Nobel Bone Regeneration System Using Carbonate Apatite-coated Carbonate Calcium in Vivo

Statement of the problem:  Effective bone regeneration system requires artificial bone substitutes to replace bone.  We developed carbonate apatite (CAp) blocks, which is a component of in vivo bone, by using a new procedure. We have already reported that CAp blocks receive absorption by osteoclasts just like in vivo bone and induce effective bone replacement[1].  Here, we further investigated modified CAp granules, which could be controlled with its absorption.

Materials and methods:  CAp granules were prepared by using dissolution-precipitation reaction from carbonate calcium (CaCO₃) as a precursor[2].  During that reaction, we were able to get the intermediate block, “carbonate apatite coated carbonate calcium”.  The degree of coating with CAp was measured by X-ray diffraction (XRD), then 10% and 30% CAp-coated CaCO₃ were prepared.

  Sixteen rabbits were subjected to bone regeneration assay.  We examined 5 groups, 1) Sham group, 2) CaCO₃ group, 2) 10% CAp-coated CaCO₃ (10% CAp) group, 3) 30% CAp-coated CaCO₃ (30% CAp) group, 4) CAp group.  Cylindrical bone defects (Φ5.0 mm×8.0 mm) of rabbit femur were formed by using dental trephine bar.  Those rabbits were sacrificed at 4 and 8 weeks after material implantation surgery and analyzed by means of microcomputed tomography (microCT; Latheta LCT-200, ALOKA).  Histological observation (MMA embedded; Villanueva-Goldner staining, Toluidine blue staining) was used to quantify new bone volume at the defect site.  

Methods of data analysis:  For quantification of the degree of absorption of those granules in rabbit bone defect study (n=3/group), the sacrificed samples at 4 and 8 weeks after operation were scanned by microCT. After standardizing the images, the area of granules was evaluated with Photoshop CS5.1 (Adobe).  For quantification of new bone volume, we calculated the area of new bone in five random images/group (40X-magnification) using ImageJ in toluidine blue stained sections.  For bone quantification analysis, a two-way analysis of variance (ANOVA) was performed.  Error is reported as the standard deviation and significance was determined using a probability value of p<0.05.

Results:  The granules in all groups showed gradually degradable behavior in time course.  CaCO₃ granules almost disappeared in bone defect site at 8 weeks.  The more CAp was coated with CaCO₃, the less absorption of granules was observed.  Furthermore, we could see newly formed bone histologically in contact with the CAp surface. As reported before, osteoclasts were also observed on the CAp surface, which induced bone remodeling, and was replaced by new bone in the bone defect area. CaCO₃ granules received degradation in vivo but did not have osteoinductive properties. In contrast, CAp surfaces induced calcified bone tissue surrounding them. Interestingly, 10% CAp and 30% CAp group induced about twice as much new bone as CAp granules alone at 4 weeks.  But, 10% CAp, 30% CAp, and CAp group showed almost equal volume of new bone at 8 weeks. Therefore, by the end of 4 weeks, 10% CAp and 30% CAp induced new bone faster than CAp granules alone.

Conclusions:  Here, we could demonstrate modified CAp granules with managed covering percentage of CAp could be induced to well-controlled absorption.  10% CAp and 30% CAp induced larger amount of new bone than CAp granules alone in the early stage.  Those CAp covered CaCO₃ might become an attractive scaffold for bone regeneration therapy in the near future.

References:

1. Fukuda M. et al. Arch BioCeramics Research 5: 75-78, 2005.

2. Matsuya S. et al. J Mater Sci Mater Med 18(7): 1361-1367, 2007.