Development of Biodegradable Osteopromotive Citrate-Based Bone Putty

Abstract

The burden of bone fractures demands development of effective biomaterial solutions, while additional acute events such as noncompressible bleeding further motivate the search for multi-functional implants to avoid complications including osseous hemorrhage, infection, and nonunion. Bone wax has been widely used in orthopedic bleeding control due to its simplicity of use and conformation to irregular defects; however, its nondegradability results in impaired bone healing, risk of infection, and significant inflammatory responses. Herein, a class of intrinsically fluorescent, osteopromotive citrate-based polymer/hydroxyapatite (HA) composites (BPLP-Ser/HA) as a highly malleable press-fit putty is designed. BPLP-Ser/HA putty displays mechanics replicating early nonmineralized bone (initial moduli from ≈2-500 kPa), hydration induced mechanical strengthening in physiological conditions, tunable degradation rates (over 2 months), low swelling ratios (<10%), clotting and hemostatic sealing potential (resistant to blood pressure for >24 h) and significant adhesion to bone (≈350-550 kPa). Simultaneously, citrate's bioactive properties result in antimicrobial (≈100% and 55% inhibition of S. aureus and E. coli) and osteopromotive effects. Finally, BPLP-Ser/HA putty demonstrates in vivo regeneration in a critical-sized rat calvaria model equivalent to gold standard autograft. BPLP-Ser/HA putty represents a simple, off-the-shelf solution to the combined challenges of acute wound management and subsequent bone regeneration.

Keywords: bone putty; bone wax; citric acid; metabonegenesis; orthopedic biomaterials.

Figure 1:. Design of multi-functional BPLP-Ser/HA bone putty.

(a) Schematic demonstration showing the synthesis of BPLP-Ser, fabrication of citrate-based fluorescent composites (BPLP-Ser/HA), and press-fit into irregular cranial defects. (b) BPLP-Ser/65%HA putty (1) is highly malleable and can be molded in different shapes (2 and 3) while displaying excellent fluorescent emission (580nm) (4). (c) Emission spectra of BPLP-Ser/50%HA putty under excitation from 330–600m, demonstrating excitation dependent fluorescent emission.

Figure 2:. Mechanical properties of commercial Bone Wax and BPLP-Ser/HA putty.

(a) Peak stress and (b) Initial modulus of Bone Wax and BPLP-Ser putty with 40%, 45%, 50%, and 55% HA when compressed to 50% strain in dry and wet (24hrs in 1x PBS) conditions. (c) Peak stress and (d) Initial modulus of the wet-conditioned (24hrs in 1x PBS, DI water, or bovine whole blood) Bone Wax and BPLP-Ser putty with 45%, 50%, and 55% HA when compressed to 50% strain, n ≥5.

Figure 3:. Physical characterization of BPLP-Ser/HA putty.

(a) Mass loss of BPLP-Ser/HA putty over 8 weeks. (b) pH evolution of BPLP-Ser/HA putty degradation media within 5 weeks. (c) swelling ratios of bone wax and BPLP-Ser/HA putty after 24h immerion in 1xPBS (d) Stability of Bone Wax and BPLP-Ser/50%HA in 1x PBS over 12 weeks. (e) Density of Bone Wax, BPLP-Ser/50%HA putty and rat calverial bone. * p<0.05, n ≥6 except for (c) (n ≥3).

Figure 4:. Liquid sealing and adhesion tests of BPLP-Ser/HA putty.

(a) Hemolytic properties of Bone Wax and BPLP-Ser/HA putty. (b) Whole blood clotting time of Bone Wax and BPLP-Ser/HA putty. (c) Schematic demonstration and photographs of sealing experiments at 37°C; a two-meter tube was placed vertically against the wall, then blue dye was injected through the upper end of the tube to simulate physiological blood pressure. The lower end of the tube was plugged with Bone Wax or BPLP-Ser/HA putty after which the lower tube end was immersed in DI water, wetting the materials from the outside. (d) Liquid sealing time of Bone Wax and BPLP-Ser/HA putty at 37°C. (e) Adhesion strength of Bone Wax and BPLP-Ser/HA putty to trabecular bone after 24 hours in PBS. (f) In vitro adhesion of separated femoral condyle and femur via Bone Wax and BPLP-Ser/50%HA putty and illustration of proposed mechanism for the adhesive property of BPLP-Ser/HA putty. * p<0.05, n ≥3.

Figure 5:. In vitro antibacterial effects of BPLP-Ser/HA putty.

Optical density of E.coli (a) and S.aureus (b) incubation broth with and without the addition of Bone Wax and BPLP-Ser/HA putties. Inhibition ratios against E.coli (c) and S.aureus (d) of Bone Wax and BPLP-Ser/HA putty for 24h. * p<0.05, n ≥4.

Figure 6:. In vitro cytotoxicity, proliferation, and osteogenic capability of BPLP-Ser/HA putty.

(a) 24hr cytotoxicity of degradation products of PLGA and BPLP-Ser/HA putty at 1x, 5x, 10x, and 100x dilution factors against L929 cells. (b) Cell proliferation of L929 cells in the presence of degradation products at 10x, 50x, and 100x dilution factors. (c) ALP production of human mesenchymal stem cells (hMSCs) cultured in OG medium and OG medium containing degradation products of BPLP-Ser/HA putty at 50x and 100x dilution factors. (d) Calcium deposition of hMSCs cultured in OG medium and BPLP-Ser/50%HA and OG medium containing BPLP-Ser/55%HA degradation products at 50x and 100x dilution factors, scale bar=500μm. * p<0.05, # p>0.05, n≥3.

Figure 7:. In vivo efficacy of BPLP-Ser/50%HA putty in critical size cranial defects.

(a) Representative micro-computed tomography (μCT) scans 1 week and 4 weeks post-surgery (red circles indicate original 8mm defect diameter). (b) μCT scans of regenerated cranial bone at 1 week and 4 weeks with remaining BPLP-Ser/50%HA and autograft removed to highlight new bone growth. (c) Ratio of new bone volume to total volume at 1 week and 4 weeks (d) Remaining fluorescent signal of explanted materials at 1 and 4 weeks post-surgery. NC(−) = negative control. n=3.

Figure 8:. Histological results of cranial defect repair.

Representative H&E-stained tissue sections and corresponding micro-computed tomography (μCT) scans at 1 week and 4 weeks. (a-b) negative control (NC(−)), (c-d) autologous bone graft, (e-f) BPLP-Ser/50%HA putty, and (g-h) bone wax 1 week (a,c,e, and g) and 4 weeks (b,d,f, and h) postoperatively. Scale bar = 300 nm. Arrows indicate BPLP-Ser/50%HA putty. (i-j) Higher magnification images of the boxed region of (d) and (f), respectively. Scale bar=100μm. “F” indicates fibrous repair tissue, “A” represents the autologous bone graft, “N” denotes new bone formation, and “BX” represents bone wax. In images (i and j), green triangles indicate osteoblasts, black triangles represent blood vessels, and yellow triangles mark residual autologous bone.