Effect of cervus and cucumis peptides on osteoblast activity and fracture healing in osteoporotic bone

Abstract

Osteoporosis is associated with delayed and/or reduced fracture healing. As cervus and cucumis are the traditional Chinese treatments for rheumatoid arthritis, we investigated the effect of supplementation of these peptides (CCP) on bone fracture healing in ovariectomized (OVX) osteoporotic rats in vitro and in vivo. CCP enhanced osteoblast proliferation and increased alkaline phosphatase activity, matrix mineralization, and expression of runt-related transcription factor 2 (Runx2), bone morphogenetic protein 4 (BMP4), and osteopontin. In vivo, female Sprague-Dawley rats underwent ovariectomy and the right femora were fractured and fixed by intramedullary nailing 3 months later. Rats received intraperitoneal injections of either CCP (1.67 mg/kg) or physiological saline every day for 30 days. Fracture healing and callus formation were evaluated by radiography, micro-CT, biomechanical testing, and histology. At 12 weeks after fracture, calluses in CCP-treated bones showed significantly higher torsional strength and greater stiffness than control-treated bones. Bones in CCP-treated rats reunified and were thoroughly remodeled, while two saline-treated rats showed no bone union and incomplete remodeling. Taken together, these results indicate that use of CCP after fracture in osteoporotic rats accelerates mineralization and osteogenesis and improves fracture healing.

Figure 1

Effect of combined cervus and cucumis peptide (CCP) extracts, cervus peptide extract from deer horn (CHDP), and cucumis extract from sweet melon (CSMP) (dose range 20–400 μg/mL) on proliferation of human osteoblast C2C12 cells. Data are means ± SD of six replicates. ^* P < 0.01 compared with DMEM.

Figure 2

Effect of CCP (dose range 1–100 μg/mL) on proliferation of mouse MC3T3E1 osteoblasts. Data are means ± SD of six replicates. ^* P < 0.05, ^** P < 0.01 compared with the control.

Figure 3

Effect of CCP on alkaline phosphatase (ALP) activity in mouse MC3T3E1 osteoblasts. Data are means ± SD of six replicates. ^** P < 0.01, ^* P < 0.05 versus control.

Figure 4

Effect of saline (a), CHDP (b), CSMP (c), BMP (d), and CCP (e) on matrix mineralization by human C2C12 osteoblasts (Von Kossa staining).

Figure 5

Effect of CCP on matrix mineralization by mouse MC3T3E1 osteoblasts (alizarin red staining). (a) Control; (b), (c), and (d) 1, 10, and 100 μg/mL CCP.

Figure 6

Effect of CCP on expression of RUNX2, OPN, and BMP-4 in MC3T3E1 osteoblasts (quantitative RT-PCR). ^** P < 0.01.

Figure 7

Micro-CT of femurs in rats with ovariectomy-induced osteoporosis. (a) Normal bone; (b) 2 weeks and (c) 4 weeks after ovariectomy.

Figure 8

Micro-CT examination of the effect of CCP treatment (intraperitoneal injection, 1.67 mg/kg, beginning on postoperative day 1 and then every day for 30 days) on healing of femoral fractures at 4, 6, 8, and 12 weeks.

Figure 9

Histological examination by H&E staining of the effect of CCP treatment (intraperitoneal injection, 1.67 mg/kg, beginning on postoperative day 1 and then every day for 30 days) on healing of femoral fractures at 12 weeks.