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A Biphasic Calcium Sulphate/Hydroxyapatite Carrier Containing Bone Morphogenic Protein-2 and Zoledronic Acid Generates Bone

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A Biphasic Calcium Sulphate/Hydroxyapatite Carrier Containing Bone Morphogenic Protein-2 and Zoledronic Acid Generates Bone

Deepak Bushan Raina et al. Sci Rep.

Abstract

In orthopedic surgery, large amount of diseased or injured bone routinely needs to be replaced. Autografts are mainly used but their availability is limited. Commercially available bone substitutes allow bone ingrowth but lack the capacity to induce bone formation. Thus, off-the-shelf osteoinductive bone substitutes that can replace bone grafts are required. We tested the carrier properties of a biphasic, calcium sulphate and hydroxyapatite ceramic material, containing a combination of recombinant human bone morphogenic protein-2 (rhBMP-2) to induce bone, and zoledronic acid (ZA) to delay early resorption. In-vitro, the biphasic material released 90% of rhBMP-2 and 10% of ZA in the first week. No major changes were found in the surface structure using scanning electron microscopy (SEM) or in the mechanical properties after adding rhBMP-2 or ZA. In-vivo bone formation was studied in an abdominal muscle pouch model in rats (n = 6/group). The mineralized volume was significantly higher when the biphasic material was combined with both rhBMP-2 and ZA (21.4 ± 5.5 mm(3)) as compared to rhBMP-2 alone (10.9 ± 2.1 mm(3)) when analyzed using micro computed tomography (μ-CT) (p < 0.01). In the clinical setting, the biphasic material combined with both rhBMP-2 and ZA can potentially regenerate large volumes of bone.

Conflict of interest statement

Prof. Lars Lidgren is a board member of Bonesupport AB, Lund, Sweden and Orthocell, Australia. None of the other authors declare any conflict of interest.

Figures

Figure 1
Figure 1. In-vitro rhBMP-2 and ZA release kinetics.
(A) Indicates rhBMP-2 release (%) from the biphasic material over a period of 7-days detected using ELISA. (B) Shows the microscopic effect of free ZA (increasing concentrations using tissue culture plates (2D TCP)) and ZA released from the biphasic material (different day fractions) on A549 lung cancer cells (Yellow arrow indicates healthy, epithelial morphology of cells, red arrow points at round, dead cells while green arrow shows floating apoptotic bodies). (C,D) Fraction of ZA released (%) from the biphasic material over a period of 7-days and the cytotoxicity induced in A549 cells by the released fraction using the MTT assay, respectively. (E) Effect of bound and free ZA on A549 cells after seeding the cells directly on the biphasic material alone, in combination with ZA and plastic control treated with free ZA using the MTT assay. **Indicates p < 0.01, #indicates non-significant. Data is expressed as mean ± SD. Scale bar represents 30 μm.
Figure 2
Figure 2. In-vitro SEM and mechanical analysis.
(A) Surface architecture and pore morphology of the biphasic material alone and with rhBMP-2 and rhBMP-2 + ZA was compared after casting (Day 0) and after 28-days of incubation in saline. SEM images on the left panels at Day 0 and day 28 have been captured at 500X while images in the right panel depict high magnification images (8000X). The lower left panel (B) shows the stiffness of the biphasic material alone or after addition of rhBMP-2 and ZA. The lower right panel (B) indicates the absorbed energy by the samples in different groups. *Indicates p < 0.05, #indicates non-significant. Data is expressed as mean ± SD. n = 5.
Figure 3
Figure 3. X-ray radiography of implanted samples in the abdominal muscle pouch.
X-ray radiographs of biphasic material alone and combined with rhBMP-2 (10 μg) and rhBMP-2 (10 μg) + ZA (10 μg) in the abdominal muscle pouch after 28-days of in-vivo implantation. Scale bar represents 1 cm.
Figure 4
Figure 4. Micro-computed tomography results 28-days post implantation.
Images in the top panel represent full 3-D rendering of the samples in the three groups (biphasic material, biphasic material + rhBMP-2 (10 μg) and biphasic material + rhBMP-2 (10 μg) + ZA (10 μg)) while images in the middle panel show sliced 2-D images in the middle of the samples in order to emphasize on the internal content of the samples across different groups. The bottom panel shows the mineralized volume in each group. **Indicates p < 0.01, #indicates non-significant. Data is expressed as mean ± SD. n =  5 for biphasic material + rhBMP-2 and rhBMP-2 + ZA groups and n = 6 for biphasic material alone. Scale bar represents 1 mm.
Figure 5
Figure 5. Histological representation and histomorphometric analysis of the samples implanted in the abdominal muscle pouch.
Images in top panels provide an overview of the whole sample (12.5X) in the three groups after 28-days of implantation. Images in the middle panel emphasizes on the periphery of the material/bone composite (100X) while images in the bottom panels indicate the innermost tissue/material construct (100X). A indicates apatite, B shows bone, F represents fibrous tissue and “BM” shows bone marrow. Data in (J) shows Histomorphometric quantification of bone area across the two groups with bone formation. Data is expressed as mean ± SD. n = 5.
Figure 6
Figure 6. SEM analysis of implanted samples after 28-days of in-vivo implantation.
Top panel provides a low magnification (50X) overview of samples in the three groups while the images in the lower panels have been taken at comparatively higher magnifications to emphasize on the appearance and surface structure of the bone/material composite. The arrows in lower left and middle panel indicate apatite particles while the arrows in the lower right panel show typical trabecular bone formation on the biphasic material loaded with rhBMP-2 and ZA.
Figure 7
Figure 7. Mechanical testing of implanted samples after 28-days of in-vivo implantation.
Left panel shows the stiffness of the bone/material composite in the three groups while right panel indicates the absorbed energy across the three groups. *Indicates p < 0.05, ***p < 0.001, #indicates non-significant. Data is expressed as mean ± SD. n = 5.
Figure 8
Figure 8. Schematic of rhBMP-2 and ZA delivery from the biphasic material and possible bone formation process.
(1) indicates a set disc of the biphasic material with ZA bound to HA while rhBMP-2 is encapsulated between the two phases. After in-vivo implantation, the material releases sulphate, rhBMP-2 and little ZA as shown in (2). Muscle stem cells interact with rhBMP-2 via BMP receptors and a change in their phenotype occurs leading to their osteogenic differentiation. Subsequently the bone formation approaches inwards into the scaffold. Due to sulphate resorbing over time, the scaffold gets more porous and the bone formation is substantiated by rhBMP-2 as shown in (3). After a significant amount of bone is formed, RANKL-RANK (Osteoblast- Osteoclast progenitor) interaction causes osteoclastogenesis as shown in (4). However, due to the presence of ZA, osteoclastic apoptosis occurs leading to a preserved bone turnover (5).

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