Collagen type I-based recombinant peptide promotes bone regeneration in rat critical-size calvarial defects by enhancing osteoclast activity at late stages of healing

Abstract

Introduction: We recently demonstrated the bone-forming potential of medium-cross-linked recombinant collagen peptide (mRCP) in animal models of bone defects. However, these studies were limited to a 4-week observation period; therefore, in the present study, we aimed to further evaluate mRCP as a suitable bone graft material for the alveolar cleft by analyzing its bone-forming potential, osteogenic-inducing ability, and biodegradation over an extended period of 12 weeks, using a rat critical-size calvarial defect model.

Methods: Using Sprague-Dawley rats, we created critical-size calvarial defects through a surgical procedure. The defects were then filled with 3 mg of mRCP (mRCP group) or 18 mg of Cytrans® (CA) granules, which has a carbonate apatite-based composition resembling natural bone, was used as a reference material (CA group). For negative control, the defects were left untreated. Bone volume, total bone volume (bone volume including CA granules), and bone mineral density (BMD) in the defect were assessed using micro-computed tomography (μ-CT) at 0, 4, 8, and 12 weeks after implantation. Using histomorphometric analyses of hematoxylin and eosin (H&E)-stained sections, we measured the amount of newly formed bone and total newly formed bone (new bone including CA granules) in the entire defect site, as well as the amount of newly formed bone in the central side, two peripheral sides (left and right), periosteal (top) side, and dura mater (bottom) side. In addition, we measured the amount of residual bone graft material in the defect. Osteoclasts and osteoblasts in the newly formed bone were detected using tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) staining, respectively.

Results: Bone volume in the mRCP group increased over time and was significantly larger at 8 and 12 weeks after surgery than at 4 weeks. The bone volume in the mRCP group was greater than that of the CA and control groups at 4, 8, and 12 weeks after implantation, and while the total bone volume was greater in the CA group after 4 and 8 weeks, the mRCP group had comparable levels of total bone volume to that of the CA group at 12 weeks after implantation. The BMD of the mRCP group reached similar levels to native calvaria bone at the same time point. H&E-stained sections revealed a larger amount of newly formed bone 12 weeks after implantation in the mRCP group compared to that of the CA and control groups. The total newly formed bone at 12 weeks after implantation was on par with that in the CA group. Furthermore, at the defect site, the area of newly formed bone was larger on the peripheral and dura mater sides. Notably, the number of osteoclasts in the mRCP group was higher than in the CA and control groups and peaked 8 weeks after implantation, which coincided with the timing of the greatest resorption of mRCP. Although the ALP-positive area was greater in the mRCP group compared to other groups, we did not detect any significant changes in the number of osteoblasts over time.

Conclusion: This study demonstrated the bone-forming potential of mRCP over an extended period of 12 weeks, suggesting that mRCP sufficiently resists resorption to promote bone formation through induction of osteoclast activation in the late stages of the healing period.

Keywords: Bone graft material; Bone regeneration; Calvaria; Osteoblast; Osteoclast; Recombinant human collagen peptide.

Fig. 1

Macroscopic appearance of the critical-size calvarial bone defect before and after implantation with bone graft materials. (A and D) 5 mm diameter critical size defects made with a trephine bur. The defects were filled with 3 mg mRCP (B) or 18 mg CA (E). Defects after the periosteal flap were repositioned and sutured in the (C) mRCP and (F) CA groups.

Fig. 2

Diagram of the critical-size bone defect in rat calvaria and histomorphometric analyses. (A) The amount of newly formed bone was calculated based on the defect width and the bone fill measurements in the peripheral area (1.25 mm × 0.8 mm) of both the sides (1.25 mm × 0.8 mm × 2) and the central area (2.5 mm × 0.8 mm). (B) The level of newly formed bone was calculated based on the defect width and the bone fill measurements on the top side (periosteum side, 5.0 mm × 0.4 mm) and the bottom side (dura mater side, 5.0 mm × 0.4 mm).

Fig. 3

Representative coronal and axial plane images of μ-CT and 3D reconstructed μ-CT analysis of the calvarial bone defects in the (A) mRCP (B) CA and (C) control groups at 0, 4, 8, and 12 weeks after implantation. Amount of bone volume (D) and total bone volume (E) in the calvarial bone defects at 4, 8, and 12 weeks after implantation with bone graft materials assessed by $\mathrm{\mu }$-CT. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. The columns and error bars in the graphs represent the mean and standard deviation (SD), respectively (n = 5 per group).

Fig. 4

Representative histological images at low ( × 20) and high ( × 400) magnification (enlarged view of boxed area) of H&E-stained sections of the critical-sized bone defects of the (A–C) mRCP, (D–F) CA, and (G–I) control groups at 4, 8, and 12 weeks after surgery. Scale bars represent 300 μm and 100 μm in the enlarged boxed area. The arrows indicate the boundary between the implanted site and the native calvaria bone. The pink-stained structures in the defect indicate newly formed bone. Scale bars represent 300 μm and 100 μm (enlarged boxed area).

Fig. 5

Histomorphometric analysis of the newly formed bone area (A) and total newly formed bone area (B) in the entire bone defect in the mRCP, CA, and control groups at 4, 8, and 12 weeks after implantation. Newly formed bone and total newly formed bone areas in the entire defect (5.0 mm × 0.8 mm) were measured using ImageJ. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. The columns and error bars in the graph represent the mean and SD (n = 3), respectively. ns: not significant.

Fig. 6

Newly formed bone areas at different locations in the defect site of the implanted groups at 4, 8 and 12 weeks after surgery. The newly formed bone in the peripheral areas on both sides (1.25 mm × 0.8 mm × 1.25 mm) were compared with the central area (5.0 mm × 0.8 mm) of the bone defect in the (A) mRCP, (B) CA, and (C) control groups. Newly formed bone in the top side (5.0 mm × 0.4 mm) and the bottom side (5.0 mm × 0.4 mm) of the entire bone defect in the mRCP (D), CA (E), and (F) control groups. The newly formed bone areas were measured using ImageJ. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. The columns and error bars in the graph represent the mean and SD (n = 3), respectively.

Fig. 7

Comparison of the newly formed bone areas between the three groups at different locations in the defect site at 4, 8, and 12 weeks after surgery. (A) central area (2.5 mm × 0.8 mm), (B) peripheral area on both sides (1.25 mm × 0.8 mm × 2), (C) top side (5.0 mm × 0.4 mm), and (D) bottom side (5.0 mm × 0.4 mm) of the defect were measured from the entire calvarial bone defect using ImageJ. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. The columns and error bars in the graph represent the mean and SD (n = 3), respectively. ns: not significant.

Fig. 8

Residual mRCP (A–C) and CA (D–F) in the implanted site after 4, 8 and 12 weeks of surgery ( × 200). Scale bar represents 100 μm. (G) The graph shows the means and SD (n = 3) of the amount of residual mRCP and CA after 4, 8, and 12 weeks of surgery measured by ImageJ. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Asterisks indicate residual bone graft. The columns and error bars in the graph represent the mean and SD (n = 3), respectively.

Fig. 9

Detection of osteoclasts in the critical-size bone defect of the mRCP, CA and control groups after 4, 8, and 12 weeks. High-magnification ( × 600) images of tartrate-resistant acid phosphatase (TRAP)-stained sections of the (A–C) mRCP, (D–F) CA, and (G–I) control groups. Scale bars represent 10 μm. Arrows indicate osteoclasts. (J) Quantification of TRAP-positive cells at 4, 8, and 12 weeks after implantation with bone graft materials. ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Asterisks indicate CA granules. The columns and error bars in the graph represent the mean and SD (n = 3), respectively.

Fig. 10

Histological analysis of high-magnification ( × 600) images of alkaline phosphatase (ALP)-stained sections of the critical-size bone defect in the mRCP, CA and control groups after 4, 8, and 12 weeks. ALP-stained sections in the (A–C) mRCP, (D–F) CA, and (G–I) control groups. Scale bars represent 10 μm. Arrows indicate osteoblasts. (J) The ALP positive area was calculated at 4, 8, and 12 weeks after implantation with bone graft materials using ImageJ software. ∗P < 0.05. Asterisks indicate CA granules. The columns and error bars in the graph represent the mean and SD (n = 3), respectively.