Table 1.
Allocation of defect sites and processing for this study.
Fig 1.
Schematic overview of where the defect was placed in the distal femur.
Fig 1 provides a schematic of where the defect was placed in the distal femur as well as where the sectioning performed for micro computed tomography as well as the PMMA and Paraffin Histology.
Fig 2.
Faxitron radiographs of the materials.
Fig 2 presents Faxitron radiographs of the materials as received in sterile packaging. Faxitron radiographs revealed the calcium phosphate mineral distributed in the material within both the materials. Both graft materials had a uniform radio-opacity along the 10 cm length in all five sample examined. No differences were detected within graft materials for grey scale for the 6 region of interest (P>0.05). The overall grey scale value for Sorrento Bone Graft Substitute was however great than that compared to Vitoss Foam Strip (P<0.05).
Fig 3.
Stereozoom and electron microscopy of the materials.
Fig 3 presents stereo-zoom and electron microscopy images revealing qualitative differences in the surface and interior of the graft materials. The Sorrento Bone Graft Substitute (A, B, C) and Vitoss Foam Strip (D,E, F) had a different surface structure based on stereozoom images (A vs D) as well as environmental scanning electron microscope image of the surface (B vs E) and the interior (C vs F).
Fig 4.
Fourier Transform Infrared Spectroscopy and X-ray Diffraction.
Fig 4a The FTIR spectra for the calcium phosphate portions of the graft are shown in the top panel. The Sorrento Bone Graft Substitute (red) and Vitoss Foam Strip (Grey) compared well to each other were both similar to the tricalcium phosphate control (Blue) and differed compared to the hydroxyapatite control (green) [55]. The FTIR spectra for the organic portions of the graft are shown in the bottom panel. The Sorrento Bone Graft Substitute (red) and Vitoss Foam Strip (Grey) compared well to each other were similar to the Collagen control (Blue). The typical amide peaks of collagen were present in all samples [54]. There was however evidence of the calcium phosphate residual material as evident by the phosphate peaks at approximately 1100–950 cm-1. Fig 4b The XRD diffraction patterns for the calcium phosphate portions of the graft are shown in the top panel. The Sorrento Bone Graft Substitute (red) and Vitoss Foam Strip (Grey) compared well to each other were both similar to the tricalcium phosphate control (Blue) and differed compared to the hydroxyapatite control (green) [55].
Fig 5.
Micro-computed tomography versus time.
Fig 5 Micro-CT provided a unique insight into the radiographic signal and performance of the two materials and healing of the defect. Increased signal intensity suggesting newly formed woven bone was seen at the defect margins as well as extending into the centre of the defects with the Sorrento BMA material at 3 weeks that progressed with time. In contrast, micro-CT for the Vitoss Foam Strip BMA group did not demonstrate new bone formation at 3 weeks while some new bone formation at the margins could be seen at 6 weeks this did not progress into the centre of the defect with time. The autograft treated defects healed well with time while the empty defects remained empty demonstrating the critical nature of the model. These findings agreed well with the histology results for all defects.
Fig 6.
Histology Fig 6 Histology confirmed the micro computed tomography findings in terms of new bone formation and implant resorption versus time.
The histology images were taken with a 1.25x objective to provide an overview of the site with the defect outlined with a black circle. PMMA histology is presented for time 0 and the 6 and 12 week time points for Sorrento and Vitoss Foam Strip and Empty defects at 12 weeks. These sections were stained with methylene blue/basic fuschin. The 3 week data is from paraffin histology stained with H&E for all groups. The 6 week histology for autograft is also from paraffin histology stained with H&E. The Sorrento BMA material supported new bone formation as early as 3 weeks with resorption of the mineral phase with time. In contrast, Vitoss Foam Strip BMA group did not demonstrate significant new bone formation at 3, 6 or 12 weeks in the middle of the defect although some new bone formation at the defect margins. The autograft treated defects healed well with time while the empty defects remained empty demonstrating the critical nature of the model.
Fig 7.
Higher magnification histology.
Fig 7 Higher magnification histology in the middle of the defect is presented at 6 weeks (4x and 10x objectives and stained with H&E) to illustrate the in vivo differences observed between Sorrento Bone Graft Substitute, Vitoss Foam Strip and Autograft. The Sorrento BMA material supported new bone formation with direct bone formation on the surface of the calcium phosphate phase (star) and newly formed bone (green arrows) marrow spaces at 6 weeks. In contrast, Vitoss Foam Strip BMA group did not demonstrate significant new bone formation 6 weeks in the middle of the defect with noted evidence of inflammatory cells (black arrows) and fibrous tissue response. The autograft treated defects demonstrate new bone formation directly on the remaining graft material present (orange arrows) in the defect as well as newly formed marrow spaces.
Fig 8.
Immunohistochemistry expression.
Fig 8 Immunohistochemistry results for Alkaline phosphatase, (ALP) is a widely accepted bone marker activity and osteogenic potential, is presented for Sorrento Bone Graft Substitute, Vitoss Foam Strip and Autograft at 3 weeks and 6 weeks. The 3 week panels are examined at a low magnification (1.25x objective) while the 6 week presents the results in the center of the defect (20x objective). Positive expression for ALP is noted by the brown staining. The results at 3 weeks demonstrate ALP expression in the osteoblasts throughout the defects for defect treated with Sorrento Bone Graft Substitute as well as Autograft while defects treated with Vitoss Foam Strip shows little to no expression apart from the defect margins. IHC for ALP at 6 weeks shows the remodeled bone in the Sorrento Bone Graft Substitute treated defects with continued expression in the osteoblasts lining the newly formed bone, the presence of some fibrous tissue and some ALP expression for Vitoss Foam Strip and expression of ALP by the active osteoblasts in the Autograft treated defects.
Table 2.
Fig 9.
Fig 9a) Mean new bone formation histomorphometry revealed Sorrento Strip + BMA outperformed Vitoss Foam Strip + BMA (P<0.05) at 6 and 12 weeks. New bone formation increased slightly for the Sorrento Strip + BMA while the Vitoss Foam Strip + BMA decreased slightly. This was consistent with additional new bone formation in the Sorrento Strip treated sights as well as remodeling of the newly formed bone. The results of the Vitoss Foam Strip + BMA for new bone formation were consistent with remodeling of the newly formed bone and lack of additional bone formation at 12 weeks. Fig 9b) Mean material histomorphometry revealed Sorrento Strip + BMA and Vitoss Foam Strip + BMA resorbed with time from 0 to 6 and to 12 weeks after surgery.