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. 2023 Apr 13;15(4):1233.
doi: 10.3390/pharmaceutics15041233.

In Vitro and In Vivo Evaluation of a Bio-Inspired Adhesive for Bone Fixation

Matthias Schlund  1   2   3 , Julien Dartus  1 , Sarah Defrançois  4 , Joël Ferri  1   2 , Jérôme Delattre  5 , Nicolas Blanchemain  1 , Patrice Woisel  4 , Joël Lyskawa  4 , Feng Chai  1
Affiliations

In Vitro and In Vivo Evaluation of a Bio-Inspired Adhesive for Bone Fixation

Matthias Schlund et al. Pharmaceutics. .

Abstract

Compared to metallic hardware, an effective bone adhesive can revolutionize the treatment of clinically challenging situations such as comminuted, articular, and pediatric fractures. The present study aims to develop such a bio-inspired bone adhesive, based upon a modified mineral-organic adhesive with tetracalcium phosphate (TTCP) and phosphoserine (OPS) by incorporating nanoparticles of polydopamine (nPDA). The optimal formulation, which was screened using in vitro instrumental tensile adhesion tests, was found to be 50%molTTCP/50%molOPS-2%wtnPDA with a liquid-to-powder ratio of 0.21 mL/g. This adhesive has a substantially stronger adhesive strength (1.0-1.6 MPa) to bovine cortical bone than the adhesive without nPDA (0.5-0.6 MPa). To simulate a clinical scenario of autograft fixation under low mechanical load, we presented the first in vivo model: a rat fibula glued to the tibia, on which the TTCP/OPS-nPDA adhesive (n = 7) was shown to be effective in stabilizing the graft without displacement (a clinical success rate of 86% and 71% at 5 and 12 weeks, respectively) compared to a sham control (0%). Significant coverage of newly formed bone was particularly observed on the surface of the adhesive, thanks to the osteoinductive property of nPDA. To conclude, the TTCP/OPS-nPDA adhesive fulfilled many clinical requirements for the bone fixation, and potentially could be functionalized via nPDA to offer more biological activities, e.g., anti-infection after antibiotic loading.

Keywords: animal model; bioresorbable adhesive; bone adhesive; bone fixation; bone glue.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Characterization of nPDA with diffusion light scattering showing an average diameter of 190 nm and a polydispersity of 0.23 (A), and with scanning electron microscopy showing spherical particles uniformly distributed (B).
Figure 2
Figure 2
Instrumental tensile adhesion test using titanium cylinder samples evaluating adhesion after 1 h immersion in a 37 °C PBS bath in order to optimize the formulation of the bone glue by varying: (A) the molar percentage (%mol) of OPS in the TTCP/OPS mixture, (B) the liquid-to-powder ratio (mL/g), and (C) the nPDA content (%wt). A statistically significant difference was shown: in (A) by * (p < 0.05) compared to the group “50%molOPS”, in (B) by “•” (p = 0.016) compared to the group “liquid-to-powder ratio 0.21 mL/g”, and in (C) by “○” (p = 0.013) between the marked groups.
Figure 3
Figure 3
Instrumental tensile adhesion tests using titanium cylinder samples comparing TTCP/OPS glue versus TTCP/OPS-nPDA glue: (A) maximum traction stress after 1 h and 24 h immersions in a 37 °C PBS bath. * stands for a statistically significant difference (p = 0.0029) between two marked groups; surfaces of titanium samples glued with TTCP/OPS (B) or with TTCP/OPS-nPDA (C) after rupture, mostly showing a cohesive failure for both.
Figure 4
Figure 4
Ex vivo tensile adhesion test comparing TTCP/OPS glue versus TTCP/OPS-nPDA glue after 1 h and 24 h immersions in a 37 °C PBS bath: (A) instrumental tensile adhesion test using cuboid bovine cortical bone samples. * stands for a statistically significant difference (p = 0.00093) between two marked groups; surfaces of bone samples glued with TTCP/OPS; (B) with TTCP/OPS-nPDA; (C) after rupture, mostly showing a cohesive failure for both; (D) experimental setup of manual tensile adhesion test on the glued rat tibia/fibula: a fibula segment sample (autograft) was glued to the tibia by a thin layer of the prepared glue, between which a Vicryl® 4/0 suture thread was deposed perpendicular to the anatomical axis of the tibia and glued together. An evaluation of the adhesive strength was performed by traction with progressively increasing standard weights attached to the Vicryl® thread (minimum N = 7 for each experiment) until rupture of adhesion. The sum of the applied weights was thus noted as the traction force of failure; (E) maximum traction stress of manual tensile adhesion test of glued fibular/tibial bone. * stands for a statistically significant difference (p = 0.029) between two marked groups.
Figure 5
Figure 5
Characterization of the bone adhesive TTCP/OPS versus TTCP/OPS-nPDA: (A) compression test using cylinder glue samples after 24 h immersion in a 37 °C PBS bath (p = 0.083); (B) measuring the setting time by Gillmore test on disk glue samples.
Figure 6
Figure 6
In vitro biological reactivity tests. Biomineralization test by soaking glue disks in simulated body fluid (SBF), with SEM images (A) showing: full mineralization layer on the TTCP/OPS-PDA surface after 4 days, with only a few small patches on TTCP/OPS disks (white arrow); after 7 days, a more abundant layer of mineralization on TTCP/OPS-nPDA disks and only a fine layer of apatite on TTCP/OPS disks; (B) grazing incidence wide-angle X-ray scattering (GIWAXS) analysis of glue disks after 0-, 4-, and 7-day immersion in SBF showing the hydroxyapatite phase in the mineralized layer (red bar for indicating the characteristic peaks of TTCP and green bar for indicating the characteristic peak of hydroxyapatite); (C) cytotoxicity test (extraction method) carried out on TTCP/OPS and TTCP/OPS-nPDA glue disks with MC3T3-E1 pre-osteoblast cells by alamarBlueTM assay (p = 0.0009). Data were normalized against the 100% negative control (cells cultured in complete culture medium without adhesive extract) to obtain a percentage of relative cell vitality representing the “survival rate”.
Figure 7
Figure 7
Photographs of in vivo rat model with glued fibula/tibia (autograft fixation) used for evaluating the fixation of the fibular graft with TTCP/OPS-nPDA glue: (A) intraoperative photograph showing a fibular autograft glued to a rat tibia with a position indicator (screw); (B) photograph of a fibular autograft glued to a rat tibia after 12 weeks, which remained stable and was without secondary displacement.
Figure 8
Figure 8
Evaluation in vivo of glued rat fibula/tibia with TTCP/OPS-nPDA glue at 5 or 12 weeks: microCT images of 2D sagittal slices (A,B) and 3D volume reconstruction (C,D) of a stably fixed fibular graft on a rat tibia showing a continuity of the interface between the graft, the adhesive, and the tibia (white arrows), with some rare fissures in the adhesive indicated by yellow arrows (C,D); non-decalcified histological sections (E,F) of glued fibular autograft to tibia with clinical success stained with hematoxylin and eosin (×10) showing the absence of inflammation and the coverage of newly formed bone over the TTCP/OPS-nPDA glue at 12 weeks ((F), black arrow).
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