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. 2020 Aug 5;13(16):3458.
doi: 10.3390/ma13163458.

Physical/Chemical Properties and Resorption Behavior of a Newly Developed Ca/P/S-Based Bone Substitute Material

Bing-Chen Yang  1 , Jing-Wei Lee  2 , Chien-Ping Ju  1 , Jiin-Huey Chern Lin  1
Affiliations

Physical/Chemical Properties and Resorption Behavior of a Newly Developed Ca/P/S-Based Bone Substitute Material

Bing-Chen Yang et al. Materials (Basel). .

Abstract

Properly regulating the resorption rate of a resorbable bone implant has long been a great challenge. This study investigates a series of physical/chemical properties, biocompatibility and the behavior of implant resorption and new bone formation of a newly developed Ca/P/S-based bone substitute material (Ezechbone® Granule CBS-400). Experimental results show that CBS-400 is comprised majorly of HA and CSD, with a Ca/P/S atomic ratio of 54.6/39.2/6.2. After immersion in Hank's solution for 7 days, the overall morphology, shape and integrity of CBS-400 granules remain similar to that of non-immersed samples without showing apparent collapse or disintegration. With immersion time, the pH value continues to increase to 6.55 after 7 days, and 7.08 after 14 days. Cytotoxicity, intracutaneous reactivity and skin sensitization tests demonstrate the good biocompatibility features of CBS-400. Rabbit implantation/histological observations indicate that the implanted granules are intimately bonded to the surrounding new bone at all times. The implant is not merely a degradable bone substitute, but its resorption and the formation of new cancellous bone proceed at the substantially same pace. After implantation for 12 weeks, about 85% of the implant has been resorbed. The newly-formed cancellous bone ratio quickly increases to >40% at 4 weeks, followed by a bone remodeling process toward normal cancellous bone, wherein the new cancellous bone ratio gradually tapers down to about 30% after 12 weeks.

Keywords: Ca-based; animal study; bone substitute; histology; resorption.

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Conflict of interest statement

J.-H.C.L. and C.-P.J. initiated the government-funded research which was later transferred to JMD. NCKU is holding the related patent rights. B.-C.Y., a graduate student, J.-H.C.L. and C.-P.J. continue to assist JMD in research.

Figures

Figure 1
Figure 1
Illustration of 5 mm dia., 10 mm deep cylindrical-shaped CBS-400 implantation site. Lines A, B, C, D and E demonstrate the specific locations of the femur bone being sectioned for histological observation.
Figure 2
Figure 2
Phase identification and composition analysis of CBS-400. (A) XRD pattern; (B) XRD-determined phase contents and SEM/EDS-determined elemental contents; (C) SEM/EDS spectrum and analyzed region.
Figure 3
Figure 3
Surface morphology, shape and integrity of CBS-400 granules without immersion (A–C) and after immersion in Hank’s solution for 1 (D–F), 3 (G–I) and 7 (J–L) days.
Figure 4
Figure 4
CBS-400 particle size distribution (A) and pore size distribution (B). Asterisk indicates the average number, and the two boundaries of the box plot above the bar graph define the 5th (left) and 95th (right) percentiles.
Figure 5
Figure 5
pH values in daily refreshed Hank’s solution, wherein CBS-400 is immersed. The significance of differences between two pH values at different immersion days are shown in the upper left table; S and NS represent p < 0.05 and p > 0.05, respectively.
Figure 6
Figure 6
Ca, P and S elements released from 1 g CBS-400 in 20 mL TRIS-HCl at 37 °C for 1, 3, and 5 days; and in 20 mL buffered citric acid solution at 37 °C for 5 days. Symbols a, b and c indicate that the mean value has a significant difference (p < 0.05) compared to Day 1, Day 3 and Day 5 groups, respectively. Asterisks indicate that there are significant differences between TRIS-HCl and citric acid groups at day 5.
Figure 7
Figure 7
Morphologies of NIH/3T3 cells before treatment (A), treated with culture medium as blank (B), treated with CBS-400 extract (C), treated with 0.3% phenol as positive control (D), treated with Al2O3 extract as negative control (E), and their O.D. values (F).
Figure 8
Figure 8
H&E-stained histological images of NZW rabbit femur condyle of CBS-400-implanted group at 4W (A,B), 8W (C,D) and 12W (E,F) post-operation. I: residual implant; NB: new bone; LM: lamellar matrix; CL: cement line; Ob: osteoblast; Oc: osteoclast; Ot: osteocyte; O: osteoid; L: lining cell. Asterisks indicate implant residues embedded in surrounding new bone. Arrow heads indicate new bone grown in implant micro-pores.
Figure 9
Figure 9
TB-stained histological images of NZW rabbit femur condyle in CBS-400-implanted group at 3D (A), 4W (B), 8W (C) and 12W (D) post-operation. I: residual implant; NB: new bone; E: epoxy.
Figure 10
Figure 10
Overall, 5 mm dia. TB-stained superimposed composite images of NZW rabbit femur condyle in CBS-400-implanted group at 3D (A), 4W (C), 8W (E) and 12W (G) post-operation and their corresponding manually drawn images (B,D,F and H), illustrating residual CBS-400 (black), new bone (green) and marrow space (blue). Moreover, 4 mm dia. inner zones are outlined by red dash circles.
Figure 11
Figure 11
Area ratios of residual CBS-400 implant and new bone at 3D, 4W, 8W and 12W post-operation. The area ratios of native bone in blank group at 12W are also included. Data from TZ and IZ zones are both presented for comparison. * Significant (p < 0.05). † Native bone area ratio instead of new bone area ratio.
Figure 12
Figure 12
Residual implant and new bone ratios. Linear regression (black lines) and quadratic regression (blue lines) are applied to new bone ratio and residual implant ratio, respectively. The regression lines and their 95% confidence intervals are drawn.
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