Bone graft material based on citric acid

Abstract

Graft materials made from citric acid-based materials are disclosed. The graft material has particular applicability in forming biodegradable scaffolds, and it generally includes a composition comprising (i) a citric acid component, (ii) a polyol, and (iii) a particulate inorganic material.

Description

[0001] background

[0002] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. provisional patent application serial number 63 / 432,420, filed on December 14, 2022, entitled “Citrate-Based Bone Grafting Materials.”

[0004] The entire contents of the aforementioned US provisional patent application are incorporated herein by reference. Technical Field

[0005] The present disclosure relates to citrate-based polymer-bioceramic compositions that have beneficial uses as synthetic implants for bone regeneration applications. Background Art

[0006] Currently, bone is the second most common transplant tissue, with over two million bone grafting procedures performed annually to repair bone defects. Although autologous grafts are considered the gold standard for bone defect repair, the use of autologous graft tissue has many disadvantages. [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, although autologous grafts are generally considered to have sufficient osteoconductivity, osteoinductivity, and bone formation, patients must endure a secondary surgery to dissect and extract the autologous bone. [See, Nalley, CC, Lieberman, IH, Morisue, H., Ferrara, LA, & Benzel, EC (2017). Bone Void Fillers. Benzel's Spine Surgery, Volume 2, Volume 2. https: / / doi.org / 10.1016 / b978-0-323-40030-5.00031-9] Up to 39% of patients experience serious complications during autologous bone extraction. Up to 25% of patients also experience persistent pain in the donor area. Furthermore, the quality of autologous bone is inconsistent, as it depends on the patient's age, sex, genetics, and overall health. [See, Nalley et al.]

[0007] Many alternatives to autologous grafts exist, including allograft tissue, decellularized extracellular matrix, and synthetic bone grafts. Allograft sources are limited, and there is evidence that allograft resorption is slow. Unwanted inflammatory responses to allograft transplantation have also been reported to impede bone regeneration. Finally, synthetic grafts composed of polymer-bioceramic composites have been developed to further enhance osteoconductivity, osteoinductivity, and osteogenesis compared to allografts and decellularized matrices.

[0008] Polymer components used 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, a mismatch with the biomechanical compliance of host tissue, and can lead 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]

[0009] To overcome the limitations of thermoplastic polymers, citric acid-based biodegradable elastomers have been developed as bioenergetic synthetic implants for bone regeneration. Citric acid is an inexpensive, nontoxic, and naturally occurring metabolic molecule that participates in bone anatomical and physiological processes by regulating the growth of apatite nanocrystals and cellular energy production. [See, Tran et al.] In addition, citric acid-based biomaterials contain bulk chemical pendant carboxylic acid and hydroxyl groups that participate in polymer chain formation and improve polymer-bioceramic interactions.

[0010] Effective synthetic grafts must be highly porous, with interconnected pore structures to facilitate nutrient transport, waste removal, and tissue penetration. [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.2020.01.007Currently, a variety of methods have been developed to produce highly porous scaffolds, including freeze-drying, gas foaming, electrospinning, phase separation, 3D printing, and leaching of porogens. [See, Abbasi et al.] Leaching of porogens is a well-established and popular method for creating porous structures because it is cost-effective, and many porogens (e.g., sodium chloride) are inert and do not interfere with the stability of the biomaterial. Leaching of porogens also allows for easy control of pore size by sieving the porogen to the target size range. Sodium chloride porogens can be easily removed by immersing the construct in deionized water. Through this leaching of porogens method, interconnected highly porous scaffolds can be prepared without compromising the chemical properties of the polymer-bioceramic composite. SUMMARY

[0011] According to the present disclosure, a highly advantageous implant material is made from a citric acid-based material. The disclosed implant material has particular applicability in forming biodegradable scaffolds.

[0012] In exemplary embodiments, the disclosed implant material comprises a composition comprising (i) a citric acid component, (ii) a polyol, and (iii) a particulate inorganic material. The citric acid component can include one or more of citric acid, a citrate, or an ester of citric. The polyol can include a diol, such as one or more of butanediol, hexanediol, octanediol, or polyethylene glycol. Other exemplary polyols contemplated according to the present disclosure include one or more of glycerol, beta-glycerol phosphate, or xylitol.

[0013] The disclosed citric acid and polyol can be reacted, for example, at a molar ratio of 1.0:1.0 to 1.0:1.5, respectively, to form a telechelic polymer, i.e., a functionalized low molecular weight polymer. In exemplary embodiments, the polyol can comprise 1-40 mol% of glycerol, based on the total polyol included in the composition. In other exemplary embodiments, the polyol can comprise 1-100 mol%, preferably 1-40 mol% of beta-glycerol phosphate, based on the total polyol included in the composition. Further, the polyol can comprise 1-100 mol%, preferably 1-40 mol% of xylitol, based on the total polyol included in the composition.

[0014] The disclosed particulate inorganic material can include one or more of hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate, calcium carbonate, carbonate apatite, and bioglass. The particulate inorganic material can also be coated with bioglass.

[0015] The particulate inorganic material may comprise a bioceramic present in an amount of 10 to 50 wt.-% of the composition.The particulate inorganic material may comprise micrometer-sized or nanometer-sized bioceramics, and / or rod-shaped bioceramics.

[0016] In exemplary embodiments, a stent can be formed at least in part from the disclosed composition. A stent can be or be defined as a cross-linked polymer network and is typically biodegradable. A stent can be 50-90% porous and is typically conformable. A stent can be configured and suitable for cutting in an operating room.

[0017] The disclosed scaffolds can be adapted to swell 500-1500% in liquid and can completely degrade in vivo within about 6-12 months or between. The scaffolds can be particulate, and the particulate scaffolds can be defined as a paste. In exemplary embodiments, one or more peptides can be bound to the scaffold.

[0018] Other features, functions, and benefits of the disclosed graft materials / scaffolds will become apparent from the following description, particularly when read in conjunction with the relevant experimental results described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] To assist those skilled in the art in making and using the disclosed materials / compositions, reference is made to the accompanying drawings, in which:

[0020] Figure 1 is an SEM image of a composite material of poly(octamethylene citrate) (POC) with 40 wt.-% hydroxyapatite (HA) and 92 wt.-% sodium chloride after salt leaching in deionized water;

[0021] Figure 2 is a graph showing the accelerated degradation of poly(octamethylene citrate) (POC) and poly(octamethylene xylitol citrate) (POXC3%) in phosphate buffered saline (PBS) at 57°C;

[0022] Figure 3is a graph showing the accelerated degradation of a composite of poly(octamethylene citrate) 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];

[0023] Figure 4 is a graph showing the pH of α-MEM after salting out POC containing 0-40% hydroxyapatite and bioglass for 72 hours;

[0024] Figure 5 is a graph showing the viability of MC3T3 mouse preosteoblasts after exposure to extracts of POC scaffolds composited with 5-40 wt.% hydroxyapatite compared to the viability of MC3T3 mouse preosteoblasts after exposure to extracts of POC scaffolds composited with 5-40 wt.% bioglass;

[0025] Figure 6 is a graph showing the comparison of the proliferation of MC3T3 mouse preosteoblasts on a POC scaffold composited with 5-40 wt.% hydroxyapatite and the proliferation of MC3T3 mouse preosteoblasts on a POC scaffold composited with 5-40 wt.% bioglass;

[0026] Figure 7 is a graph showing the pH of the extraction medium after salting out a POC scaffold containing 0-60 wt.-% bioglass for 72 hours;

[0027] Figure 8 is a graph showing the activity of MG-63 human preosteoblasts after exposure to extracts of POC scaffolds composited with 10-60 wt.% bioglass;

[0028] Figure 9 is a graph showing the proliferation of MG-63 human preosteoblasts on POC scaffolds composited with 10-60 wt.% bioglass;

[0029] Figure 10 is a graph showing the alkaline phosphatase activity of MG-63 cells seeded on a tissue control plate and a POC scaffold composited with 40 wt.% bioglass;

[0030] Figure 11AThis is a SEM image of the POC scaffold composited with 40 wt.% bioglass before culture in simulated body fluid;

[0031] Figure 11B is a SEM image of the POC scaffold composited with 40 wt.% bioglass after being cultured in simulated body fluid for seven (7) days;

[0032] Figure 12 is an X-ray diffraction (XRD) pattern showing a POC scaffold composited with 40 wt.% bioglass before being cultured in simulated body fluid;

[0033] Figure 13 is an XRD pattern showing the POC scaffold composited with 40 wt.% bioglass after being cultured in simulated body fluid for seven (7) days;

[0034] Figure 14 is a graph showing compressive peak stress of poly(octamethylene citrate) POC porous scaffolds compounded with 10, 20, 30, 40 and 50 wt-% of hydroxyapatite (HA); and

[0035] Figure 15 is a photograph of a poly(octamethylene citrate) POC porous scaffold composited with 60 wt.-% hydroxyapatite (HA); when salt-leached in water, the scaffold exhibited brittle behavior and broke upon handling. DETAILED DESCRIPTION

[0036] As described above, the presently disclosed highly advantageous graft material is made from a citric acid-based material. The disclosed graft material has particular applicability in forming biodegradable scaffolds. In an exemplary embodiment, the disclosed graft material comprises a composition comprising (i) a citric acid component, (ii) a polyol, and (iii) a particulate inorganic material. Additional features, functions, and benefits of the disclosed graft material / scaffold are described below, at least in part, with reference to experimental results.

[0037] a. Porosity

[0038] To evaluate the porosity of biodegradable scaffolds made according to the present disclosure, a citric acid-based polymer (e.g., poly(octamethylene citrate) (POC)) was composited with 40 wt.-% hydroxyapatite (HA) and 92 wt.-% sodium chloride. SEM images of the scaffold cross section are shown in FIG. Figure 1 As shown, it shows a porous interconnected structure.

[0039] The porosity of the POC-HA scaffold containing 92 wt.-% sodium chloride was also evaluated using mercury porosimetry (i.e., mercury porosimetry of a composite of poly(octamethylene citrate) (POC) with 40 wt.-% hydroxyapatite and 92 wt.-% sodium chloride after salt leaching). As shown in Table 1, the addition of 92 wt.-% sodium chloride resulted in a scaffold with a porosity of 86%.

[0040] Table 1

[0041]

[0042] b. Bone remodeling

[0043] During the bone remodeling process, bone tissue formation may take about 4 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 period, as osteoblasts infiltrate and adhere to the scaffold matrix, it is important that the degradation of the bone graft coincides with the formation of new bone. One benefit of the disclosed citric acid-based polymers is the ability to control the polymer degradation rate to meet the requirements of specific regenerative engineering applications. Since the degradation of citric acid-based polymers occurs primarily through the hydrolysis of polyesters, the polymer degradation rate can be fine-tuned by the hydrophobicity and hydrophilicity of the polyols used to react with citric acid (e.g., selecting aliphatic diol chain lengths and introducing hydrophilic polyols). For example, according to the present disclosure, xylitol can be advantageously used as a hydrophilic polyol.

[0044] like Figure 2 As shown, increasing the hydrophilicity of the biomaterial by incorporating xylitol into poly(octamethylene citrate) (POXC 1% and 3%) significantly increased the degradation rate of the resulting porous synthetic graft when compared to a citric acid-based graft (POC) synthesized without xylitol.

[0045] c. Bioceramics

[0046] According to the present disclosure, synthetic polymer-based implants can increase cell infiltration, proliferation, and differentiation by incorporating bone-stimulating and bioactive bioceramics. Exemplary bioceramics include inorganic materials containing calcium and phosphate, whose chemical properties are 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 provide a framework for the scaffold structure and have been shown to increase the compressive strength and hardness of the resulting composites. Many bioceramics are osteoconductive, which allows bone to grow on the surface of the implant. [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 are able to form hydroxyapatite mineralization on the implant surface. Additionally, many calcium phosphate bioceramics are resorbable, resulting in gradual degradation and absorption into the body, meaning surgical removal is not required.

[0047] Hydroxyapatite (HA) is a bioceramic that has been incorporated into a variety of commercial bone void fillers because it is present in the extracellular matrix of natural bone tissue. Although HA is relatively bioactive when compared to inert implants, its reactivity with existing bone is low. HA implants also exhibit relatively slow degradation, which results in less bone formation because the failure of HA-based implants comes from fracture 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]

[0048] Bioglass 45S5 is an alternative bioceramic according to the present disclosure and is widely used due to its many beneficial properties for bone grafts. Bioglass 45S5 is composed of 43-47% silicon dioxide, 22.5-26.5% calcium oxide, 5-7% phosphorus pentoxide, and 22.5-26.5% sodium oxide, and when compared to HA, bioglass 45S5 absorbs faster and increases the bioactivity of the synthetic graft. [Safety Data Sheet - mo-SCI corporation Mo-SCICorporation. (nd). Retrieved on May 13, 2022 https: / / mo-sci.com / wp-content / uploads / product-docs / biomaterials / GL0811-SDS.pdf ]

[0049] like Figure 3 As shown, the POC composite containing 40 wt.-% bioglass (POC B4) degraded significantly faster when compared to the POC composite containing 40 wt.-% HA (POC H4).

[0050] In addition to a faster absorption rate, bioglass is more bioactive than HA. When bioglass is hydrated in liquid, the alkali ions on the surface react with hydrogen ions (H + ) fast exchange. With H + The exchange of ions and subsequent increase in the solution's pH results in the formation of a hydroxycarbonate apatite (HCA) layer on the material's surface, mimicking the inorganic components of bone tissue. This HCA layer establishes a bond with the surrounding bone, stimulating its 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]

[0051] Utilizing the aforementioned pH phenomenon, bioglass was compounded into POC polymer at concentrations ranging from 0 to 40 wt.% to determine whether bioglass could buffer the acidity of the POC polymer. ISO 10993 cytotoxicity testing was performed on POC-bioglass composite scaffolds and compared to POC-HA composite scaffolds fabricated using similar concentrations.

[0052] like Figure 4As shown, bioglass concentrations above 5 wt.-% in POC scaffolds increased the extraction medium pH to create an alkaline environment (pH > 7.4), which has been shown to promote osteoblast differentiation and proliferation.

[0053] Due to the alkaline pH benefit of the bioglass containing POC composites, the viability of MC3T3 mouse preosteoblasts in response to 72 h α-MEM leaching extract exceeded 90% when 20, 30, and 40 wt.-% of bioglass were composited into POC polymers, as shown in Figure 5 shown.

[0054] Bioglass also exhibits enhanced osteogenic and osteostimulatory properties by increasing 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. April 2006, 27(11): 2414-25] MC3T3 mouse preosteoblasts were seeded onto POC composites containing bioglass and compared to hydroxyapatite composites. At 5 wt.-% composite loading, there was insufficient bioceramic to provide an osteoconductive surface for MC3T3 proliferation.

[0055] like Figure 6 As shown, POC composites containing 20, 30 and 40 wt.-% bioglass allowed significantly higher cell proliferation when compared to the HA control.

[0056] Although increasing bioglass concentrations resulted in enhanced preosteoblast proliferation, there may be an upper limit to the concentration of bioglass incorporated into POCs. ISO 10993 cytotoxicity and cell proliferation tests were repeated using MG-63 human preosteoblasts on POC scaffolds containing 10-60 wt.-% (in 10% increments) of bioglass. The human osteoblast cell line MG-63 provided insight into cell-material interactions. Bioglass concentrations exceeding 40 wt.-% resulted in extraction media pH values ​​exceeding 9, as Figure 7 shown.

[0057] POC scaffolds containing bioglass at concentrations higher than 40% resulted in decreased cell viability due to increased pH (see Figure 8 ) and decreased proliferation (see Figure 9 ).

[0058] Based on these results, the alkaline phosphatase (ALP) activity of POC scaffolds containing 40 wt.% bioglass was measured to determine whether this composition would increase ALP in MG63 cells compared to tissue culture plate controls. The results showed that the ALP of the scaffolds in vitro was significantly increased, as shown in Figure 2. Figure 10 shown.

[0059] In vitro apatite growth was measured on POC scaffolds containing 40 wt.% bioglass by SEM imaging and XRD analysis according to ISO 23317. The scaffolds were imaged and scanned before and after incubation in simulated body fluid at 37°C. The results showed the presence of apatite crystals after incubation, indicating that the scaffolds were bioactive (see Figure 11A 、 Figure 11B 、 Figure 12 and Figure 13 ).

[0060] d. Stent implantation

[0061] Prefabricated and readily available polymer-bioceramic composite implants can also be fine-tuned to impart ductility for ease of implantation and to match the mechanical properties of native tissue. As previously mentioned, calcium phosphate bioceramics can be added to composites to increase the material's compressive strength. While bioceramics alone can be brittle, when incorporated into synthetic polymers, they can improve the stiffness and strength of the composite. Typically, higher ceramic content is utilized to enhance the osteoconductivity of the resulting composite. However, due to the porous and interconnected structure of citric acid-based synthetic implants, increasing the bioceramic concentration can weaken the resulting construct.

[0062] like Figure 14 As shown in Figure 3, the compressive peak stress of the POC-HA composite material reaches its peak at 40 wt.-% HA concentration. Increasing the HA concentration to 50 wt.-% significantly reduces the compressive peak stress of the scaffold.

[0063] Furthermore, 60 wt.% hydroxyapatite scaffolds are brittle and non-ductile when made into highly porous structures and cannot be used as bone void fillers because surgeons should be able to handle and manipulate the material easily. Figure 15 As shown, the POC-HA scaffolds exhibited brittle behavior and broke immediately after salt immersion.

[0064] In an exemplary embodiment of the present disclosure, the stent is adapted to expand 500-1500% in liquid.In another exemplary embodiment, the stent completely degrades in vivo between 6-12 months.

[0065] As described herein, advantageous bone graft materials / compositions are provided that are particularly advantageous for use in forming scaffolds.While the present disclosure has provided exemplary embodiments thereof, the present disclosure is not limited by or to such exemplary embodiments.

Claims

1. A composition for use as a bone graft material, comprising: a. Citric acid component, b. polyols, and c. Granular inorganic materials.

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

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

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

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

6. The composition of claim 1, wherein the citric acid and the polyol are reacted in a molar ratio of 1.0:1.0 to 1.0:1.5, respectively, to form a telechelic polymer.

7. The composition according to claim 1, wherein the polyol comprises 1-100 mol%, preferably 1-40 mol% of glycerol based on the total polyols contained in the composition.

8. The composition according to claim 1, wherein the polyol comprises 1-100 mol%, preferably 1-40 mol% of beta-glycerophosphate based on the total polyols contained in the composition.

9. The composition according to claim 1, wherein the polyol comprises 1-100 mol%, preferably 1-40 mol% of xylitol based on the total polyols contained in the composition.

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

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

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

13. The composition of claim 1, wherein the particulate inorganic material comprises a micro-scale or nano-scale bioceramic.

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

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

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

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

18. The stent of claim 15, wherein the stent is conformable.

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

20. The stent of claim 15, wherein the stent is adapted to expand 500-1500% in a liquid. The stent according to claim 15 , wherein the stent completely degrades in vivo within 6-12 months.

22. The stent of claim 15, wherein the stent is particulate.

23. The stent of claim 22, wherein the particulate stent is in the form of a paste.

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

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