Titania nanotube-based protein delivery system to inhibit cranial bone regeneration in Crouzon model of craniosynostosis

Abstract

Background: Craniosynostosis is a developmental disorder characterized by the premature fusion of skull sutures, necessitating repetitive, high-risk neurosurgical interventions throughout infancy. This study used protein-releasing Titania nanotubular implant (TNT/Ti) loaded with glypican 3 (GPC3) in the cranial critical-sized defects (CSDs) in Crouzon murine model (Fgfr2^c342y/+ knock-in mutation) to address a key challenge of delaying post-operative bone regeneration in craniosynostosis.

Materials and methods: A 3 mm wide circular CSD was created in two murine models of Crouzon syndrome: (i) surgical control (CSDs without TNT/Ti or any protein, n=6) and (ii) experimental groups with TNT/Ti loaded with GPC3, further subdivided into the presence or absence of chitosan coating (on nanotubes) (n=12 in each group). The bone volume percentage in CSDs was assessed 90 days post-implantation using micro-computed tomography (micro-CT) and histological analysis.

Results: Nano-implants retrieved after 90 days post-operatively depicted well-adhered, hexagonally arranged, and densely packed nanotubes with average diameter of 120±10 nm. The nanotubular architecture was generally well-preserved. Compared with the control bone volume percentage data (without GPC3), GPC3-loaded TNT/Ti without chitosan coating displayed a significantly lower volume percent in cranial CSDs (P<0.001). Histological assessment showed relatively less bone regeneration (healing) in GPC3-loaded CSDs than control CSDs.

Conclusion: The finding of inhibition of cranial bone regeneration by GPC3-loaded TNT/Ti in vivo is an important advance in the novel field of minimally-invasive craniosynostosis therapy and holds the prospect of altering the whole paradigm of treatment for affected children. Future animal studies on a larger sample are indicated to refine the dosage and duration of drug delivery across different ages and both sexes with the view to undertake human clinical trials.

Keywords: craniosynostosis; glypican; murine; protein delivery; titania nanotube.

Figure 1

An electrochemically anodized Titania nanotubular implant (TNT/Ti) for sustained delivery of a bone antagonizing protein (glypican 3, GPC3) and a control protein (bone serum albumin; BSA) in surgically created critical-sized defect (CSD) as part of craniosynostosis therapy. Abbreviations: Ti, titanium; TNT, Titania nanotube.

Figure 2

Flowchart showing the experimental layout (study design) for different TNT/Ti treatment groups at two different stages: (A) first stage of implant testing in Wildtype mice (proof of principle) to refine the delivery method, and (B) second stage of translating the refined drug delivery system into the Crouzon murine model (study aim). Abbreviations: TNT/Ti, Titania nanotubular implant; BSA, bovine serum albumin; GPC3, glypican 3 protein.

Figure 3

SEM images showing the surface topography of representative TNT/Ti delivery platforms after day 90 of the in vivo study showing (A) the whole fabricated implant disc with TNT structures, and (B–C) only the TNT structures (top view) at different magnifications. Abbreviations: TNT/Ti, Titania nanotubular implant; TNT, Titania nanotube.

Figure 4

Micro-CT images and bone volume percentage data for newly formed bone in wildtype mice treated with non-functional BSA protein (either with chitosan coating [TNT-BSA-CH] or no coating [TNT-BSA]), and GPC3 protein (TNT-GPC3) at day 90 post-operatively; (A) Top view of the 3D micro-CT reconstructions showing surgically created critical-sized defects (CSDs), (B) Sagittal sections through the middle of the CSDs , and (C) bone volume percentage within a 3 mm wide cylindrical volume of interest around the CSDs. * and ** indicate significant differences at P<0.01 between the TNT-GPC3 group and the TNT-BSA-CH group, and the TNT-GPC3 group and the TNT-BSA group, respectively. There was no significant difference in bone volume percentage between the TNT-BSA-CH and TNT-BSA groups. Effect size: TNT-GPC3 vs TNT-BSA-CH=1.13 (large); TNT-GPC3 vs TNT-BSA=1.20 (large). Abbreviations: BSA, bovine serum albumin; BV, bone volume; CH, chitosan; GPC3, glypican 3 protein; Ti, titanium; TNT, Titania nanotube; TV, total volume; WT, wildtype.

Figure 5

Histological (H&E) images showing bone regeneration in the critical-sized defects (CSDs) of the three wildtype groups at day 90 post-operatively at (A) a low magnification (×4) and (B) a high magnification (×20). Images A and B were prepared from different slices of the same CSD (for each group) that displayed the features most clearly at each magnification. Black arrows mark the new bone edge.Notes:  WT-TNT-BSA-CH, wildtype mice in which Titania nanotubes were loaded with bovine serum albumin and also coated with chitosan; WT-TNT-BSA, Wildtype mice in which Titania nanotubes were loaded with bovine serum albumin but not coated with chitosan; WT-TNT-GPC3, Wildtype mice in which Titania nanotubes were loaded with glypican 3 protein. Abbreviations: nb, new bone; ft, fibrous tissue; TNT, Titania nanotube (delaminated); WT, wildtype; BSA, bovine serum albumin; TNT, Titania nanotube; CH, chitosan; GPC3, glypican 3 protein.

Figure 6

Micro-CT images and bone volume percentage data for newly formed bone in three groups of the Crouzon murine model (with Fgfr2^c342y/+ knock-in mutation) including the surgical control (craniectomy only) and the two experimental groups, including Titania nanotubes loaded with glypican 3 protein and then either coated with chitosan (TNT-GPC3-CH) or not (TNT-GPC3), at day 90 post-operatively; (A) Top view of the 3D reconstructed skulls from micro-CT scans showing surgically created critical-sized defects (CSDs), (B) Sagittal sections through the middle of the CDSs, and (C) bone volume percentage within a 3 mm wide cylindrical volume of interest around the CSDs. *Significant difference between the TNT-GPC3 group and the control group at P<0.001, and **Significant difference between the TNT-GPC3 group and the TNT-GPC3-CH group at P<0.05. There was no significant difference in bone volume percentage between the control and TNT-GPC3-CH groups. Effect size: TNT-GPC3 vs control=0.86 (large); TNT-GPC3 vs TNT-GPC3-CH=0.57 (large). CZ represents Crouzon murine model in image A. Abbreviations: BV, bone volume; CH, chitosan; GPC3, glypican 3 protein; TNT, Titania nanotube; TV, total volume.

Figure 7

Histological images showing bone regeneration in the critical-sized defects (CSDs) of three groups of Crouzon murine model (with Fgfr2^c342y/+ knock-in mutation) at day 90 post-operatively for (A) H&E staining at a low magnification (×4), (B) H&E staining at a high magnification (×20), and (C) Movat Pentachrome (MOV) staining at a low magnification (×4). Images A and B were prepared from different slices of the same CSD (for each group) that displayed the features most clearly at each magnification. Black arrows mark the new bone in the CSDs. Notes: CZ-Control, Crouzon murine model without Titania nanoimplants or proteins (i.e. surgical control); CZ-TNT-GPC3-CH, Crouzon murine model with Titania nanotubes loaded with glypican 3 protein and also coated with chitosan; CZ-TNT-GPC3, Crouzon murine model with Titania nanotubes loaded with glypican 3 protein (but no chitosan coating). Abbreviations: nb, new bone; ft, fibrous tissue; CH, chitosan; GPC3, glypican 3 protein; TNT, Titania nanotube.