Effect of Hydroxyapatite Fiber Material with Magnesium on Bone Regeneration:
in vivo and in vitro study
Wataru Kozuma, Kazuhiro Kon, Sawako Kawakami, A. BO BO THIKE, Masahiro Shimogishi, Makoto Shiota, Shohei Kasugai
Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University
Oral implant therapy is now widely accepted1. However, implant placement is often limited by bone resorption. Fujii et al. have previously developed Hydroxyapatite fiber (HAf), which is 100% pure hydroxyapatite fiber of 5-10 mm in diameter2. HAf is biodegradable and can be easily manipulated in bone augmentation site. Magnesium (Mg) which is present in teeth and bone plays an important role in bone metabolism. According to the report, Mg ions promoted osteoblast proliferation and differentiation in bone marrow stromal cells (BMSCs)3.
The aim of this study was to evaluate the effect of HAf with Mg on bone regeneration.
Materials and Methods
We used a previously described HAf material. It looks like cotton having fibrous 3-D structure (Fig.1a, b). Mg-applied HAf were prepared by dropping in an aqueous solution containing different amounts of MgCl2(H2O)6 (0, 5.7, 11.4, 28.5 mol%).
Cell Culture Experiment
Bone marrow cells were prepared from the femurs of male Wistar Rat and cultured in a-MEM supplemented with 10% FBS. The cells of the 3rd passage were used and seeded in 24-well culture plate at 5.0 x 104 cells/well. They were incorporated into Mg-applied HAf (0, 5.7, 11.4, 28.5 mol%) and cultured in osteogenic medium (n=6). After 3, 5, and 7 days, gene expression of osteogenic genes (alkaline phosphatase (Alp), collagen 1 alpha 1 (Col1a1) and osteocalcin (Oc)) was examined by RT-qPCR, with Gapdh selected as the reference gene. These data were statistically compared using Dunnett's T3. Differences were considered statistically significant at p < 0.05.
Three male Japanese white rabbits (3.1–3.4 kg in weight) were used in vivo study. Polytetrafluoroethylene chambers, 5 mm-diameter and 3mm-hight, were fixed bilaterally with stainless-steel screws on the cranial bone. They were filled with 30 mg HAf containing the different amount of Mg (0, 5.7, 11.4, 28.5 mol%). A chamber was left empty as the negative control. Animals were sacrificed at 4 weeks. The samples were radiologically analyzed with micro-CT, and histologically analyzed. Cranial bones were embedded in polyester resin to obtain non-decalcified specimens. The ratio of new bone formation was calculated by micro-CT.
Chambers were fixed bilaterally on the cranial bone (a) and filled with HAf containing the different amount of Mg (b).
Results & Discussion
Gene expression results
The expression of all osteogenic markers was significantly more pronounced in the test groups (5.7 mol% and 11.4 mol%) compared to the control group on day 3 (p < 0.05). Alp and Col1a1 of test groups (5.7 mol% and 11.4 mol%) showed significantly higher on day 5. On the other hand, Oc of 28.5 mol% group showed significantly lower on day 7.
Osteogenic gene expression of bone marrow cells cultured on each sample type. Expression of the osteogenic genes measured by RT-qPCR at different time points. (n=6), *p < 0.05
Fig.4 Histological images of empty group at 4 weeks (a, b). The height of newly formed bone was approximately one-third of the chamber height. The newly formed bone was found around the bottom of the chamber, close to the host bone. (a) Magnification x 4. (b) Magnification x 50.
Fig.5 Histological images of HAf only group at 4 weeks (a, b). The height of newly formed bone reached almost half of the chamber height. (a) Magnification x 4. (b) Magnification x 50.
HAf + Mg 5.7 mol%
Fig.6 Histological images of Mg 5.7 mol% group at 4 weeks(a, b). The newly formed bone was more calcified than HAf only group. (a) Magnification x 4. (b) Magnification x 50.
HAf + Mg 11.4 mol%
Fig.7 Histological images of Mg 11.4 mol% group at 4 weeks (a, b). The newly formed bone was observed as a net-like structure. (a) Magnification x 4. (b) Magnification x 50.
HAf + Mg 28.5 mol%
Fig.8 Histological images of Mg 28.5 mol% group at 4 weeks (a, b). The newly formed bone clearly showed trabecular structure, but HAf seemed not to be absorbed much compared to 5.7 mol% group. (a) Magnification x 4. (b) Magnification x 50.
Fig.9 Micro CT images (a, b, c, d, e).
Fig.10 Ratio of new bone formation at 4 weeks post-operation from micro-CT data.
In animal study, both of histologic observation and micro-CT in all test groups showed more new bone formation than empty group. The newly formed bone was found around the bottom of the chamber, close to the host bone. In HAf only group and all Mg-applied HAf groups, the height of newly formed bone reached almost half of the chamber height. The newly formed bone of all Mg-applied HAf groups was more calcified than HAf only group. Mg could make bone more calcified. However, the high concentration may interfere with bone formation.
Mg-applied HAf has the potentiality to promote osteogenic differentiation of osteogenic cells in the early stages in vitro study. This material was effective in vertical bone augmentation in vivo study.
1. Ferrigno N et al. A long-term follow-up study of nonsubmerged ITI implants in the treatment of totally edentulous jaws. Part I: ten-year life table analysis of a prospective multicenter study with 1286 implants. Clin Oral Implants Res 2002; 13: 260-273.
2. S. Fujii. Fibrous apatite and method for producing the same. Shoichi Mori JT, editor. United States Patent, Patent number: 4659617; 1987.
3. S. Yoshizawa et al. Role of magnesium ions on osteogenic response in bone marrow stromal cells, Connect Tissue Res 2014; 55: 155-159.