The effect of depression on fracture healing and osteoblast differentiation in rats

Abstract

Background: Depressive disorder has been proven to be associated with disturbed bone metabolism. However, the effect of depression on fracture healing still lacks evidence.

Materials and methods: A rat depressive model was first established by exposing the animals to chronic unpredictable stress, which was assessed using the sucrose preference test, forced swimming test, and open field test. Subsequently, the bone repairing ability was detected by micro-computed tomography and histological analysis of the femoral condyle defect rats with or without depression. To further investigate the potential mechanisms of depression on fracture healing, the osteogenic differentiation and autophagic level were compared between the bone marrow mesenchymal stem cells (BMSCs) derived from depressive and normal rats.

Results: Our results showed that rats with depressive disorder significantly slowed the healing process at 4 and 8 weeks postinjury. Furthermore, the osteogenic potential and autophagy remarkably decreased in BMSCs from the depressive rats, suggesting an inherent relationship between autophagy and osteogenic differentiation. Finally, rapamycin, an autophagic agonist, significantly improved osteogenic differentiation of depressive BMSCs through autophagy activation.

Conclusion: The present study indicated that depression had a negative effect on fracture healing with low osteoblast differentiation of BMSCs. Also, autophagy activation in BMSCs offers a novel therapeutic target for depressive patients with poor fracture healing.

Keywords: autophagy; bone marrow mesenchymal stem cells; depression; fracture healing; osteoblast differentiation.

Figure 1

CUS caused depression-like behavior in rats. Notes: (A) Experimental flowchart. (B) The sucrose preference and sucrose consumption were detected by SPT. (C) The immobility time in a beaker of water was detected by FST. (D) The total distance, time spent in center, and number of rearing behaviors were detected by OFT. The values are expressed as the mean ± SD. *P<0.05 vs normal group. ^#P>0.05 vs normal group. Abbreviations: CUS, chronic unpredictable stress; SPT, sucrose preference test; OFT, open field test; FST, forced swimming test; CT, computed tomography; HE, hematoxylin and eosin; BMSCs, bone marrow mesenchymal stem cells.

Figure 2

Depression attenuated bone healing in a rat femur defect model. Notes: The bone defect healing was assessed by micro-CT analysis and histological observation at 4 and 8 weeks postsurgery. (A) 3D reconstruction images were used to evaluate the new bone formation within femur defect in rats. Red bar =200 μm. (B) Micro-CT parameters like BV/TV, Tb.N, Tb.Th, and Tb.Sp of the VOI were determined, respectively. (C) Histologic evaluation of HE stained paraffin-embedded tissue sections (amplification 200×). The values are expressed as the mean ± SD. *P<0.05 vs normal group. Abbreviations: CUS, chronic unpredictable stress; CT, computed tomography; BV/TV, bone volume/trabecular volume ratio; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; VOI, volume of interest; HE, hematoxylin and eosin.

Figure 3

BMSCs derived from depressive rats had low osteogenic potential and autophagic level. Notes: (A) Representative picture of ALP and ARS staining to detect osteogenic differentiation (amplification 200×). (B) The gene expressions of osteogenic markers COL I, OCN, OPN, and RUNX2 were examined by qPCR. Relative mRNA expressions were normalized to GAPDH. (C) Morphological observation of autophagy under TEM (amplification 15,000×). Red arrows represent the characteristic double-membranous ultrastructural morphology of autophagic vacuoles. N represents cell nucleus. (D) Western blot for the autophagy-related protein levels of LC3 and P62. The rate of LC3 II/I and P62/β-actin represent the relative expressions. The values are expressed as the mean ± SD. *P<0.05 vs normal group. Abbreviations: ALP, alkaline phosphatase; ARS, Alizarin red S; CUS, chronic unpredictable stress; BMSCs, bone marrow mesenchymal stem cells; qPCR, quantitative polymerase chain reaction; TEM, transmission electron microscopy.

Figure 4

Autophagy activation promoted osteogenic differentiation of depressive BMSCs. Notes: The depressive BMSCs were cultured in osteogenic differentiation medium in the presence of autophagy activator RAP. (A) Autophagic levels were detected by Western blot. (B) The levels of osteogenic differentiation were detected by ARS and ALP staining (amplification 200×). (C) The gene expressions of osteogenic marker COL I, OCN, OPN, and RUNX2 were examined by qPCR. Relative mRNA expressions were normalized to GAPDH. The values are expressed as the mean ± SD. *P<0.05 vs control group. Abbreviations: RAP, rapamycin; ALP, alkaline phosphatase; ARS, Alizarin red S; BMSCs, bone marrow mesenchymal stem cells; qPCR, quantitative polymerase chain reaction.