HIF-1α transgenic bone marrow cells can promote tissue repair in cases of corticosteroid-induced osteonecrosis of the femoral head in rabbits

Abstract

Although corticosteroid-induced osteonecrosis of the femoral head (ONFH) is common, the treatment for it remains limited and largely ineffective. We examined whether implantation of hypoxia inducible factor-1α (HIF-1α) transgenic bone marrow cells (BMCs) can promote the repair of the necrotic area of corticosteroid-induced ONFH. In this study, we confirmed that HIF-1α gene transfection could enhance mRNA expression of osteogenic genes in BMCs in vitro. Alkaline phosphatase activity assay and alizarin red-S staining indicated HIF-1α transgenic BMCs had enhanced osteogenic differentiation capacity in vitro. Furthermore, enzyme linked immunosorbent assay (ELISA) for VEGF revealed HIF-1α transgenic BMCs secreted more VEGF as compared to normal BMCs. An experimental rabbit model of early-stage corticosteroid-induced ONFH was established and used for an evaluation of cytotherapy. Transplantation of HIF-1α transgenic BMCs dramatically improved the bone regeneration of the necrotic area of the femoral head. The number and volume of blood vessel were significantly increased in the necrotic area of the femoral head compared to the control groups. These results support HIF-1α transgenic BMCs have enhanced osteogenic and angiogenic activity in vitro and in vivo. Transplantation of HIF-1α transgenic BMCs can potentially promote the repair of the necrotic area of corticosteroid-induced ONFH.

Figure 1. The transduced BMCs and the western blotting to dectect the expression of HIF-1a.

(A): The transduced BMCs were sorted with FACS and checked by fluorescence microscope. (B): The western blotting confirmed the overexpression of HIF-1a in Lenti-HIF-1a-transduced BMCs and there was nearly no expression in Lenti-GFP-transduced BMCs and normal BMCs.

Figure 2. Gene expression of related factors in transduced and normal BMCs.

The mRNA expression level of HIF-1a (A), VEGF (B), OCN (C) and ALP (D) at day 4, 7, 14, 21 and 28 after transduction. #, p<0.01 (compared with Lenti-GFP-transduced BMCs) and *, p<0.01 (compared with normal BMCs). Abbreviations: HIF-1a, hypoxia-inducible factor-1 alpha; VEGF, vascular endothelial growth factor; OCN, osteocalcin; ALP, alkaline phosphatase.

Figure 3. VEGF production, Alkaline Phosphatase assay and Alizarin red-s staining in vitro.

(A): VEGF production in Lenti-HIF-1a-transduced BMCs was significantly increased as compared to that in Lenti-GFP-transduced BMCs and normal BMCs for at least 28 days. (B): Alkaline Phosphatase activity in Lenti-HIF-1a transfected BMCs was about 2-fold higher than that in Lenti-GFP transduced BMCs and normal BMCs 14 days after osteogenic induction. (C): Alizarin red-S staining showed more calcium deposits in Lenti-HIF-1a-transduced BMCs 21 days after osteogenic induction. (D): Quantitative analysis revealed an increase of about 1.5-fold in extracellular matrix mineralization in Lenti-HIF-1a-transduced BMCs compared to Lenti-GFP-transduced BMCs and normal BMCs. #, p<0.01 (compared with Lenti-GFP BMCs) and *, p<0.01 (compared with normal BMCs).

Figure 4. Representative three-dimensional images of the femoral head obtained by microCT.

The subchondral trabeculae of the femoral head appeared intact and well distributed In Group I and II (A, B). The subchondral trabeculae became thin and sparse In Group III (C). In Group IV, local destroyed trabeculae could be observed with a fewer amount (D). The quantitative analysis of microCT showed significantly higher values in Group I than other three groups in BV/TV and BMD (p<0.01) (E and F). BV, bone volume; TV, trabecular volume; BMD, bone mineral density.

Figure 5. Assessment of new bone regeneration in the necrotic area.

Representative histological photomicrograph of the necrotic area of the femoral head showed massive trabecular tissue had formed in Group I and there were few granulation tissue (A). In Group II, there were fewer trabecular tissue and more granulation tissue as compared to that in Group I (B). In Group III, a few osteocyte filled lacunae were replaced by empty lacunae. The marrow fat cells were increased compared to that of Group I and II (C). In Group IV, there were rarely any trabecular tissue, and a lot of empty lacunae were observed (D). Scale bars = 50 um. Arrowheads indicate empty lacunae. (E) Histomorphometric analysis showed new bone density in Group I was significantly more than that of the other three groups (p<0.01).

Figure 6. Assessment of femoral head neovascularization.

(A): Representative images of microCT 2-D sections and reconstructed 3-D microangiography of the femoral head from various groups. Quantitative analysis of MicroCT showed mean number of blood vessels (B) and vessel volume (C) of Group I were significantly higher than that of the other three groups (p<0.01).

Figure 7. Immunohistochemistry for CD31.

There were more CD31-positive vessels observed in Group I (A), compared to that in Group II (B). Few CD31-positive vessels were observed in Group III (C), while nearly none were present in Group IV (D). White arrowheads indicate CD31-positive vessels. Scale bars = 500 um.

Figure 8. Immunofluorescence of the necrotic area of the femoral head.

(A): DAPI and GFP merged pictures revealed the transplanted GFP-positive BMCs existed in the necrotic area of the femoral head in Group I. (B): Immunofluorescence for OCN showed some GFP-positive cells in the necrotic area expressed OCN. White arrowheads indicate colocalization. Scale bars = 25 um. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; OCN, osteocalcin.