Gene Therapy (2008) 15, 1523–1535 & 2008 Macmillan Publishers Limited All rights reserved 0969-7128/08 $32.00 www.nature.com/gt ORIGINAL ARTICLE Treatment of avascular necrosis of the femoral head by hepatocyte growth factor-transgenic bone marrow stromal stem cells QWen1,LMa1, Y-P Chen2, L Yang2, W Luo1 and X-N Wang3 1Institute of Molecular Immunology, Southern Medical University, Guangzhou, People’s Republic of China; 2Department of Imageology, Nanfang Hospital, Guangzhou, People’s Republic of China and 3School of Biosciences and Bioengineering, South China University of Technology, Guangzhou, People’s Republic of China The treatment of hormone-induced early-stage avascular pathological sections. A regular arrangement of trabeculae necrosis of the femoral head (ANFH) with transplantation of and obvious bone regeneration were observed in the hepatocyte growth factor (HGF)-transgenic bone marrow animals receiving transplanted transgenic BMSCs with stromal stem cells (BMSCs) was examined. A rabbit model of FG. Newly generated capillaries were visible on the hormone-induced early ANFH was first established. BMSCs bone plates of the trabeculae, and the bone marrow was were transplanted by core decompression under the rich in hematopoietic tissue. These results demonstrate guidance of computed tomography (CT). A supportive that the combination of core decompression and trans- fibrinogen drug delivery mixture (FG) was tested for plantation of HGF transgenic autologous BMSCs enhanced mechanical enhancement of stem cell delivery. Therapeutic blood vessel regeneration and bone reconstruction in efficacy was evaluated by CT, magnetic resonance imaging the ANFH model. This study provides experimental data that (MRI), CT perfusion imaging, ink artery infusion angiography, motivate possible clinical use of this therapeutic strategy. hematoxylin-and-eosin staining and immunohistochemical Gene Therapy (2008) 15, 1523–1535; doi:10.1038/gt.2008.110; staining for extracellular signal-regulated kinase-1/2 of published online 17 July 2008 Keywords: avascular necrosis of the femoral head; hepatocyte growth factor; bone marrow stromal stem cells; fibrin glue Introduction The most widespread procedure used to treat early stages of ANFH is core decompression of the hip, which Avascular necrosis of the femoral head (ANFH) is a involves drilling a small hole in diseased, necrotic bone. progressive pathological process that primarily afflicts The goal of core decompression is to decrease the people under 40 years of age. Without timely effective intraosseous pressure and allow the regrowth of healthy treatment, ANFH causes in situ avascular necrosis, and bone tissue.4 Although the efficacy of this procedure ultimately deforms the bone. The consequence is remains controversial, it has been used for more than impaired hip joint function and permanent disability.1–3 three decades.5,6 Reconstruction of blood circulation and Whether the etiology of ANFH is traumatic or aseptic stimulation of bone repair are important aspects of nontraumatic, the basic cause is a vicious cycle of healing that support this surgical intervention. Thus, the elevated intraosseous pressure and obstructed blood combination of core decompression with the delivery of supply in the femoral head. ANFH is considered angiogenic or osteoinductive agents may improve the irreversible, and any diagnostic or therapeutic strategy results of this surgical procedure in the future.7 for ANFH is best introduced in the early stage. Early Vasodilatation and drug intervention therapy to intervention will reduce intraosseous pressure and activate blood and remove stasis are currently used to improve the blood supply to the necrotic femoral treat existing blood vessels, which results in disease head. Osseous repair should be performed alongside remission but are not permanent solutions. However, the supplemental interventions. induction of blood vessel regeneration and the construc- tion of a collateral circulation are the most effective ways Correspondence: Professor L Ma, Institute of Molecular Immuno- to break the vicious pathological cycle of arterial disease logy, Southern Medical University, No. 1838, Northern Guangzhou or necrosis. Avenue, Guangzhou, Guangdong, 510515, People’s Republic of Cytokine supplement therapy is a potential treatment China. for peripheral arterial disease. Hepatocyte growth factor E-mail: [email protected] or X-N Wang, School of Biosciences and (HGF), originally purified and cloned as a mitogen for Bioengineering, South China University of Technology, Guangzhou, hepatocytes,8,9 is a novel member of the endothelium- Guangdong, 510641, People’s Republic of China. E-mail: [email protected]. specific growth factors, and it can stimulate angiogen- Received 14 January 2008; revised 14 May 2008; accepted 16 May esis. Interestingly, HGF can stimulate a significantly 2008; published online 17 July 2008 higher level of DNA synthesis than basic fibroblast Treating ANFH with HGF-transgenic BMSCs QWenet al 1524 growth factor or vascular endothelial growth factor In a rabbit model of hormone-induced early ANFH, the (VEGF). Comparatively, HGF is the most potent stimu- BMSC–FG mixture was transferred into the necrotic lator of endothelial cell growth.10,11 Laboratory animals femoral head under computed tomography (CT) control with induced acute hepatic or renal injury showed much during core decompression. To evaluate therapeutic less histological damage and more satisfactory organ effects, pathological changes were observed with multi- function after treatment with HGF. This putative anti-cell ple imaging techniques. This study provides evidence for death function of HGF was further supported by motivating ultimate clinical use of this stem-cell strategy. subsequent studies with renal epithelial cells and neurons,12,13 as well as hepatocytes.14,15 Due to its ability to prevent a decrease in DNA synthesis and cell death in Results endothelial cells under serum-free treatment,16 HGF should be reclassified as a new growth factor with anti- Transfection of recombinant adenovirus and cell death actions. With these characteristic effects on cell expression of HGF in BMSCs survival, HGF therapy may be a useful therapeutic The infectious titer of adenoviral vectors carrying human 10 À1 strategy for ANFH. HGF gene (Ad-HGF) was 2.6 Â 10 TCID50 ml and the Bone marrow stromal stem cells (BMSCs) are pluripo- high-performance liquid chromatography purity was tent cells17 that secrete large amounts of growth factors and greater than 95% (Figure 1a). HGF expression in BMSCs angiogenic factors. BMSCs are bone progenitor cells with was confirmed at the mRNA level by a single 2.2-kb excellent potential for differentiation into osteoblasts18,19 band after reverse transcription-PCR assay. There was no that can restore bone tissue.20 BMSCs can be easily isolated corresponding band amplified from nontransfected from bone and transfected with an exogenous gene. BMSCs (Figure 1b). The transfection efficacy of Ad-GFP Moreover, autotransplantation of BMSCs induces no was about 98% at a multiplicity of infection of 300, immunological rejection. For these reasons, BMSCs are assayed by FACS (Figure 1c). regarded as the seed cells with the most potential for bone The HGF protein expressed by HGF transgenic BMSCs tissue engineering in the clinical context.21,22 was secreted into the culture supernatant. The expres- Our study used recombinant adenovirus carrying the sion peak occurred at 48 h after transfection, and the human HGF gene in the AdMax adenovirus vector expression level was 133 ng mlÀ1. HGF protein expres- system to transfect the second generation of autologous sion and secretion was observed for 2 weeks (Figure 2). BMSCs in vitro. Transgenic BMSCs strongly expressing Cell-cycle analysis demonstrated that Ad vector transfec- HGF protein were used as seed cells, and mixed with tion does not affect the cell cycle of the BMSCs, medical fibrin glue (FG), which functioned as a scaffold. regardless of the exogenous gene (Table 1). These results a mAU 28.451 1200 b 123 1000 15000 10000 7500 800 5000 2500 1000 600 250 400 200 0 0 5 1015202530 min c 400 400 12 320 320 240 1.07% 240 98.00% M1 M1 Counts 160 Counts 160 80 80 0 0 100 101 102 103 104 100 101 102 103 104 GFP GFP Figure 1 Ad vector construction and identification. (a) High-performance liquid chromatography analysis of Ad-HGF purity after CsCl ultracentrifuge. The purity was more than 95%. (b) Reverse transcription-PCR analysis of Ad-HGF after transfection into BMSCs. Lane 1: DL15000 molecular marker ladder; lane 2: BMSCs without transfection; lane 3: BMSCs transfected with Ad-HGF. There is a specific 2.2-kb band reflecting transcription of the HGF gene (mRNA). (c) Flow cytometry analysis of transfection efficiency of Ad-GFP into BMSCs. (1) BMSCs without transfection; (2) BMSCs transfected with Ad-GFP, multiplicity of infection (MOI) ¼ 300. Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1525 demonstrated that Ad-GFP will not affect the HGF epiphyseal line was clear. By contrast, femoral heads expression or BMSC cell cycle. subjected to induced ANFH group showed multiple spot-like low-density areas of structural change. The bone cortex was thinner, the epiphyseal line was blurred Evaluation of therapeutic efficacy and osteoporosis was evident. In the transgenic BMSC Radiology evaluations group, the articular cavity was reduced and the spot-like Computed tomography. Core decompression was then or line-like high-signal area in the femoral head was conducted