ABSTRACT
Objective To investigate the effect of gefitinib, an inhibitor of epidermal growth factor receptor ( EGFR ) , on the proliferation and osteogenic differentiation of endosteum-derived stem cells ( EDSCs ) in rats. Methods Femoral fracture models were established in healthy male 4-week old SD rats. They were randomly divided into 2 groups. The experimental group was subjected to intragastric lavage with gefitinib, an EGFR signaling inhibitor ( 100 mg/kg·d ) while the control group to intragastric lavage with an isodose of methyl cellulose. Bilateral femurs and tibias were harvested one week after lavage for separation of EDSCs and bone marrow mesenchymal stem cells ( BMSCs ) respectively using density gradient centrifuga-tion. After proliferative cloning in vitro, expression of the cell surface antigens ( CD29, CD34, CD44 and CD45) of the third passage cells was detected by flow cytometry (FCM). Proliferation of the cells was detected by BrdU, cell cycle was measured by FCM, and expression of the genes related to cell cycle inhibitory factors (p15, p16, p21 and p27) was determined by PCR. ALP staining was performed 14 days after osteogenesis induction. After 21 days of chondrogenic induction, von Kossa staining was conducted. qRT-PCR of the mRNA obtained was used to detect expression of osteogenic differentiation of related genes ( osteocalcin, bsp, runx2 and osterix ). Results CD29 and CD44 were positively expressed while CD34 and CD45 negatively expressed in EDSCs and BMSCs. After the EGFR signaling pathway was blocked by gefitinib, BrdU detection found that gefitinib inhibited BMSCs ( 11.15%) much more than EDSCs ( 0.25%). Cell cycle detection showed that the volume of EDSCs was increased in phases G0/G1 and S but decreased significantly in phase G2-M. ALP staining showed that the increase of EDSCs ALP+ cells (53.31% ) was significantly higher than that of BMSCs (25.04% ) . The increased expression percentages of the genes related to cell cycle inhibitors in EDSCs (103.9%, 58.0%, 117.3% and 105.1%, respectively) were significantly higher than those in BMSCs (39.3%, 38.4%, 24.5% and 83.4%, respectively) ( P ＜0.05). The increased expression percentages of the genes related to osteogenic differentiation in EDSCs (247.0%, 289.9%, 66.1% and 233.2%, respectively) were significantly higher than those in BMSCs (106.5%, 186.4%, 41.7% and 190.8%, respectively). All the above differences were statistically significant ( P ＜0.05) . Conclusions Gefitinib, an EGFR inhibitor, can inhibit proliferation of EDSCs and BMSCs but promote their osteogenic differentiation. It inhibits proliferation of BMSCs more significantly as it promotes osteogenic differentiation of EDSCs.
ABSTRACT
Objective To observe the effect of epidermal growth factor receptor (EGFR) signal pathway inhibitor gefitinib on femoral fracture healing in rats.Methods Eighty SD rats were divided into experimental group (n =40) and control group (n =40) by random number table.An open femur fractured model was established by cutting the middle part of the femoral shaft and placing 1 mm Kirschner wire through medial and lateral femoral patella incision.Rats in the experimental group were given gefitinib (100 mg · kg-1 · d-1),an inhibitor of epidermal growth factor receptor (EGFR) signaling pathway,and rats in the control group were given equal doses of methylcellulose.The X-ray examination was performed before femoral and serum specimens were collected at 1,2,3,4,and 6 weeks.Bone fracture healing was evaluated by Lane and Sandhu X ray scoring and biomechanical test.Then the tissue morphology was observed by HE staining,safranin fast green staining,and microCT detection.The bone volume (BV),bone volume fracture (BV/TV),trabecular number (Tb.N),trabecular thickness (Tb.Th),and trabecular separation (Tb.Sp) were measured by MicroCT.The levels of serum BALP,PINP,TRACP5b,and CTX were measured by Elisa.Results The X-ray scores of the experimental group (1.26 ±0.54,4.94 ± 1.16,and 8.23 ± 1.17) were significantly higher than those in the control group (0.91 ± 0.52,4.11 ± 1.18,and 7.08 ± 0.86) at 1,2 and 3 weeks after the fracture (P ＜ 0.05).The failure load of experimental group at 3 and 4 weeks after fracture [(38.65 ± 1.07) N,(63.63 ± 7.74)N] were significantly larger than those of the control group [(29.47 + 1.00)N,(45.42 + 3.26) N] (P ＜ 0.05).The callus formation in the experimental group was obvious and the healing process of the fracture was faster than that of the control group according to HE and safranin fast green staining.The results of microCT showed that in the experimental group,BV was significantly higher at 2 and 3 weeks after fracture [(59.30 ± 6.54) mm3 and (43.08 ± 2.33) mm3] than those in control group [(42.39 ± 7.82) mm3 and (33.43 ± 5.94) mm3];BV / TV at 2,3,and 4 weeks after fracture [(0.61 ±0.06)％,(0.55 ±0.05)％,and (0.53 ±0.04)％] were significantly higher than those in the control group [(0.48±0.07)％,(0.44±0.07)％,and (0.43±0.03)％];Tb.N at 2,3,and 4 weeks after fracture [(2.05 ± 0.11)/mm,(1.86 ± 0.18)/mm,and (2.034 ± 0.26)/mm] were significantly higher than those in control group [(1.63 ±0.21)/rmm,(1.32 ±0.21)/rmm,and (1.65 ± 0.08)/mm)];Tb.Th at 2,3,and 4 weeks after fracture [(0.33 ± 0.02) mm,(0.33 ± 0.03) mm,and (0.27 ± 0.02) mm] were significantly higher than those in the control group [(0.27 ±0.03)rmm,(0.28 ± 0.02)mm,and 0.23 ±0.01)mm];Tb.Sp at 3 weeks after fracture [(0.39 ±0.07)mm] was significantly higher than that in the control group [(0.30 ± 0.03) mm] (P ＜ 0.05).The levels of serum BALP in experimental group at 2 and 3 weeks after fracture [(2.57 ±0.14) ng/ml,(3.47 ±0.26) ng/ml] were significantly higher than those of control group [(2.07 ±0.19)ng/ml,(2.77 ±0.29)ng/ml];the PINP at 1,2,and 3 weeks after fracture in the experimental group [(2 216.50 ±96.68)pg/ml,(2 692.33 ± 136.76)pg/ml,and (3 196.75 ± 221.90)pg/ml] were significantly higher than those of the control group [(1 969.50 ± 89.34) pg/ml,(2 241.33 ± 107.86) pg/ml,and (2 603.25 ± 361.60) pg/ml];the levels of TRACP5b [(11.58 ± 0.47) ng/ml] and CTX [(8.02 ± 0.40) ng/ml] at 1 week after fracture were higher than those of the control group [(10.33 ± 0.53) ng/ml,(7.11 ± 0.36) ng/ml] (P ＜ 0.05).Conclusion Gefitinib can promote the maturation of osteoblasts and the early reabsorption function of osteoclasts to induce bone formation in advance and accelerate the fracture healing.