Chitosan Scaffold Containing Periostin Enhances Sternum Bone Healing and Decreases Serum Level of TNF-α and IL-6 after Sternotomy in Rat

Abstract

Background: In the aftermath of bone injuries, such as cranium and sternum, bone wax (BW) is used to control bleeding from the bone surfaces during surgery. Made up of artificial substances, however, it is associated with many complications such as inflammation, increased risk for infection, and bone repair delay. We, therefore, in this study set out to design and evaluate a novel BW without the above-mentioned side-effects reported for other therapies.

Methods: The pastes (new BW(s)) were prepared in the laboratory and examined by MTT, MIC, MBC, and degradability tests. Then, 60 adult male Wistar rats, divided into six equal groups including chitosan (CT), CT-octacalcium phosphate (OCP), CT-periostin (Post), CT-OCP-Post, Control (Ctrl), and BW, underwent sternotomy surgery. Once the surgeries were completed, the bone repair was assessed radiologically and thereafter clinically in vivo and in vitro using CT-scan, H&E, ELISA, and qRT-PCR.

Results: All pastes displayed antibacterial properties and the CT-Post group had the highest cell viability compared to the control group. In contrast to the BW, CT-Post group demonstrated weight changes in the degradability test. In the CT-Post group, more number of osteocyte cells, high trabeculae percentage, and the least fibrous connective tissue were observed compared to other groups. Additionally, in comparison to the CT and Ctrl groups, higher alkaline phosphatase activity, as well as decreased level of serum tumor necrosis factor-α, interleukin-6, and OCN in the CT-Post group was evident. Finally, Runx2, OPG, and RANKL genes' expression was significantly higher in the CT-Post group than in other groups.

Conclusion: Our results provide insights into the desirability of pastes in terms of cellular viability, degradability, antibacterial properties, and surgical site restoration compared to the BW group. Besides, Periostin could enhance the osteogenic properties of bone tissue defect site.

Keywords: Bone healing; Chitosan; OPG and RANKL; Periostin; Runx2.

Fig. 1

Experimental design for the different groups

Fig. 2

In vitro degradation rate of various pastes and BW in PBS. After 25 days, in the BW, no weight loss was observed but higher weight change was observed in the CT-Post. Data represent mean ± S.D (n = 4)

Fig. 3

The MTT assay of BMSCs on pastes and BW after 24 h of incubation. The highest viability was related to the CT-Post (p = 0.1 vs Ctrl). But the lowest viability was related to the BW (p = 0.0001 vs Ctrl). Data represent mean ± S.D (n = 4)

Fig. 4

A Radiographic imaging of the sternum bone defect at the days of 7th, 21st and 56th of injury. Bone tissues filled the defects of CT-Post and Ctrl groups. The defects in other groups were filled with remnant substance and variable soft to hard tissues. After eight weeks, the CT-Post followed by the Ctrl group had the highest percentage of bone volume (p < 0.05). B The BW had the minimum bone volume compared with the CT-Post (***, p = 0.0001), Ctrl (***, p = 0.0006) and other treated groups. The CT-Post had the highest bone volume compared with the CT (**, p = 0.0024), CT-OCP (**, p = 0.0032) and CT-OCP-Post (*, p = 0.01) groups. Ctrl had the highest bone volume than CT and CT-OCP groups (*, p < 0.05) (n = 3)

Fig. 5

Horizontal histopathological sections of the sternum defects in rats after 8 weeks of bone injury. There are still most of remnants of the BW and were degraded partially of the CT, CT-OCP, CT-OCP-Post. While the CT-Post was almost completely degraded and replaced mostly with woven bone. BM bone marrow, TB trabecular bone, FCT fibrous connective tissue, R remnants of paste, Ctrl control, BW bone wax, CT chitosan, OCP octacalcium phosphate, Post periostin. Stained with H&E

Fig. 6

Histomorphometric findings of healed area after 8 weeks of bone injury. A Indicates the mean of osteocytes per field (×20) (*, p < 0.05) (**, p = 0.0065) (***, p = 0.0007) (****, p < 0.0001). B Indicates the percent of TBV than healed area (*, p < 0.05) (***, p = 0.0006), (****, p < 0.0001). C Indicates the percent of FCT than healed area (*, p = 0.02) (**, p = 0.0016), (***, p = 0.0008). D Indicates the representative image containing OC, FCT and, TBV markers that were used to count osteocyte number and determine the density (%) of FCT and TBV, respectively. FCT fibrous connective tissue, TBV trabecular bone volume, OC osteocyte. Data represent mean ± SD (n = 3)

Fig. 7

Effects of BW and pastes on mRNA expression of A Runx2, B RANKL and C OPG on the 7th, 21st and 56th days. Runx2 runt-related transcription factor 2, RANKL receptor activator of nuclear factor kappa-Β ligand, OPG osteoprotegerin. Data represent Mean ± SD (On the day 7, n = 4; on the days 21 and 56, n = 3)

Fig. 8

Serum concentration of A OCN, B IL-6 and C TNF-α at 7th, 21th and 56th days detected by ELISA. OCN osteocalcin, IL-6 interleukin 6, TNF-α tumor necrosis factor alpha. Data represent mean ± SD (On the day 7, n = 6; on the day 21, n = 4; on the day 56, n = 3)

Fig. 9

ALP activity on days 7, 21 and 56. By Colorimetry test. On day 7, the highest ALP activity was related to the CT-Post and it was significantly different than the Ctrl (***, p = 0.0003), BW (****, p < 0.0001), CT (**, p = 0.0021), and the CT-OCP-Post (*, p < 0.05). ALP alkaline phosphatase. Data represent mean ± SD (On the day 7, n = 6; on the day 21, n = 4; on the day 56, n = 3)