Citrate-based bone graft material

Abstract

Implant materials fabricated from citrate-based materials are disclosed, which have particular applicability in the formation of biodegradable scaffolds and generally comprise compositions including (i) a citrate component, (ii) a polyol, and (iii) a particulate inorganic material.

Description

[Technical Field]

[0001] background 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 432,420, filed December 14, 2022, and entitled "Citrate-Based Bone Graft Material," the entire contents of which are incorporated herein by reference.

[0002] 2.Technical Field The present disclosure refers to citrate-based polymer bioceramic compositions that have beneficial utility as synthetic grafts for bone regeneration applications. [Background technology]

[0003] 3.Background technology Currently, bone is the second most commonly transplanted tissue, with over 2 million bone graft procedures performed annually to repair bone defects. While autografts are considered the gold standard for bone defect repair, there are many drawbacks associated with the use of autograft tissue (see Wang, W., & Yeung, KWK (2017). Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioactive Materials, 2(4), 224–247. https: / / doi.org / 10.1016 / j.bioactmat.2017.05.007). For example, while autografts are generally considered to be sufficiently osteoconductive, osteoinductive, and to promote new bone formation, patients must endure secondary surgery for the cutting and harvesting of the autologous bone (see Nalley, CC, Lieberman, IH, Morisue, H., Ferrara, LA, & Benzel, EC (2017). Bone Void Fillers. Benzel's Spine Surgery, 2-Volume Set. https: / / doi.org / 10.1016 / b978-0-323-40030-5.00031-9). When autologous bone is harvested, major complications can occur in up to 39% of patients. Up to 25% of patients also experience persistent pain in the donor area. Furthermore, the quality of autologous bone is inconsistent, depending on the patient's age, sex, genetics, and overall health (see Nalley et al.).

[0004] Many alternatives to autografts exist, including allograft tissue, decellularized extracellular matrix, and synthetic bone grafts. Allograft sources are limited, and there is evidence that allograft resorption is slow. Similarly, undesirable inflammatory responses to allograft implantation have been reported to hinder bone regeneration. Finally, synthetic grafts composed of polymer-bioceramic composites have been developed to further improve osteoconductivity, osteoinduction, and bone formation compared to allografts and decellularized matrices.

[0005] Polymeric components for synthetic bone grafts have historically been limited to thermoplastic polymers, such as polylactic acid (PLA) or polyglycolic acid (PGA). However, these polymers exhibit slow degradation, limited cellular response, poor biomechanical compatibility with host tissue, and can contribute to chronic inflammation (see Tran, RT, Yang, J., & Ameer, GA (2015). Citrate-Based Biomaterials and Their Applications in Regenerative Engineering. Annual review of materials research, 45, 277–310. https: / / doi.org / 10.1146 / annurev-matsci-070214-0208153).

[0006] To overcome the limitations of thermoplastic polymers, citrate-based biodegradable elastomers have been developed as bioenergetic synthetic implants for bone regeneration. Citrate is an inexpensive, nontoxic, and naturally occurring metabolic molecule that contributes to bone anatomy and physiology by regulating apatite nanocrystal growth and cellular energy production (see Tran et al.). Furthermore, citrate-based biomaterials contain pendant carboxylic acid and hydroxyl groups in the bulk chemistry, which participate in polymer chain formation and improve polymer-bioceramic interactions.

[0007] Effective synthetic implants must be highly porous, with interconnected pore structures to facilitate nutrient transport, waste removal, and tissue infiltration (see Abbasi, N., Hamlet, S., Love, RM, & Nguyen, N.-T. (2020). Porous scaffolds for Bone Regeneration. Journal of Science: Advanced Materials and Devices, 5(1), 1–9. https: / / doi.org / 10.1016 / j.jsamd.2020.01.007). Currently, various methods have been developed to generate highly porous scaffolds, including freeze-drying, gas foaming, electrospinning, phase separation, 3D printing, and porogen leaching (see Abbasi et al.). Porogen leaching is a well-established and popular method for creating porous structures because it is cost-effective and many porogens, such as sodium chloride, are inert and do not interfere with the stability of biomaterials. Porogen leaching also allows for easy control of pore size by screening the porogen to a target size range. Sodium chloride porogen can be easily removed by submerging the structure in deionized water. Through this porogen leaching method, interconnected, highly porous scaffolds can be fabricated without compromising the chemistry of the polymer-bioceramic composite. Summary of the Invention [Problem to be solved by the invention]

[0008] In accordance with the present disclosure, highly advantageous implant materials have been fabricated from citrate-based materials. The disclosed implant materials have significant applicability in the formation of biodegradable scaffolds. [Means for solving the problem]

[0009] In an exemplary embodiment, the disclosed implant material comprises a composition including (i) a citrate component, (ii) a polyol, and (iii) a particulate inorganic material. The citrate component can include one or more of citric acid, citrate salts, or esters of citric acid. The polyol can include one or more of a diol, such as butanediol, hexanediol, octanediol, or polyethylene glycol. Other exemplary polyols contemplated according to the present disclosure include one or more of glycerol, β-glycerol phosphate, or xylitol.

[0010] The disclosed citrates and polyols can be reacted, for example, in a molar ratio of 1.0:1.0 to 1.0:1.5, respectively, to form telechelomers, i.e., functionalized low molecular weight polymers. In an exemplary embodiment, the polyol can include glycerol at 1 to 40 mol% of the total polyols contained in the composition. In another exemplary embodiment, the polyol can include β-glycerol phosphate at 1 to 100 mol%, preferably 1 to 40 mol%, of the total polyols contained in the composition. Furthermore, the polyol can include xylitol at 1 to 100 mol%, preferably 1 to 40 mol%, of the total polyols contained in the composition.

[0011] The disclosed particulate inorganic materials can include one or more of hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate, calcium carbonate, carbonated apatite, and bioglass. The particulate inorganic materials can also be coated with bioglass.

[0012] The particulate inorganic material may comprise bioceramics present in an amount of 10-50% by weight of the composition. The particulate inorganic material may comprise micro-sized or nano-sized bioceramics and / or rod-shaped bioceramics.

[0013] In exemplary embodiments, a scaffold can be at least partially formed from the disclosed compositions. The scaffold can be or define a crosslinked polymer network and is generally biodegradable. The scaffold can be 50-90% porous and is generally conformable. The scaffold can be configured and adapted to be cut in an operating room.

[0014] The disclosed scaffolds can be adapted to swell 500-1500% in liquid and can be completely degraded in vivo within about 6-12 months or thereafter. The scaffolds can be microparticulate, and the microparticulate scaffolds can define a paste. In exemplary embodiments, one or more peptides can be conjugated to the scaffold.

[0015] Additional properties, functions, and advantages of the disclosed implant materials / scaffolds will become apparent when read in conjunction with the following description, particularly the associated experimental results described herein. [Brief explanation of the drawings]

[0016] To assist those skilled in the art in making and using the disclosed materials / compositions, reference is made to the accompanying drawings. [Figure 1] Figure 1 shows an SEM image of poly(octamethylene citrate) (POC) composited with 40 wt % hydroxyapatite (HA) and 92 wt % sodium chloride after salt leaching in deionized water. [Figure 2] FIG. 2 is a plot showing the accelerated degradation of poly(octamethylene citrate) (POC) and poly(octamethylene xylitol citrate) (POXC 3%) in phosphate buffered saline (PBS) at 57° C. [Figure 3]Figure 3 shows the accelerated degradation of poly(octamethylene citrate) composites with 40 wt% hydroxyapatite (POC H4) and 40 wt% bioglass (POC B4) in phosphate-buffered saline (PBS) at 57°C. (See Ma, C. et al. (2018). In vitro cytocompatibility evaluation of poly(octamethylene citrate) monomers toward their use in orthopedic regenerative engineering. Bioactive Materials, 3(1), 19-27.) [Figure 4] FIG. 4 is a chart showing the pH of α-MEM after 72 hours of leaching of hydroxyapatite and bioglass with POCs of 0-40%. [Figure 5] Figure 5 is a chart showing MC3T3 mouse preosteoblast viability after exposure to extracts of POC scaffolds composited with 5–40 wt% hydroxyapatite compared to those of 5–40 wt% Bioglass. [Figure 6] FIG. 6 is a chart showing MC3T3 mouse preosteoblast proliferation on POC scaffolds composited with 5-40 wt% hydroxyapatite compared to those with 5-40 wt% bioglass. [Figure 7] FIG. 7 is a chart depicting the pH of the extraction medium after 72 hours of leaching of POC scaffolds containing 0-60 wt% bioglass. [Figure 8] FIG. 8 is a chart depicting MG-63 human preosteoblast cell viability after exposure to extracts of POC scaffolds composited with 10-60 wt% Bioglass. [Figure 9] FIG. 9 is a chart depicting MG-63 human preosteoblast proliferation on POC scaffolds composited with 10-60 wt% bioglass. [Figure 10]FIG. 10 is a chart depicting alkaline phosphatase activity of MG-63 cells seeded on tissue control plates versus POC scaffolds composited with 40 wt % Bioglass. [Figure 11A] FIG. 11A is an SEM image of a POC scaffold composited with 40 wt % bioglass before incubation in simulated body fluid. [Figure 11B] FIG. 11B is an SEM image of a POC scaffold composited with 40 wt % Bioglass after 7 days of incubation in simulated body fluid. [Figure 12] FIG. 12 is a plot representing the X-ray diffraction (XRD) of a POC scaffold composited with 40 wt % bioglass before incubation in simulated body fluid. [Figure 13] FIG. 13 is a plot showing the XRD of a POC scaffold composited with 40 wt % bioglass after 7 days of incubation in simulated body fluid. [Figure 14] FIG. 14 is a chart showing the compressive peak stress of porous scaffolds of poly(octamethylene citrate) POC composited with 10, 20, 30, 40, and 50 wt % hydroxyapatite (HA). [Figure 15] Figure 15 shows images of a porous scaffold of poly(octamethylene citrate) POC composited with 60 wt% hydroxyapatite (HA), and upon salt leaching in water, the scaffold exhibits brittle behavior and disintegrates immediately upon handling. DETAILED DESCRIPTION OF THE INVENTION

[0017] Description of Exemplary Embodiments As noted above, the presently disclosed highly advantageous implant materials are processed from citrate-based materials. The disclosed implant materials have significant applicability in the formation of biodegradable scaffolds. In exemplary embodiments, the disclosed implant materials comprise compositions including (i) a citrate component, (ii) a polyol, and (iii) a particulate inorganic material. Additional properties, functions, and advantages of the disclosed implant materials / scaffolds are described herein below, at least in part, with reference to experimental results. [Example]

[0018] a.Porosity (porosity) To evaluate the porosity of biodegradable scaffolds fabricated according to the present disclosure, a citrate-based polymer, e.g., poly(octamethylene citrate) (POC), was composited with 40 wt% hydroxyapatite (HA) and 92 wt% sodium chloride. Figure 1 shows an SEM image of the cross section of the scaffold, revealing a porous interconnected structure.

[0019] The porosity of the 92 wt% sodium chloride POC-HA scaffold was further evaluated using mercury porosimetry (mercury intrusion), i.e., the mercury porosimetry results after salt leaching of poly(octamethylene citrate) (POC) composited with 40 wt% hydroxyapatite and 92 wt% sodium chloride. As shown in Table 1, the blend of 92 wt% sodium chloride results in a scaffold with a porosity of 86%.

[0020] [Table 1]

[0021] b. Bone reconstruction During bone remodeling, bone tissue formation can take approximately four months (16 weeks) (Kenkre, JS; Bassett, JHD (2018). The bone remodeling cycle. Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, 55(3), 308-327). During this time, it is important that the degradation of the bone graft be matched with new bone formation as osteoblasts infiltrate and attach to the scaffold matrix. One advantage of the disclosed citrate-based polymers is the ability to control the degradation rate of the polymer to meet the needs of a particular tissue engineering application. Because degradation of citrate-based polymers occurs primarily through hydrolysis of the polyester, the degradation rate of the polymer can be fine-tuned by the hydrophobicity and hydrophilicity of the polyol used in the reaction with citric acid (e.g., by selecting the aliphatic diol chain length and incorporating a hydrophilic polyol). For example, xylitol can be advantageously utilized as a hydrophilic polyol in accordance with the present disclosure.

[0022] As shown in Figure 2, increasing the hydrophilicity of the biomaterial through the incorporation of xylitol into poly(octamethylene citrate) (POXC 1% and 3%) significantly increases the degradation rate of the resulting porous synthetic implant when compared to a citrate-based implant synthesized without xylitol (POC).

[0023] c. Bioceramics According to the present disclosure, synthetic polymer-based implants can be engineered to increase cell infiltration, proliferation, and differentiation through the incorporation of osteostimulatory and bioactive bioceramics. Exemplary bioceramics include calcium- and phosphate-containing inorganic materials that are chemically similar to the mineral phase of natural bone and can be used to reinforce orthopedic implants (Mala, R.; Ruby Celsia, A.S. (2018). Bioceramics in orthopaedics: A Review. Fundamental Biomaterials: Ceramics, 195-221). Bioceramics have been shown to provide structure to scaffold construction and increase the compressive strength and stiffness of the resulting composite. Many bioceramics are osteoconductive, allowing bone growth on the implant surface (Huang, Y.-Z., Xie, H.-Q.; Li, X. (2020). Scaffolds in Bone Tissue Engineering: Research Progress and Current Applications. Encyclopedia of Bone Biology, 204-215). Specifically, bioactive bioceramics allow for the formation of hydroxyapatite mineralization on the surface of the implant. Furthermore, many calcium phosphate bioceramics are resorbable, resulting in gradual degradation and absorption by the body, meaning that surgical removal is not required.

[0024] Hydroxyapatite (HA) is a bioceramic that has been incorporated into various commercially available bone void fillers because it is found in the extracellular matrix of native bone tissue. HA is relatively bioactive compared to inert implants, but its reactivity with existing bone is low. HA implants also exhibit relatively slow degradation, which results in less bone formation because failure of HA-based implants is due to the breakdown of the HA-bone interface (Devis Bellucci, Antonella Sola, Alexandre Anesi, Roberta Salvatori, Luigi Chiarini, Valeria Cannillo, Bioactive glass / hydroxyapatite composites: Mechanical properties and biological evaluation, Materials Science and Engineering: C, Vol. 51, 2015, pp. 196-205).

[0025] Bioglass 45S5 is an alternative bioceramic according to the present disclosure that is widely used for its many beneficial properties in bone grafts. Bioglass 45S5 is composed of 43-47% silica, 22.5-26.5% calcium oxide, 5-7% phosphorus pentoxide, and 22.5-26.5% sodium oxide. Compared to HA, Bioglass 45S5 is rapidly absorbed and increases the bioactivity of synthetic grafts (Safety Data Sheet - mo-SCI Corporation (n.d.). Retrieved May 13, 2022, from https: / / mo-sci.com / wp-content / uploads / product-docs / biomaterials / GL0811-SDS.pdf).

[0026] As shown in Figure 3, the POC composite containing 40 wt% bioglass (POC B4) degrades significantly faster when compared to the POC composite containing 40 wt% HA (POC H4).

[0027] In addition to a faster resorption rate, bioglass is bioactive when compared to HA. When bioglass hydrates in liquid, alkali ions on the surface transfer hydrogen ions (H) from the surrounding liquid. + ) is rapidly exchanged with H + As ions are exchanged, the pH of the solution subsequently increases, allowing the formation of a hydroxycarbonate apatite (HCA) layer on the material surface, which mimics the inorganic component of bone tissue. This HCA layer establishes local connections with the surrounding bone and stimulates bone growth. (Sayed Mahmood Rabiee, Neda Nazparvar, Misaq Azizian, Daryoosh Vashaee, Lobat Tayebi, Effect of ion substitution on properties of bioactive glasses: A review, Ceramics International, Vol. 41, No. 6, 2015, pp. 7241-7251)

[0028] Taking advantage of the pH phenomenon described above, bioglass was composited into POC polymer at concentrations of 0-40 wt% to determine whether bioglass could buffer the acidic nature of the POC polymer. ISO 10993 cytotoxicity testing was performed on the POC-bioglass composite scaffolds and compared with POC-HA composite scaffolds fabricated using similar concentrations.

[0029] As shown in Figure 4, bioglass concentrations above 5 wt% in the POC scaffolds increased the pH of the extraction medium, creating an alkaline environment (pH > 7.4) that has been demonstrated to promote osteoblast differentiation and proliferation.

[0030] Due to the alkaline pH advantage of the POC composite containing bioglass, the viability of MC3T3 mouse preosteoblasts against the 72-hour α-MEM leaching extract exceeded 90% when 20, 30, and 40 wt% bioglass was composited into the POC polymer, as shown in Figure 5.

[0031] Bioglass also exhibits enhanced osteogenic and osteostimulatory properties through increased alkaline phosphatase (ALP) production, DNA synthesis, and osteoblast proliferation (Hu, Yong-cheng; Zhong, Ji-pin. Osteostimulation of bioglass, Chinese Medical Journal: October 2009 - Vol. 122 - No. 19 - pp. 2386-2389; Chen QZ, Thompson ID, Boccaccini AR. 45S5 Bioglass-derived glass-ceramic scaffolds for bone tissue engineering. Biomaterials. 2006 Apr 27(11):2414-2425). MC3T3 mouse preosteoblasts were seeded on POC composites containing bioglass and compared with hydroxyapatite composites. At a composite loading of 5 wt%, there was insufficient bioceramic to provide an osteoconductive surface for MC3T3 cell proliferation.

[0032] As shown in Figure 6, POC composites containing 20, 30, and 40 wt% Bioglass allowed significantly higher cell proliferation when compared to the HA control.

[0033] While increasing bioglass concentration resulted in enhanced proliferation of preosteoblasts, there may be an upper limit to the bioglass concentration that can be compounded into POC. ISO 10993 cytotoxicity and cell proliferation studies were repeated using MG-63 human preosteoblasts on POC scaffolds containing 10 to 60 wt% bioglass (in 10% increments). The human osteoblast cell line MG-63 is useful in providing insight into cell-material interactions. As shown in Figure 7, bioglass concentrations greater than 40 wt% result in an extraction medium with a pH greater than 9.

[0034] Due to the increased pH value, POC scaffolds containing bioglass concentrations above 40% resulted in decreased cell viability (see Figure 8) and proliferation (see Figure 9).

[0035] Based on these results, alkaline phosphatase (ALP) activity was measured for POC scaffolds with 40 wt% bioglass to determine whether the composition increased ALP for MG63 cells compared to tissue culture plate controls. The results, as shown in Figure 10, demonstrated a significant increase in ALP for the scaffolds in vitro.

[0036] In vitro apatite growth was measured for POC scaffolds with 40 wt% Bioglass by SEM photography and XRD analysis according to ISO 23317. Scaffolds were photographed and examined before and after incubation in simulated body fluid at 37°C. Results showed the presence of apatite crystals after incubation, suggesting that the scaffolds were bioactive (see Figures 11A, 11B, 12, and 13).

[0037] d. Scaffold implantation Pre-fabricated and readily available polymer-bioceramic composite implants are also adaptable for ease of implantation and can be fine-tuned to match the mechanical properties of native tissue. As previously mentioned, calcium phosphate bioceramics can be added to composites to increase the compressive strength of the material. Bioceramics alone can be brittle, but when incorporated into a synthetic polymer, they can improve the stiffness and strength of the composite. Typically, higher ceramic contents are utilized to improve the osteoconductive capabilities of the resulting composite. However, due to the porous and interconnected structure of citrate-based synthetic implants, increasing the concentration of bioceramics can weaken the resulting structure.

[0038] As shown in Figure 14, the compressive peak stress of the POC-HA composite peaked at an HA concentration of 40 wt%. Increasing the HA concentration to 50 wt% significantly reduced the compressive peak stress of the scaffold.

[0039] Additionally, 60 wt% hydroxyapatite scaffolds cannot function as bone void fillers because they are brittle and non-compliant when fabricated into highly porous structures, and surgeons need to be able to easily handle and manipulate the material. As shown in Figure 15, POC-HA scaffolds exhibit brittle behavior and collapse immediately after salt leaching.

[0040] In exemplary embodiments of the present disclosure, the scaffold is adapted to swell 500-1500% in liquid, hi further exemplary embodiments, the scaffold completely degrades in vivo in 6-12 months.

[0041] As described herein, advantageous bone graft materials / compositions are provided that may be advantageously utilized, inter alia, in scaffold formation. While the present disclosure is provided with exemplary implementations thereof, the present disclosure is not limited by or to such exemplary implementations.

Claims

1. a. Citrate ingredient b. polyol, and c. Granular inorganic material 1. A composition for use as a bone graft material comprising:

2. 10. The composition of claim 1, wherein the citrate component comprises one or more of citric acid, a citrate salt, or an ester of citric acid.

3. The composition of claim 1 , wherein the polyol comprises a diol.

4. 4. The composition of claim 3, wherein the diol comprises one or more of butanediol, hexanediol, octanediol, or polyethylene glycol.

5. 10. The composition of claim 1, wherein the polyol comprises one or more of glycerol, β-glycerol phosphate, or xylitol.

6. 10. The composition of claim 1, wherein the citrate and polyol are reacted in a molar ratio of 1.0:1.0 to 1.0:1.5, respectively, to form a telechelomer.

7. 2. The composition of claim 1, wherein the polyol comprises glycerol in an amount of 1 to 100 mol %, preferably 1 to 40 mol %, of the total polyol contained in the composition.

8. 2. The composition of claim 1, wherein the polyol comprises β-glycerol phosphate in an amount of 1 to 100 mol %, preferably 1 to 40 mol %, of the total polyol contained in the composition.

9. 2. The composition of claim 1, wherein the polyol comprises xylitol in an amount of 1 to 100 mol %, preferably 1 to 40 mol %, of the total polyols contained in the composition.

10. 10. The composition of claim 1, wherein the particulate inorganic material comprises one or more of hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate, calcium carbonate, carbonate apatite, and bioglass.

11. The composition of claim 1 , wherein the particulate inorganic material is coated with bioglass.

12. 10. The composition of claim 1, wherein the particulate inorganic material comprises a bioceramic present in an amount of 10 to 50% by weight of the composition.

13. The composition of claim 1 , wherein the particulate inorganic material comprises bioceramics that are microsized or nanosized.

14. The composition of claim 1 , wherein the particulate inorganic material comprises a bioceramic that is rod-shaped.

15. 10. A scaffold formed at least in part from the composition of claim 1, wherein the scaffold is a crosslinked polymer network.

16. 16. The scaffold of claim 15, wherein the scaffold is biodegradable.

17. 16. The scaffold of claim 15, wherein the scaffold is 50-90% porous.

18. 16. The scaffold of claim 15, wherein the scaffold is malleable.

19. 16. The scaffold of claim 15, wherein the scaffold is configured and adapted to be cut in an operating room.

20. 16. The scaffold of claim 15, wherein the scaffold is adapted to expand in a liquid by 500 to 1500%.

21. 16. The scaffold of claim 15, wherein the scaffold completely degrades in vivo between 6 and 12 months.

22. 16. The scaffold of claim 15, wherein the scaffold is particulate.

23. 23. The scaffold of claim 22, wherein the particulate scaffold is a paste.

24. 16. The scaffold of claim 15, further comprising a peptide conjugated to the scaffold.

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