matrix of the body includes collagen. In some embodiments, the formation of the CML includes synthesizing a glyoxating buffer, submerging the body in the glyoxating buffer for about 72 hours, and dialyzing the body in phosphate buffer saline after the about 72 hours. In some embodiments, the body, the calcium solution and the phosphate solution are incubated for about 7 days at 37ºC while agitating.

[0010] In some embodiments, the reservoir of calcium solution and the reservoir of phosphatesolution are separated from the body using at least one semi-permeable membrane. In some embodiments, the separation of the body from the calcium and phosphate solutions includes securing the at least one semi-permeable membrane to an opening of a vial containing the reservoir of calcium solution. In some embodiments, the vial is inverted and submerged into the reservoir of phosphate solution. In some embodiments, the at least one semi-permeable membrane at least partially surrounds the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings show embodiments of the disclosed subject matter for the purpose ofillustrating the invention. However, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

[0012] FIG 1 is a chart of a method of tuning mineralization of a bone matrix with proteinmodification according to some embodiments of the present disclosure;

[0013] FIG. 2 is a more detailed chart of a step of the method of tuning mineralization of a bonematrix with protein modification according to some embodiments of the present disclosure;

[0014] FIG.3 is a schematic depiction of an apparatus used in the method of tuning mineralizationof a bone matrix with protein modification according to some embodiments of the present disclosure;

[0015] FIG. 4A is a schematic depiction of a mineralization process using a bone matrix that wasnot modified according to some embodiments of the present disclosure;

[0016] FIG. 4B is a schematic depiction of a mineralization process using a bone matrix that wasmodified according to some embodiments of the present disclosure; 3 29252478.1Attorney Docket No.: 105026-101

[0017] FIG. 5 is a chart of a method of tuning mineralization of a medical implant with proteinmodification according to some embodiments of the present disclosure;

[0018] FIG. 6A is a graph of Raman spectroscopy results showing that following in vitromineralization, glyoxal-treated bone samples prepared according to some embodiments of the present disclosure exhibited a heightened mineral-to-matrix ratio relative to untreated bone samples;

[0019] FIG. 6B is a graph of Raman spectroscopy results showing that following in vitromineralization, glyoxal-treated bone samples prepared according to some embodiments of the present disclosure exhibited a heightened crystallinity parameter relative to untreated bone samples;

[0020] FIG. 6C is a graph of Raman spectroscopy results showing that following in vitromineralization, glyoxal-treated bone samples prepared according to some embodiments of the present disclosure also possessed a lower carbonate-to-phosphate ratio than untreated bone samples;

[0021] FIG. 7A is a graph showing that the treatment of bone samples with glyoxal according tosome embodiments of the present disclosure did not alter collagen maturity pre-mineralization and post-mineralization;

[0022] FIG. 7B is a graph showing that the treatment of bone samples with glyoxal according tosome embodiments of the present disclosure did not alter matrix surface rigidity pre-mineralization and post-mineralization; and

[0023] FIG. 7C is a graph showing the results of a biochemical assessment of untreated bonesamples (control) and glyoxal-treated bone samples prepared according to some embodiments of the present disclosure indicating that CML content in glyoxal-treated bone samples was marginally increased following mineralization. DETAILED DESCRIPTION

[0024] The following discussion relates to various embodiments of a method of tuning bonemineralization with protein modification. It will be understood that the herein described versions are examples that embody certain inventive concepts as detailed herein. To that end, other variations and modifications will be readily apparent to those of sufficient skill. The terms “about” 4 29252478.1Attorney Docket No.: 105026-101 or “approximately” as may be used herein may refer to a range of 80%-125% of the claimed or disclosed value.

[0025] Bone growth and bone remodeling is a critical process that occurs during the course ofhuman life and is responsible for increasing the size of bones, healing bones, and for overall bone maintenance. However, it has been shown that individuals suffering from type 2 diabetes and / or from obesity have an elevated risk of bone fracture due to skeletal fragility while bone mineral density measurements in the same individuals are often normal or even elevated. Bone mineral density (BMD) is a parameter that is closely tied to bone strength and often used to predict bone fracture risk for individuals with osteoporosis. Accordingly, the stark discrepancy between the continuum of BMD values and the increased propensity of bone fractures in individuals with type 2 diabetes and obesity indicate that these conditions impair bone quality rather than bone quantity. Furthermore, the underperformance of BMD in individuals with type 2 diabetes and obesity also suggest a defect in the bone matrix.

[0026] One common feature of obesity and type 2 diabetes is chronic, low-grade inflammation,which may be caused by visceral fat accumulation in obesity and by hyperglycemia in type 2 diabetes, respectively. The release of inflammatory factors is known to perturb the bone remodeling cycle, which leads to skeletal fragility in obese individuals as well as those with type 2 diabetes. The same chronic inflammation is further known to exacerbate oxidative stress, which may promote the formation of advanced glycation and glycoxidation end-products (AGEs / AGOEs). The AGEs and AGOEs are a class of compounds that have been shown to accumulate in skeletal tissue and play a role in skeletal fragility by directly modifying the extracellular matrix of bone. Moreover, there is increasing evidence that AGEs and AGOEs, such as carboxymethyl-lysine (CML) and pentosidine, are associated with a heightened risk of bone fracture. Accordingly, known glycation methods promote AGE formation including CML formation at a level that is much smaller relative to other AGEs, which subsequently obscures detection of this adduct in bone. This type of modification is also known to impact collagen maturity and is associated to increased embrittlement of the organic bone matrix.

[0027] Some embodiments of the present disclosure are directed to a method 100 of tuning themineralization of a bone matrix with protein modification as shown in FIG. 1. At 102, a bone matrix is provided. In some embodiments, the bone matrix may be a bone matrix sample or may comprise at least a portion of a medical implant, such as a bone organoid, a dental implant or an orthopedic implant. In some embodiments, the bone matrix may be a demineralized bone matrix, a partially demineralized bone matrix, or a mineralized bone matrix. In some embodiments, the 5 29252478.1Attorney Docket No.: 105026-101 bone matrix comprises collagen. In some embodiments, the bone matrix comprises one or more biomolecules comprising carboxylic acid moieties.

[0028] At 104, carboxylmethyl-lysine (CML) formation is enhanced on the surface of the bonematrix and / or in the bone matrix. In some embodiments, CML enhancement is performed in vitro. The CML enhancement, in some embodiments, begins with the synthesizing of a glyoxating buffer comprised of 0.2M disodium phosphate, 0.2M sodium chloride, 3g / L sodium cyanoborohydride, and 0.15M glyoxal. In some embodiments, the pH of the glyoxating buffer is adjusted to be greater than 7, for example from 8-8.5. The bone matrix is submerged in the glyoxating buffer and incubated at 37ºC for about 72 hours in order to enhance CML formation on the surface of the bone matrix and / or in the bone matrix. After incubation, the bone matrix is dialyzed with phosphate buffer saline (PBS) at a pH of about 7.4 and then stored in fresh PBS at about 4ºC until needed.

[0029] Still referring to FIG. 1, at 106 the bone matrix is positioned between a reservoir of acalcium solution and a reservoir of a phosphate solution. In some embodiments, the calcium solution is comprised of 89 mM of CaCl2 and the phosphate solution is comprised of 55 mM NaH2PO4. In some embodiments, the pH of both the calcium solution and the phosphate solution are adjusted to 7.4 by the addition of 0.1M NaOH. In some embodiments, the 0.1 M NaOH is added to each of the calcium and phosphate solutions in a dropwise manner until the pH of each solution is 7.4. At 108, the bone matrix, the calcium solution, and the phosphate solution are incubated for a period of time to enable calcium and phosphate ions to spontaneously combine and form mineral precursors that precipitate onto and / or within the bone matrix that is enhanced with CML.

[0030] An exemplary embodiment of step 106 of FIG. 1 will now be described in more detail withreference to FIG.2 and the apparatus 112 shown in FIG. 3. At 106a, the bone matrix 120 is at least partially surrounded by at least one semipermeable membrane 130. In some embodiments, the semi-permeable membrane is a polymeric membrane structured to enable certain molecules or ions to pass through by osmosis. In some embodiments, the semi-permeable membrane is a type of dialysis membrane. At 106b, a vial 140 is provided which defines an opening 142 across which the at least one semipermeable membrane 130 is secured. The reservoir of calcium solution 144 is contained in the vial 140 which, at 106c, is inverted and submerged into the reservoir of phosphate solution 154. In some embodiments, the reservoir of calcium solution 144 comprises a volume of 2mL and the reservoir of phosphate solution 154 comprises a volume of 100mL. In some embodiments, the bone matrix 120 and the at least one semi-permeable membrane 130 form 6 29252478.1Attorney Docket No.: 105026-101 an interface between the reservoir of calcium solution 144 and the reservoir of phosphate solution 154.

[0031] At 106d, in some embodiments, the apparatus 112 or setup is then incubated for about 7days at 37°C while agitating. In some embodiments, the agitation is performed with a shaking plate or a rocking plate in order to facilitate mineral growth on the surface of the bone matrix 120 or in the bone matrix 120. A schematic representation of unmodified bone matrix versus bone matrix treated with the glyoxating buffer according to some embodiments of the present disclosure is shown in FIGS. 4A and 4B, respectively. The bone matrix treated with the glyoxating buffer according to some embodiments of the present disclosure changes the balance of electric charges in the bone matrix. In some embodiments and as shown in FIG.4B, the bone matrix treated with the glyoxating buffer includes carboxylic acid moieties, which attract cations, such as calcium and magnesium, to the bone matrix. Accordingly, the glyoxation of collagen and other components of the extracellular matrix (ECM) of the bone bolsters mineralization by attracting mineral crystallites. As a result, there may be portions of collagen that have a greater affinity to the mineral and would subsequently be more insoluble than other unmodified proteins.

[0032] As shown in FIG. 5, some embodiments of the present disclosure are directed to a method200 of producing a medical implant at least partially comprised of a bone matrix. At 202, a body of the medical implant is provided that is at least partially comprised of a bone matrix. In some embodiments, the body may comprise at least a portion of a bone organoid, a dental implant or an orthopedic implant. In some embodiments, the bone matrix may be a demineralized bone matrix, a partially demineralized bone matrix, or a mineralized bone matrix. In some embodiments, the bone matrix comprises collagen.

[0033] At 204, CML formation on the bone matrix of the body is enhanced in a similar manner aspreviously described in relation to 104. Accordingly, in some embodiments, CML enhancement is performed in vitro. In some embodiments, the CML enhancement, in some embodiments, begins with the synthesizing of a glyoxating buffer comprised of 0.2M disodium phosphate, 0.2M sodium chloride, 3g / L sodium cyanoborohydride, and 0.15M glyoxal. In some embodiments, the pH of the glyoxating buffer is adjusted to be greater than 7, for example from 8-8.5. The body of the medical implant is submerged in the glyoxating buffer and incubated at 37ºC for about 72 hours in order to enhance CML formation on the bone matrix. In some embodiments, the body of the medical implant is dialyzed with phosphate buffer saline (PBS) at a pH of about 7.4 after incubation and then stored in fresh PBS at about 4ºC until needed. 7 29252478.1Attorney Docket No.: 105026-101

[0034] Still referring to FIG. 5, at 206 the body of the medical implant is positioned between areservoir of a calcium solution and a reservoir of a phosphate solution. In some embodiments, the calcium solution is comprised of 89 mM of CaCl2and the phosphate solution is comprised of 55 mM NaH2PO4. In some embodiments, the pH of both the calcium solution and the phosphate solution are adjusted to 7.4 by the addition of 0.1M NaOH. In some embodiments, the 0.1 M NaOH is added to each of the calcium and phosphate solutions in a dropwise manner until the pH of each solution is 7.4. In some embodiments, step 206 may be performed using a similar apparatus 112 as previously described in steps 106a-d and shown in FIGS.2 and 3. However, in such a case, the bone matrix 120 previously described and shown in the figures would be replaced with the body of the medical implant.

[0035] Accordingly, at 106a, the body of the medical implant is at least partially surrounded by atleast one semipermeable membrane 130. In some embodiments, the semi-permeable membrane is a polymeric membrane structured to enable certain molecules or ions to pass through by osmosis. In some embodiments, the semi-permeable membrane is a type of dialysis membrane. At 106b, a vial 140 is provided which defines an opening 142 across which the at least one semipermeable membrane 130 is secured. The reservoir of calcium solution 144 is contained in the vial 140 which, at 106c, is inverted and submerged into the reservoir of phosphate solution 154. In some embodiments, the reservoir of calcium solution 144 comprises a volume of 2mL and the reservoir of phosphate solution 154 comprises a volume of 100mL. In some embodiments, the body of the medical implant and the at least one semi-permeable membrane 130 form an interface between the reservoir of calcium solution 144 and the reservoir of phosphate solution 154.

[0036] At 208 (and 106d), in some embodiments, the apparatus 112 or setup is then incubated forabout 7 days at 37°C while agitating. In some embodiments, the agitation is performed with a shaking plate or a rocking plate in order to facilitate mineral growth. During the incubation calcium ions and phosphate ions are enabled to form mineral precursors and precipitate onto and / or in the bone matrix of the medical implant that is enhanced with CML.

[0037] The methodology previously described utilizes the reactive carbonyl species (RCS)glyoxal to induce preferential formation of CML that subsequently promotes mineral growth on and / or in the bone matrix. However, in some embodiments, one or more alternative RCS molecules, RCS-derived compounds, and metabolites from various biological pathways such as methylglyoxal, glyoxylic acid, glucuronic acid may be employed to induce CML formation in bone or prime DBMs for mineral growth. The findings suggest that the carboxyl group of CML facilitates mineral growth by attracting cations such as calcium and magnesium to the bone matrix. 8 29252478.1Attorney Docket No.: 105026-101 Therefore, in some embodiments, biomolecules with carboxylic acid moieties may be utilized to control and enhance mineralization through the same molecular mechanism as CML. For example, citrate is a tricarboxylic structure that has been shown to regulate mineral growth by either interacting with the organic matrix of bone or stabilizing the calcium-phosphate crystallite precursors.

[0038] Accordingly, an alternate method of tuning mineralization of bone matrix with proteinmodification may comprise treating the bone matrix, specifically a DBM, with citrate prior to surgical implantation in order to accelerate mineralization and subsequent osteointegration of the bone matrix. In some embodiments, amino acids with carboxyl groups, such as glutamic acid or aspartic acid, may be added to the bone matrix of DBMs to promote mineralization using the same mechanism as CML or citrate. In dome embodiments, the DBMs may be functionalized with negatively-charged amino acids to improve biocompatibility and osteointegration since these compounds will be readily metabolized by the cells in the bone matrix.

[0039] In some embodiments, peptides may also be integrated into the DBMs to bolster thenumber of negatively-charged amino acids in the bone matrix. In some embodiments, this may be facilitated by modifying the amino acid sequence of type-1 collagen to increase the number of glutamic or aspartic acid residues. In some embodiments, the DBMs may then be incubated with these synthesized collagen peptides to promote adsorption onto the bone matrix surface to functionalize the DBMs and augment their capacity to support mineral growth. Accordingly, reagents like methylglyoxal, glyoxylic acid, glucuronic acid, and citrate, along with tools such as engineered peptides, may be used to functionalize DBMs and improve osteointegration by enhancing mineralization. EXAMPLE I

[0040] In an exemplary study, the tibiae of nine donors (ages 23 – 89, Caucasian male / female)were sectioned into 10 x 10 x 0.5 mm blocks or samples (n = 69 total). Twenty-four of the samples were treated in the same manner previously described in order to promote the formation of carboxymethyl-lysine (CML) while remaining samples were kept untreated and left in phosphate buffered saline (PBS) prior to experimentation. In order to investigate how the disclosed glyoxation methodology influences the mineralization of the bone matrix, the bone samples were suspended inside a plastic vial containing 2 mL of 89 mM of CaCl2. The vial was then inverted and submerged in a larger container filled with 100 mL of 55 mM NaH2PO4. Both solutions were pH-adjusted to 7.4 with dropwise addition of 0.1 M HCL and NaOH. The apparatus was incubated 9 29252478.1Attorney Docket No.: 105026-101 at 37°C for 7 days on a rocking plate to facilitate mineral growth on the surface of the bone samples and throughout the bone samples.

[0041] After incubation, the bone samples were washed in dionized water and then analyzed withRaman spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, nanoindentation, and biochemistry methods were used to measure the composition and structural integrity of the bone matrix before and after mineralization. All the bone samples were analyzed with Raman and nanoindentation, whereas a subset of the bone samples were tested with FTIR (n = 4 / group) and biochemistry (n = 5 for untreated, n = 6 for glyoxal-treated). Metrics of interest which can be calculated from Raman spectroscopy include the mineral-to-matrix ratio, crystallinity parameter and the carbonate-to-phosphate ratio. The mineral-to-matrix ratio captures the relative degree of mineralization, whereas the crystallinity parameter and carbonate-to-phosphate ratio can reflect the degree of perfection and relative amount of carbonation of the mineral crystal, respectively. FTIR allows for the assessment of collagen quality through the determination of collagen maturity, or the ratio of mature to immature crosslinks in the collagen. Crosslink formation is required for the structural integrity of collagen, so alterations to this ratio can reflect an impairment in the collagen network. Nanoindentation was used to capture the stiffness or rigidity at the matrix surface

[0042] The amount of CML content in the bone matrix, for example in the collagen of thebone matrix, may be verified with multiple biochemistry methods, including enzyme-linked immunosorbent assay (ELISA), a commonly used biochemistry technique that has routinely been applied to mineralized tissue. For example, an ELISA can be used to measure CML content before and after mineralization.

[0043] The results of the analysis indicated that bone samples treated with glyoxating bufferaccording to some embodiments of the present disclosure exhibited a heightened mineral-to-matrix ratio (+18.6%) and crystallinity parameter (+2.6%) when compared to the untreated samples (FIGS. 6A-B). The carbonate-to-phosphate ratio was decreased in bone samples treated with glyoxating buffer according to some embodiments of the present disclosure, which was indicative of more phosphate ions in the newly formed mineral (FIG.6C). FTIR and nanoindentation testing of the bone samples showed that neither the collagen maturity nor matrix surface rigidity were altered in both the untreated bone samples and the bone samples treated with glyoxating buffer according to some embodiments of the present disclosure (FIGS.7A-B). ELISA results showed that CML content post-mineralization in both the soluble and insoluble fractions of extracted proteins were not significantly different between groups (FIG.7C). Spectroscopic assessments of 10 29252478.1Attorney Docket No.: 105026-101 the bone matrix composition of the bone samples revealed that the degree of mineralization (mineral-to-matrix ratio) was profoundly increased in CML enhanced bone samples after being subjected to the mineralization solutions according to some embodiments of the present disclosure.

[0044] Accordingly, the disclosed methodology applied to all proteins in the ECM was successfulin promoting mineral growth in bone samples. The observed trends for the crystallinity parameter and carbonate-to-phosphate ratio indicated that a matured mineral crystallite was the predominant species in bone samples treated with glyoxating buffer according to some embodiments of the present disclosure, suggesting that the glyoxation accelerated the mineral maturation process. In addition, the finding that collagen maturity was unchanged in both the untreated control bone samples and the bone samples treated with glyoxating buffer according to some embodiments of the present disclosure both before and after mineralization indicates that the formation of CML and the formation of mineral crystallites did not impair the integrity of the collagen network. The preservation of collagen maturity following glyoxation is a notable advantage of the disclosed methodology when compared to currently used glycation methods which do not increase CML specifically over other AGEs. The currently used glycation methods negatively impact collagen maturity and are associated with an increased embrittlement of the organic bone matrix. The disclosed method of glyoxal treatment to modify the bone matrix can preserve the integrity of the collagen network while heightening CML content and subsequently promoting mineral growth. Glyoxal-treated bone samples prepared according to some embodiments of the present disclosure also possessed an unaltered surface rigidity, which corroborates the notion that the glyoxation preserves the mechanical compliance of the bone matrix.

[0045] The observed increase in mineralization and mineral maturation highlight a promising wayto improve the osteoconductivity of DBMs by functionalizing the collagen of the bone matrix with CML without impairing the integrity of the organic bone matrix. Some embodiments of the method according to the present disclosure are superior than other forms of collagen modification, namely standard non-enzymatic glycation, which is non-specific and can disrupt the collagen integrity and ultimately weaken the rigidity of the bone matrix.

[0046] The study showed that CML content was marginally, but not significantly, increased inglyoxal-treated samples prepared according to some embodiments of the present disclosure after mineralization. The subtle differences in CML content shown suggest that some of the methods of the present disclosure related to bone protein extraction and isolation may be better optimized for mineralized bone tissue. Moreover, the observation that a greater amount of isolated CML content was from the soluble protein fraction suggest that some of the methods of the present 11 29252478.1Attorney Docket No.: 105026-101 disclosure may be improved to further enhance mineral formation in bone. There are several factors of some of the methods of the present disclosure that may be assayed and modulated to maximize the growth of mineral in the bone matrix, including the source of calcium and phosphate, pH range, incubation time, choice of crystallization technique, and the inclusion of additives to regulate crystal aggregation. Exploring these parameters may provide an ability to personalize the mineralization capacity of DBMs, allowing for DBMs to be optimized for the age, sex, and health of the patient. In addition, the inclusion of agents that regulate mineralization and crystal growth to DBMs functionalized with glyoxation introduces a new area of personalized medicine where DMBs can be optimized for the patient.

[0047] Moreover, it has been shown that the mineralization of bone, a process crucial for bonedevelopment, may be mediated and therefore accelerated through a modification of the collagen matrix. The glyoxation of collagen and other non-collagenous bone matrix proteins utilizes lysine, an amino acid residue in proteins, to form structurally small in size adducts, such as carboxymethyl-lysine (CML), in high quantity while preserving the structural and mechanical integrity of the ECM. A diffusion-based mineralization experiment described in the example revealed that glyoxation may allow for an 18.6% increase in mineral quantity and a 2.6% increase in mineral crystal perfection within 7 days of incubation. Accordingly, glyoxation may be a practical way to improve tissue mineralization beyond its native capacity. Furthermore, biochemical results suggest that the disclosed method may be tuned to the personal needs of a patient, again by modulating several factors of our developed methodology such as the inclusion of additives that can further regulate crystal growth. Taken together, these findings illustrate a method to functionalize and even personalize DBMs to bolster its ability to promote mineralization following implantation.

[0048] Although the invention has been described and illustrated with respect to exemplaryembodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions may be made therein and thereto, without parting from the spirit and scope of the present invention. 12 29252478.1

Claims

Attorney Docket No.: 105026-101 CLAIMS What is claimed is:

1. A method of tuning mineralization of a bone matrix, the method comprising:enhancing formation of carboxymethyl-lysine (CML) on the bone matrix; positioning the bone matrix between a reservoir of a calcium solution and a reservoir of a phosphate solution; and incubating the bone matrix, the calcium solution and the phosphate solution for a period of time, wherein calcium and phosphate ions are enabled to form mineral precursors and deposit on the bone matrix enhanced with CML.

2. The method of claim 1, further comprising washing the bone matrix after the incubating.

3. The method of claim 1, further comprising structuring the bone matrix to comprise collagen.

4. The method of claim 1, further comprising adjusting a pH of the calcium solution and the phosphatesolution to about 7.

4.

5. The method of claim 1, wherein the enhancing the formation of the CML further comprises:synthesizing a glyoxating buffer; submerging the bone matrix in the glyoxating buffer and incubating for about 72 hours; and dialyzing the bone matrix in phosphate buffer saline (PBS) after the about 72 hours of incubation.

6. The method of claim 1, wherein the period of time is about 7 days and the incubating occurs whileagitating at 37ºC. 13 29252478.1Attorney Docket No.: 105026-101 7. The method of claim 1, further comprising separating the reservoir of calcium solution and thereservoir of phosphate solution from the bone matrix using at least one semi-permeable membrane.

8. The method of claim 1, wherein the bone matrix comprises one of: (i) a demineralized bone matrix;(ii) a partially demineralized bone matrix; or (iii) a mineralized bone matrix.

9. The method of claim 7 further comprising:securing the at least one semi-permeable membrane to an opening of a vial containing the reservoir of calcium solution; and inverting and submerging the vial into the reservoir of phosphate solution, wherein the at least one semi-permeable membrane at least partially surrounds the bone matrix.

10. The method of claim 1, further comprising forming the calcium solution from 89 mM CaCl2.

11. The method of claim 1, further comprising forming the phosphate solution from 55 mM NaH2PO4.

12. A method of forming a medical implant, comprising:structuring a body to be least partially comprised of a bone matrix; enhancing formation of carboxymethyl-lysine (CML) on the bone matrix of the body; positioning the body between a reservoir of calcium solution and a reservoir of phosphate solution; and incubating the body, the calcium solution and the phosphate solution for a period of time, wherein calcium and phosphate ions are enabled to form mineral precursors and deposit on the bone matrix enhanced with CML. 14 29252478.1Attorney Docket No.: 105026-101 13. The method of claim 12, wherein the medical implant comprises one of an autograft and anallograft.

14. The method of claim 12, further comprising washing the body after the incubating.

15. The method of claim 12, further comprising structuring the bone matrix to comprise collagen.

16. The method of claim 12, wherein the enhancing formation of the CML further comprises:synthesizing a glyoxating buffer; submerging the body in the glyoxating buffer and incubating for about 72 hours; and dialyzing the body in phosphate buffer saline (PBS) after the about 72 hours of incubation.

17. The method of claim 12, wherein the period of time is about 7 days and the incubating occurs whileagitating at 37ºC.

18. The method of claim 12, further comprising separating the reservoir of calcium solution and thereservoir of phosphate solution from the body using at least one semi-permeable membrane.

19. The method of claim 18 further comprising:securing the at least one semi-permeable membrane to an opening of a vial containing the reservoir of calcium solution; and inverting and submerging the vial into the reservoir of phosphate solution, wherein the at least one semi-permeable membrane at least partially surrounds the body.

20. The method of claim 12, further comprising:15 29252478.1Attorney Docket No.: 105026-101 forming the calcium solution from 89 mM CaCl2; and forming the phosphate solution from 55 mM NaH2PO4. 16 29252478.1

Images