Application of Low Crystalline Carbonate Apatite Combined With BMP-2 to Bone Reconstruction
Carbonated apatite (CO3Ap) is the inorganic component of bone and is shown to be a good candidate for bioresorbable bone substitutes in the treatment of bony defects. We have previously proposed a new method to fabricate low crystalline CO3Ap block based on dissolution-reprecipitation reaction [1]. We have demonstrated that CO3Ap granules were resorbed and partially replaced with new bone when implanted into the surgically created bone defects [2], and that CO3Ap promoted osteoblastic differentiation of human bone marrow cells [3].
Bone morphogenetic protein-2 (BMP-2) regulates osteoblast differentiation and bone formation. It was extensively used in bone tissue engineering. Sustained release of BMP-2 is very important during the process of bone formation. Therefore, scaffold materials are also designed as carriers to release BMP-2 in bone tissue engineering.
In the present study, we used CO3Ap granules as a carrier for the delivery of BMP-2 and examined in vivo behavior of CO3Ap combined with BMP-2 implanted into the subcutaneous tissue of rats.
Materials and methods
Preparation of CO3Ap granules: CO3Ap granules with 300-600 μm particle size were prepared, as reported previously [1]. Briefly, CO3Ap block was prepared by phase transformation of calcite block. The resultant CO3Ap block was crushed to granule.
Animals and in vivo experiment: Eight-week-old male Wistar rats were used for the implantation study. CO3Ap granules combined with BMP-2 (0, 5, 50 µg) implanted into the back subcutaneous tissue of rats. At 2, 4, 8, 14, and 28 weeks after implantation, the rats were sacrificed and the tissue samples were harvested. The samples were used in micro-CT analysis and histological analysis.
Methods and data analysis
Micro-CT analysis: The samples were subjected to micro-CT analysis. The particle size of the CO3Ap granules was measured. Quantitative analysis of granular resorption was carried out using ImageJ software (NIH, MD, US). Statistical evaluation included standard deviation and student’s t-test.
Histological analysis: The samples were decalcified, dehydrated and embedded in paraffin. Longitudinal sections were used for hematoxylin and eosin (H&E) staining, immunohistochemical staining of Runx-2 and Osterix, and TRAP (tartrate-resistant acid phosphatase) staining.
Results
New bone formation: The ectopic bone formation was observed in the rats implanted CO3Ap granules combined with 50 µg of BMP-2 at two weeks after implantation and new bone area increased with time. In the rats implanted CO3Ap granules combined with 5 µg of BMP-2 and without BMP-2, ectopic bone formation was not observed.
Expression of Runx-2 and Osterix: After 2 to 4 weeks, Runx-2 and Osterix were expressed in the cells surrounding CO3Ap granules. Runx2-expressing cells decreased markedly in number. On the other hand, Osterix were expressed in the surrounding tissues of the CO3Ap granules and the newly-formed osteoid tissues. Its expression was continued until 28 weeks.
Resorption of CO3Ap granule: The size of the CO3Ap granules decreased with time and became about 30% of the initial size. TRAP positive cells were observed in the surface of the particles.
Conclusions
CO3Ap granules combined with BMP-2 stimulated the ectopic new bone formation by promoting osteoblastic differentiation of mesenchymal cells in the surrounding tissue, which may be useful in improving bone regeneration therapy.
References
[1] Matsuya S. et al.: Fabrication of porous low crystalline calcite block by carbonation of calcium hydroxide compact. J Mater Sci Mater Med. 2007, 18, 1361–1367.
[2] Nagai H. et al.: Effects of low crystalline carbonate apatite on proliferation and osteoblastic differentiation of human bone marrow cells. Arch BioCeramics Research. 2005, 5, 83-86.
[3] Fukuda M. et al.: Application of carbonate apatite to bone reconstruction: fabrication and histological evaluation of low crystalline carbonate apatite. Arch BioCeramics Research. 2005, 5, 75-78.