Elsevier

Bone

Volume 81, December 2015, Pages 544-553
Bone

Original Full Length Article
Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis

https://doi.org/10.1016/j.bone.2015.09.005Get rights and content

Highlights

  • The function of BMMSC was impaired during steroid-related ONFH.

  • Hypoxic preconditioning of BMMSCs provided an effective strategy for treatment of ONFH.

  • The mechanism involves: enhanced growth factors releasing, stimulated angiogenesis and osteogenesis.

Abstract

Purpose

Osteonecrosis of the femoral head may be a disease resulting from abnormal proliferation or differentiation of mesenchymal stem cells. The present investigation explored the novel strategy of hypoxia-preconditioned BMMSCs to reverse the impairment of osteonecrosis BMMSCs and enhance the therapeutic potential of hypoxia-treated BMMSC transplantation.

Methods

BMMSCs from the anterior superior iliac spine region of osteonecrosis rabbit were cultured under 20% O2 or 2% O2 conditions. Normal BMMSCs were cultured under 20% O2 condition as control. Growth factors secreted were examined by enzyme-linked immunosorbent assay. 20% O2 or 2% O2 BMMSCs were injected into the femoral head of rabbits after core decompression. Cell viability and apoptosis were assessed in vitro, and TUNEL staining of the femoral head was analyzed after transplantation. Angiogenesis (capillary-like structure formation, CD31 immunohistochemical staining and ink infusion angiography) and osteogenesis (Alizarin red-S staining, micro-CT scanning and OCN immunohistochemical staining) tests were conducted as well.

Results

2% O2 exposure up-regulated growth factor secretion in BMMSCs. Apoptosis in 2% O2 group was lower when compared with that in 20% O2 osteonecrosis group. Cell viability in 2% O2 was significantly higher when compared with that in 20% O2 osteonecrosis group. Growth factor secretion, cell viability, apoptosis, capillary-like structure formation, Alizarin red-S staining, and ALP staining showed no difference between the 2% O2 BMMSC and normal BMMSC groups. Transplantation of 2% O2 versus 20% O2 mesenchymal stem cells after core decompression resulted in an increase in angiogenesis function and a decrease in local tissue apoptosis. Our study also found that osteogenesis function was improved after hypoxic stem cell transplantation.

Conclusion

Hypoxic preconditioning of BMMSCs is an effective means of reversing the impairment of osteonecrosis BMMSCs, promoting their regenerative capability and therapeutic potential for the treatment of osteonecrosis.

Introduction

Osteonecrosis of the femoral head (ONFH) is a difficult and complicated orthopedic disorder that often leads to femoral head collapse with activity-related hip pain [1]. Several treatment methods for ONFH have been attempted, including drug therapy, surgical core decompression, vascularized bone grafting, and osteotomy. However, these methods have barely been effective in treating the disease. Although the pathogenesis and etiology of ONFH have not yet been completely understood, the interrupted blood circulation in the femoral head that leads to osseous tissue necrosis seems to be the major factor [2], [3], [4]. Determining how to promote vascular repair and angiogenesis in ONFH becomes a crucial part of the treatment.

Bone marrow mesenchymal stem cells (BMMSCs) are multipotent adult stem cells that are able to differentiate into several cell lineages, including endothelial cells and osteocytic cells. Both animal and clinical studies have provided evidence that BMMSC transplantation has potency in the treatment of ischemic diseases, such as myocardial infarction and ONFH [5], [6]. A major limitation in stem cell therapy for ischemic diseases is the low survival rate of transplanted cells in the ischemic and peri-necrosis region [7]. Thus, improving grafted cell survival is vital for enhancing the efficacy and efficiency of stem cell therapy. However, Hernigou and Beaujean demonstrated that abnormal proliferation and differentiation of MSCs led to osteonecrosis of the femoral head [8]. Later, Wei et al. suggested that glucocorticoid has a direct effect on impairing the proliferation and function of BMMSCs [9]. It would be helpful if we discovered a new way to reverse the impairment of osteonecrosis BMMSCs for treating ONFH.

Hypoxic preconditioning (HP) by sublethal hypoxic insult stimulates endogenous mechanisms resulting in compensatory regulation including protein expressions that protect against future lethal hypoxia and other insults [10]. On the basis of the well-documented manifold benefits of HP, in the present investigation, we hypothesize that the HP approach in BMMSC transplantation therapy can lead to a set of multiple responses that increase trophic factor support, reduce cell apoptosis, stimulate endothelial and osteogenic differentiation, and finally reverse the impairment of osteonecrosis BMMSCs. A combination of these effects will result in increased angiogenesis, osteogenesis, and ultimately better bone tissue repair after osteonecrosis.

Section snippets

The rabbit model of early ONFH

Twenty-eight 26–28-week old male mature New Zealand White rabbits with body weight of 3.2–4.0 kg for each group (model group, 20% O2 BMMSC group, and 2% O2 BMMSC group) were used in this experiment. The rabbits were randomly assigned to the groups in our study. All investigators were blinded to the treatment group for all analyses.

The ONFH induction procedure was performed using the following method. First, lipopolysaccharide (LPS; Sigma, USA) (10 μg/kg) was injected through an ear vein.

Characterization of rabbit BMMSCs

BMMSCs were successfully expanded three days after initial seeding, and they rapidly expanded into colonies of confluent spindle cells at ten to fourteen days. The third-passage cells were incubated with antibodies of both CD105 and CD34. They were chosen as markers in flow cytometry. The results showed that the cells were positive to CD105 (99.12%) and negative to CD34 (0.88%). The cultured cells were effectively BMMSCs.

Histological analysis of the ONFH model group and core decompression groups

The model group showed discrete trabecular bone with many empty lacunae,

Discussion

Osteonecrosis of the femoral head (ONFH) is a relatively common disease. Because the pathogenesis and etiology of nontraumatic osteonecrosis of the femoral head (ONFH) has not been completely understood, current treatment of ONFH simply focuses on preventing irreversible complications, such as the biomechanical collapse of the femoral head and osteoarthritis of the hip joint. However, in many cases, these treatments have not been effective in preventing the disease [17].

Previous studies

Conflicts of interest

Lihong Fan, Chen Zhang, Zefeng Yu, Zhibin Shi, Xiaoqian Dang, and Kunzheng Wang declare that they have no conflict of interest.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Grant nos. 81101363, 81371944, 81301562 and 81572145) and the Fundamental Research Funds for the Central Universities.

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