Abstract
Bone loss resulting from infections, periodontal diseases, trauma, or genetic abnormalities present significant challenges for oral and cranio-maxillofacial surgeons, especially when combined with tooth loss. Restoring the bone structure and dental function often requires the use of dental implants, necessitating the restoration of adequate bony support. In cases of large bony defects, bone grafts and substitutes in the form of particulates are not enough and these require bone ‘block’ grafts with physical robustness and osteo-regenerative properties to facilitate bone healing and regeneration. Bovine bone is widely accepted as a suitable source for xenograft material; however, in order to prepare it for human use it must undergo processing and sterilisation which can significantly reduce the mechanical strength and biological compatibility of the grafts. This study aimed to develop a bovine bone block graft by assessing the effects of two different processing techniques, sintering or supercritical fluid extraction (SCF) on bovine bone blocks. Material characterisation, physicochemical properties, mechanical strength, biological safety and biocompatibility both in vitro and in vivo were tested.
Bone blocks were prepared from the condyles of bovine femurs purchased locally from a butchery. The sintering study was conducted on bone blocks divided into four groups; Group 1: Raw bone, Group 2: Boil (6 h), Group 3: boil (6 h) followed by 550°C sintering and Group 4: boil (6 h) followed by 1100°C sintering. The SCF processing study was conducted on bone blocks divided into six groups; Group 1: Raw bone, Group 2: SCF-CO2, Groups 3: SCF-CO2-H2O2, Group 4: SCF-CO2-H2O2 + Pepsin and Group 5: SCF-CO2-H2O2 + Pepsin + TMSB10.
Initially material characterisation was undertaken which included testing for remaining organic contents (thermogravimetric analysis, TGA), crystallinity using X-ray diffraction (XRD), surface topography was observed using scanning electron microscopy (SEM), and chemical composition was assessed using Fourier-transform infrared spectroscopy (FTIR). Mechanical testing was conducted using compression testing, clinically relevant handling was assessed by conducting a bench-top drill test by placing a bone screw through the block and fixating it on a wooden block. Biological testing was carried out in vitro and then in vivo assessing the biosafety of the bone blocks in rat model, and the clinical feasibility and regeneration potential with a sheep mandibular diastema onlay model.
The results showed that the sintered bone at higher temperature removed most of the organic components (only 0.02% organic components and 0.02% residual organic components remained), and improved biocompatibility of the bone blocks at 24 h (1525 ± 314.2 fluorescence units [FU]), 48 h (1632 ± 232.9 FU), 72 h (1868 ± 404.6 FU) and 96 h (1527 ± 519.9 FU) as compared to the raw bone group (86.0 ± 101.0 FU, 250.3 ±333.6 FU, 240.8 ± 478.0 FU, and 184.8 ± 462.2 FU, respectively (p<0.05); but on the other hand, increased crystallinity (95.33%), reduced mechanical strength (MPa: 4.21 ± 1.97, 3.07 ± 1.21, 5.14 ± 1.86, respectively) compared with raw bone (Group 1) (MPa: 23.22 ± 5.24, p<0.05) compromised the clinical handling. The SCF treated bone blocks showed more promising results in terms of reducing organic contents (raw bone = 17.55%, SCF-CO2-H2O2 + Pepsin = 12.42%), enhancing mechanical strength (raw bone (mean = 23.09 ± 8.92 MPa), SCF-CO2-H2O2 (mean = 48.85 ± 11.60 MPa), p<0.0001) and cellular metabolic activity when seeded with mesenchymal stem cells (p<0.05). Moreover, the bench-top clinical handling was improved. Rat antigenicity testing showed that SCF treated bone blocks were safe to use and later were tested in a pilot study with a sheep onlay model. The sheep study showed the feasibility of the bone blocks in terms of clinical handling, however, severe resorption was observed for most of the bone block grafts with slight remnants visible for few of the blocks. In conclusion, SCF-treated bone samples have shown promising results in in vitro mechanical and biological testing, and in vivo biosafety testing. However, the in vivo biological testing was inconclusive and requires further investigation. Future studies would be aimed at optimising resorption rates and improve fixation techniques, for example, two screws or non-oral site. Overall, the SCF-CO2 treatment along with post treatments has shown potential for further full-scale trials due to enhanced mechanical strength and biological safety.