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 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 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 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

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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 under CT control (Figure 3). Normal (control) lower than that in the core decompression-alone group. femoral heads were symmetrical, the joint surface was In the transgenic BMSC–FG group, femoral heads were distinct and the cortex was smooth and intact. There was symmetrical and the high-signal area was even lower no change in the texture of cancellous bone, and the than that in the transgenic BMSC group (Figure 4).

160 Magnetic resonance imaging. In normal bone, femoral * Control 140 heads were symmetrical. In FS-T2WI, the high-level HGF signal of fat under the cortex of the femoral head was 120 inhibited and showed a low-level signal. In the ANFH 100 group, magnetic resonance imaging (MRI) examination of * the femoral head showed a larger articular cavity than in 80 normal bone. The high-level FS-T2WI signal at the * metaphyses suggested edema in the bone marrow. The 60 cancellous bone of the femoral head appeared with 40 irregular spot-like or line-like patterns of structural HGF expression (ng/ml) changes in the high-level signal image. In the core 20 * decompression-alone group, the spot-like or line-like high-signal area was relatively obvious. In the transgenic 0 0 2468101214 BMSC group, the articular cavity and the spot-like or line- Time (day) like high-signal area was somewhat reduced. The spot- like low-density area in the transgenic BMSC–FG group Figure 2 HGF protein expression in Ad-HGF transfected and was lower than that in the transgenic BMSC group, but Ad-GFP-transfected BMSCs over 14 days. Black bars, Ad-HGF at not significant (P40.05). Both the transgenic BMSC group a multiplicity of infection (MOI) of 300. White bars, Ad-GFP transfected cells. The supernatant was collected at time points and the transgenic BMSC–FG group showed a reduced indicated on the x axis (days), and HGF expression was determined femoral head necrosis volume (o5%) compared with the by enzyme-linked immunosorbent assay. Statistical difference ANFH group (10%), and this difference was significant between Ad-HGF-transfected BMSCs and Ad-GFP control BMSCs (Po0.05). Core decompression alone did not show any were calculated, and significance is indicated with an asterisk (*), difference from ANFH (Figure 5). Po0.05, n ¼ 3.

CT perfusion imaging. In the normal group, femoral heads appeared symmetrical. Blood volume (BV) was Table 1 Cell-cycle analysis by FACS (%) ðx Æ s; n ¼ 3Þ also relatively high and became red. Femoral heads in the ANFH group were also highly symmetrical, but the Group Cell cycle BV was reduced to about 35% of that the normal group (Po0.05) and was blue. The femoral heads of the core G G S 1 2 decompression-alone group had increased BV compared with that of the ANFH group, and the proportion of Ad-HGF transfection 79.17±0.35 6.33±0.06 14.57±0.35 Ad-GFP transfection 80.50±3.58 5.47±1.36 14.03±2.31 the red/green area was increased, but there was no Nontransfection 81.00±1.08 7.28±3.72 13.20±0.69 significant difference between these two groups. The BV of the femoral heads in the transgenic BMSC group was

Figure 3 Core decompression guided by CT. The hips were visualized by CT (left). After the animals were anesthetized, the trocar was applied to puncture through skin and along the medial side of the great trochanter on the femoral head (right). The tip of the needle is located in the femoral head. Core decompression and delivery of BMSCs ensued.

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1526

Figure 4 Conventional CT examination of hormone-induced ANFH in the rabbit (coronal reconstruction). (a) Normal group. (b) ANFH group. (c) [L], transgenic BMSCs group; [R], core decompression-alone group. (d) Transgenic BMSCs-FG group. Arrows indicate the location of the femoral head.

Figure 5 MRI examination of hormone-induced ANFH in the rabbit and semi-quantitative analysis. (a) Normal group. (b) ANFH group. (c) [L] transgenic BMSCs group; [R], core decompression-alone group. (d) Transgenic BMSC–FG group. Circles show the location of the femoral head. (e) Bar graph represents quantification of necrosis volume in the femoral head. * ¼ Po0.05, as compared with normal group; # ¼ Po0.05 as compared with the core decompression-alone group. In the bar chart, treatment groups are represented by letters in this and subsequent figures: group A ¼ normal; group B ¼ ANFH+no therapy; group C ¼ ANFH+core decompression; group D ¼ ANFH+core decompression+ transgenic BMSC; group E ¼ ANFH+core decompression+transgenic BMSC–FG.

higher than that of the core decompression-alone group decreased by 82%. In the core decompression-alone (Po0.05), and was red. The BV of both femoral heads in group, there were twofold more unobstructed blood the transgenic BMSC–FG group was similar to that of the vessels than in the ANFH group. However, there was normal group (P40.05) and was not significantly still obvious thrombosis in the core decompression-alone different from the transgenic BMSC group (P40.05) group. In the transgenic BMSC group, the blood (Figure 6). vessels under the were reconstructed and there were large recovery blood vessels in the metaphyses. In the transgenic BMSC–FG group, blood vessels Histopathological assessment were almost fully reconstructed and were distributed Ink artery infusion angiography. In the normal femoral evenly and abundantly (Figure 7). Interestingly, the head, the was rich in capillaries that number of unobstructed blood vessels in the BMSC–FG formed a network. The vessels in the network were group was more than threefold greater than those in patent, and small vessels were abundant. In contrast, the ANFH group (Po0.05). There was no significant femoral heads in the ANFH group showed fewer difference in the number of unobstructed blood blood vessels, and the capillary network was sparse. vessels between the two BMSC transplantation groups The blood vessels appeared obstructed, and the shallow- (P40.05). layer vessels under the joint surface of the femoral head had almost disappeared. Compared with the normal Histopathological observations. The of group, the number of unobstructed blood vessels the normal group femoral heads was smooth, and the

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1527

Figure 6 Computer tomography perfusion image of hormone-induced ANFH in the rabbit (blood volume (BV) image) and semiquantitative analysis. The left color bar denotes the BV level. Blue to red represents increasing BV. (a) Normal group. (b) ANFH group. (c) Core decompression-alone group. (d) Transgenic BMSC group. (e) Transgenic BMSC–FG group. (f) Quantitative comparison between treatment groups; asterisk (*) shows statistically significant difference from the normal group (Po0.05).

Figure 7 Ink artery infusion angiography of hormone-induced ANFH in the rabbit and semiquantitative analysis. (a) Normal group. (b) ANFH group. (c) Core decompression-alone group. (d) Transgenic BMSC group. (e) Transgenic BMSC–FG group. (f) Bar graph shows the ratio of the area of ink artery to the whole area of femoral head, observed at least in five fields per section. Asterisk (*) shows statistically significant difference from the normal group (Po0.05).

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1528 cartilage cells were in a tidy arrangement. The trabeculae Discussion were intact, and their arrangement was regular, compact and full. The bone cells in the trabeculae were clearly Taken together, our findings suggest that animals at an visible and there were few empty bone lacunae. The early stage of ANFH may recover healthy tissue with were columnar, cuboidal or spindle shaped core decompression therapy in conjunction with BMSC and distributed along the trabeculae in strings. There transplantation. CT and MRI examination showed the were abundant medullary hematopoietic cells and small differences in femoral head configuration among the fat cells, and the cell morphology was normal. different treatment groups. Radiology findings sug- The ANFH group showed multiple differences from gested that the animals in ANFH groups had stage-I or the normal group. The periosteum of the femoral head -II osteonecrosis of the femoral head, according to the was incomplete and the cartilage cells were partly shed. system of the Association Research Circulation Osseous. The bone marrow cells were also decreased to nearly CT perfusion imaging demonstrated changes in BV, 30% of values seen in the normal group (Po0.05). There with increases in the ANFH group, and comparative were few trabeculae, which were thin, showed disor- improvements in the two transgenic BMSC groups. dered texture and were often broken into fragments. The Consistent with the changes in the femoral head nuclei of the bone cells were condensed. The empty configuration shown by radiological scans, histopatho- lacunae in the trabeculae increased nearly onefold logical assays demonstrated a profound change in the compared with the normal group (Po0.05). Few spin- blood flow, hematopoietic cell number in the bone dle-shaped osteoblasts were distributed along the trabe- marrow and bone cells in the trabeculae of BMSC-treated culae. The medullary hematopoietic areas were poorly groups. The results showed that serum- and hormone- organized and had fewer cells, a sparse capillary induced pathologies in the ANFH model can be network and partly obstructed blood vessels. The fat alleviated by core decompressions. Such pathologies cells were larger and had fused together into bubbles. include blood vessel obstruction, proportions of hema- Compared with the ANFH group, the core decom- topoietic cells in the bone marrow and bone cells in the pression-alone group showed a somewhat decreased trabeculae. These ANFH-associated changes may be number of empty lacunae, but the effect was not rectified to some extent after core decompression treat- significant for this observation period (P40.05). Meta- ment, and particularly when it is supplemented with physeal osteoblasts had proliferated greatly and new implantation of transgenic BMSCs. bone had been generated. The hematopoietic tissue was In 2002, Vale´rie Gangji et al.23 applied core decom- scarce, predominantly in fat cells, and had no significant pression and implantation of autologous bone-marrow difference compared with the ANFH group (P40.05). mononuclear cells to treat 13 patients (18 hips) with the In the transgenic BMSC group, the trabeculae were Association Research Circulation Osseous stage-I or II generally in a tidy arrangement; compared with the necrosis of the femoral head. After 24 months, there was ANFH group, the bone matrix was increased, and the a significant reduction in pain measured with visual number of empty bone lacunae had decreased. Newly analog scale (P ¼ 0.021) and in joint symptoms measured generated capillaries were visible on the bone plate, and with the Lequesne index (P ¼ 0.001) and the Western there were many osteoblasts at the edges of the trabeculae. Ontario and McMaster Universities (WOMAC) In the transgenic BMSC–FG group, the trabeculae Osteoarthritis index (P ¼ 0.013). The ratio of the volume were also in a tidy arrangement and there was obvious of necrotic lesion to that of the whole femoral head was new bone. Newly generated capillaries were visible on significantly decreased by a mean of 35% in the bone- the bone plate of the trabeculae. There was abundant marrow-graft group after 24 months (P ¼ 0.001). The core hematopoietic tissue in the bone marrow and expansion decompression-alone group increased by a mean of 23%. of new blood vessels (Figure 8). In general, transgenic The researchers thought this finding suggested that BMSC groups showed a significant decrease in the necrotic lesions might be reversible to some extent. On number of empty lacunae (Po0.05, compared with the basis of this successful case, we further tested the ANFH group). Although the level of empty lacunae therapeutic improvements for human ANFH and eval- in the BMSC groups was not the same as those in the uated it on the rabbit model of early-stage ANFH. Gene normal group, they were very low. The number of bone therapy, stem cell transplantation and tissue engineering marrow hematopoietic cells in both the transgenic BMSC have been rationally combined in our study. Simulta- group and the transgenic BMSC–FG group were also neous with physical decompression, the HGF secreted by very close to measures in the normal group (P40.05). the transplanted BMSCs was released in a sustained way Extracellular signal-regulated kinase (ERK)-1/2-posi- from the FG, and induced the local regeneration of new tive bone marrow cells in the ANFH group were only blood vessels and the construction of a collateral 37.5% of measures in the normal group. In the core circulation. Blood supply was improved in the femoral decompression-alone group, the number of bone marrow head. Locally, BMSC could be induced to differentiate cells expressing ERK1/2 was nearly twofold higher than into osteoblasts to repair the necrotic bone tissue. those in the ANFH group, slightly higher than the Our study integrates core decompression, vascular normal groups. However, there was no significant regeneration and bone reconstruction, and provides a difference between these three groups (P40.05). Inter- new therapeutic mode for ANFH. To date, no other estingly, both transgenic BMSC groups showed a report of a similar therapeutic model exists. The significantly increased number of ERK1/2-positive bone therapeutic efficacy of transgenic BMSC treatment is marrow cells compared with the three other groups. In evidenced by the following three sets of data. (1) addition, FG compound made no difference in this Radiological scans showed a difference in the normal, measure, as confirmed by statistical comparison ANFH and treated femoral heads of the rabbit early (P40.05) (Figure 9). ANFH model. But after treatment, the signals in the

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1529

Figure 8 Histopathological images of hormone-induced ANFH in the rabbit and semiquantitative analysis. Panels a–e show hematoxylin- and-eosin stain in sample tissue, scale bar ¼ 100 mm. (a) Normal group. (b) ANFH group. (c) Core decompression-alone group. (d) Transgenic BMSC group. (e) Transgenic BMSC–FG group. (f) Bar graph represents the ratio of the bone marrow cells to the area of bone marrow observed in at least in five fields per section. (g) Bar graph represents the ratio of the empty lacunae to the area of trabeculae observed in at least in five fields per section. Asterisk (*) shows statistically significant difference from the normal group (Po0.05). femoral head of transgenic BMSC groups changed in the These processes led eventually to avascular necrosis in direction of normal bone tissue. (2) The blood vessels the bone. Our rabbit model-induced ANFH results are under the cartilage appeared reconstructed. The blood consistent with the imaging characteristics of early vessels in the transgenic BMSC–FG group were almost ANFH according the system of the Association Research fully reconstructed and were distributed evenly and Circulation Osseous, and can also be classified as stages I abundantly. (3) Osteogenesis activity is vigorous. Com- and II according to the clinical Ficat stage criteria. Our pared with the ANFH group, there were obvious model resembles the clinical disease progress of human newborn blood cells and new bone formation in both hormone-induced ANFH, because large doses of hor- transgenic BMSC-treatment groups. mone used to treat patients with other diseases such as As early diagnosis and intervention for ANFH is arthritis or vasculitis can eventually cause ANFH. For extremely important, we were motivated to establish the example, systemic lupus erythematosus is associated rabbit model of hormone-induced early ANFH. We used with a high incidence rate of ANFH due to the horse serum to cause vasculitis, and combined it with a consequence of necessary hormone therapy.25 large dose of prednisolone acetate to inhibit the synthesis Angiogenic factors are a group of cytokines that of collagen and elastic protein.24 The subsequent aggra- induce the generation of blood vessels. Of the numerous vated contraction of the blood vessels with vasculitis, angiogenic factors, VEGF is known to be the most potent platelet aggregation and necrosis of endothelial cells and is used most frequently in the clinical context. Some causes the breakdown and obstruction of small arteries. researchers have tried to introduce VEGF to induce new

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1530

Figure 9 Extracellular signal-regulated kinase (ERK)-1/2 labeling in the femoral head, and semiquantitative analysis. Panels a–e show immunohistochemical labeling for a sample tissue from each treatment group (scale bar ¼ 50 mm). (a) Normal group. (b) ANFH group. (c) Core decompression-alone group. (d) Transgenic BMSC group. (e) Transgenic BMSC–FG group. (f) Bar graph represents the percentage of ERK1/2-positive bone marrow cells observed at least in five fields per section. Asterisk (*) shows statistically significant difference from the normal group (Po0.05).

blood vessels in ANFH, but have been unsuccessful, increased somewhat in the core decompression-alone possibly for two reasons. First, no effective means of group and significantly increased in the two transgenic decompression has been adopted for these schemes. BMSC groups. This result suggests that in the normal Therefore, it is difficult for an ideal network of new blood condition without stimulation, ERK1/2 is not popularly vessels to be generated, in the same way that it is difficult activated. Once some kind of interference such as core for bamboo shoots under a huge stone to emerge from decompression was taken, and the intraosseous pressure the ground. Second, VEGF also increases vascular was relatively decreased, ERK1/2 was stimulated, but permeability, which in turn causes tissue edema, and the level was not enough to support the recovery of the further increases intraosseous pressure.26,27 This in- entire femoral head. Overexpression of HGF by trans- creased pressure undermines the necessary effort toward genic BMSCs likely increased the activation level of decompression in ANFH. Another angiogenic factor ERK1/2. In these treatment groups, the recovered blood often used in the clinical setting is basic fibroblast circulation and new bone formation were significant. growth factor. However, it enhances the proliferation of Therefore, activation of ERK1/2 by HGF in endothelial smooth muscle in blood vessels, as well as that of cells and supports a hypothesis that HGF vascular endothelial cells, which may cause arterial signaling promotes the proliferation of endothelial cells stenosis.28 Consequently, it is critical to choose an and osteocytes. In 2004, Yang Cao et al.36 alleviated appropriate angiogenic factor for ANFH treatment. necrosis in rabbit femoral heads after transferring a Hepatocyte growth factor is a multifunctional cytokine mixture containing collagen and a eukaryotic expression produced by mesenchymal cells, which also has a potent plasmid with VEGF165 and basic fibroblast growth angiogenic function. HGF can also inhibit cellular factor genes. However, there is still no report on the apoptosis and alleviate tissue fibrosis.29–31 As HGF has treatment of ANFH with HGF. shown excellent potential for application in therapies for A technique for local drug delivery with high avascular cardiopathy and peripheral arterial occlu- efficiency is required for the reconstruction of new sion,32–34 it is likely to be an excellent angiogenic factor vascular networks in femoral heads. A local sustained in therapy for ANFH. The main signal pathway of HGF delivery technique can partly achieve this goal, but a function is the activation of ERK1/2, which furthers large amount of drug still enters the systemic circulation, reduction of necrotic cell death, as well as apoptotic cell and it is difficult to maintain an effective working death.35 In our study, the level of ERK1/2 activation was concentration at the desired site. Stem cell gene therapy relatively low in the normal group and the ANFH group, technology has developed rapidly in recent years and

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1531 provides a new approach to this problem. The trans- Both the study of Peng Song-Lin et al.42 and our plantation of transgenic autologous stem cells into the primary study found that the mixture of BMSCs and FG body allows the donor cells to secrete the target protein in vitro had no effect on the growth, proliferation or locally and in a sustained way. Stem cell delivery also differentiation of the BMSCs. In our study, the concen- improves the bioavailability and therapeutic efficacy of tration of fibrinogen was adjusted to 80 mg mlÀ1, mixed the therapeutic molecule. Bone marrow and cancellous with 2000 kIU mlÀ1 aprotinin, which slowed the rate of bone trabeculae are the major differentiation environ- FG degradation and maintained the effective period at 4 ments in which BMSCs can proliferate and differentiate weeks, consistent with the time necessary for exogenous into osteogenic cells, from preosteoblasts into osteoblasts. gene expression by Ad-HGF-transfected BMSCs (data Such differentiation is the basis for the restoration of not shown). Although in our study there was no bone tissue.37 significant difference between the transgenic BMSC There is precedent for autologous BMSC transplanta- group and the transgenic BMSC–FG group, the trans- tion in humans with ANFH. In 2006, Wang Wu-Zhou et genic BMSC–FG group achieved relatively better ther- al.38 treated 19 patients suffering from grades I–III ANFH apeutic effects. This finding suggests that FG could be with a combination of core decompression and auto- used as an effective alternative measure for transplanta- logous BMSC transplantation. The postoperative follow- tion of BMSCs. up was 12 months. Reexamination with MRI showed that In our rabbit animal model study, the surgical the average necrotic area of the femoral heads decreased therapeutic intervention was conducted only once. from 31.88 to 13.18%. The improvement in patients with Although the Ad vector could not recombine into the grades I and II ANFH was especially notable; the pain in BMSC genome for long-term expression, our study the hip joint was relieved 3 weeks after the operation, showed that it could persist for at least 1 month (data peripheral function began to recover 6 months after not shown). As the therapeutic goal of our study is transplantation and the Harris score for the hip joint the early-stage ANFH, the disease condition is not increased from 58.74 to 86.76. This finding in humans very severe. Our results demonstrated that a single demonstrates the efficiency and safety of this therapy delivery of Ad-HGF-transgenic BMSCs could still using autologous BMSCs for the treatment of ANFH. At achieve relatively good effects. However, multiple present, results obtained with the treatment of early stem-cell deliveries may deliver better therapeutic ANFH with autologous BMSCs are encouraging. results. We are currently considering improvements in Our study elaborates on these clinical findings with the transplantation therapy. the use of transgenic BMSCs in an animal model. There Our demonstration in a rabbit model of hormone- are significant advantages with the use of HGF trans- induced early ANFH demonstrated the feasibility and genic BMSCs over BMSCs without exogenous gene safety of the transgenic BMSC therapeutic strategy. The transfer. One is that transgenic BMSCs have the capacity combined results from CT, MRI, CT perfusion imaging, to promote bone generation and at the same time secrete ink artery infusion angiography and histology showed HGF that induces blood vessel generation and improves that this method can effectively promote vascular blood supply in the femoral head. Another is that HGF regeneration and bone reconstruction in necrotic femoral has chemotactic effects on BMSCs, and locally secreted heads. In addition, therapeutic efficacy with transgenic high HGF concentrations can not only sustain BMSCs BMSC was better than in the core decompression-alone in the necrotic area, but also promote BMSC differentia- group. This study provides the basis for the development tion into osteoblasts. A third advantage is that HGF of an original drug for the early treatment of ANFH and inhibits BMSC apoptosis, and the blood vessels induced constitutes original independent intellectual property. by HGF can improve the environment for BMSCs, by On the basis of these results, we are further studying the increasing the survival rate of transplanted cells. All of efficacy of this method in a beagle dog model. We are these HGF advantages support an increased therapeutic tracking the location and differentiation of BMSCs to efficiency. better understand the path they follow in vivo. There are some mechanics to consider when develop- ing the BMSC transplantation model. If HGF transgenic BMSCs are transplanted into the high-pressure femoral Materials and methods necrotic area through the tunnel of the core decompres- sion, the cells may drain away with the release of the Animals and treatment groups intraosseous pressure. However, mixing the cells with Thirty healthy adult male New Zealand rabbits with an medical FG before transplantation can effectively solve average weight of 3 kg were provided by the Experi- this problem. FG has excellent histocompatibility and mental Animal Center of Nanfang Hospital (Guangzhou, plasticity and its molecular structure favors cell adhesion. It Guangdong, China) and maintained under specific can quickly mold in vivo and is easily degraded and pathogen-free conditions. Animals were randomly as- absorbed. The rate of FG degradation can be regulated by signed to five treatment groups. Five rabbits were set modulating the concentration of fibrinogen or adding aside as no-treatment unoperated normal controls. The aprotinin. For these reasons, FG is an excellent supporting remaining 25 animals were induced with ANFH (meth- material for seed cells in tissue engineering,39–41 and ods below). Of those 25, sixteen survived and were particularly for ANFH, which may require added support randomly assigned to two groups of 8. Five received no for transplanted BMSCs. When HGF-transgenic BMSCs therapy (ANFH group). Eight animals received different are mixed with FG in an appropriate proportion, the gel treatment in each hip. One set of hips were treated with structure formed also functions as a sponge-like drug only core decompression (core decompression-alone vehicle that could release secreted HGF protein slowly and group). The other set was treated with a combination maintain a desired local concentration. of core decompression and transplantation of HGF

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1532 Table 2 Animals treatment groups

Treatment group Total number of hips Number of hips (animals) in analysis groups (animals) per group Radiology Histology

Ink artery infusion angiography HE/IHC

Normal 10 (5) 6 (3) 4 (2) 6 (3) ANFH 10 (5) 6 (3) 4 (2) 6 (3) Core decompression-alonea 8 (8) 6 (6) 4 (4) 4 (4) Transgenic BMSCa 8 (8) 6 (6) 4 (4) 4 (4) Transgenic BMSC–FG 16 (8) 6 (3) 8 (4) 8 (4)

Total number of hips (animals) 52 (26)b

aEight animals received different treatment in each hip. One set of hips were treated with only core decompression (core decompression-only group). The other set was treated with a combination of core decompression and transplantation of HGF transgenic autologous BMSCs (transgenic BMSC group). bFour of 30 animals were died of infection during establishment of ANFH model.

transgenic autologous BMSCs (transgenic BMSC group). reverse transcription-PCR. The HGF protein expression The remaining eight animals were treated with the was periodically assayed at over a 14-day period with combination of core decompression and transplantation an enzyme-linked immunosorbent assay kit (BioSource of HGF transgenic autologous BMSCs mixed with FG in International Inc., Camarillo, CA, USA), according to the both hips (transgenic BMSC–FG group) (Table 2). manufacturer method. Concentration of HGF protein was measured in optical density units by an Absorbance Adenoviral vector preparation Microplate Reader ELx800 (Bio-Tek Instruments Inc., Replication-deficient recombinant adenoviral vectors Winooski, VT, USA) at 450-nm wavelength. carrying either human HGF gene (Ad-HGF) or the green fluorescent protein gene (Ad-GFP) were constructed Detection of BMSC cell cycle. To detect the cell-cycle with AdMax system (Microbix Biosystems Inc., Toronto, stage of BMSCs, a total of 1 Â106 transfected cells were Ontario, Canada). Both vectors were purified with treated with RNase A for 30 min at 37 1C, then stained cesium chloride (SINOJIE (HK) Ltd, Shanghai, China) with propidium iodided at a final concentration of gradient centrifugation.43 A tissue culture infectious dose 20 mgmlÀ1 for another 30 min at 4 1C. After staining,

(TCID50) assay was used to determine the virus titer. The cell-cycle analysis was immediately performed using purity of Ad-HGF was determined by a high-perfor- flow cytometry (FACSCalibur). mance liquid chromatography assay. The rabbit model of early ANFH. Artificial ANFH was BMSC preparation induced in rabbits by the following method. A combina- BMSC culture and transfection. Bone marrow (3 ml) tion of horse serum (Hyclone) and prednisolone acetate was aspirated from the bilateral posterior superior iliac (Pharmacia & Upjohn Co., Kalamazoo, MI, USA) was spine and placed into high-glucose Dulbecco’s modified injected over time to create a model of early ANFH. First, Eagle’s medium (GIBCO BRL, Gaithersburg, MD, USA) horse serum (10 ml kgÀ1) was injected through an ear containing 50 U mlÀ1 of heparin sodium (Xian Tianyi vein. Two weeks later, 5 ml kgÀ1 of horse serum was Biotechnology Co. Ltd., LinTong, Shanxi, China) and 10% injected in the same way once a day for 2 days. Two fetal calf serum (Hyclone Ltd, Logan, UT, USA). The weeks later, 7.5 mg kgÀ1 of prednisolone acetate was dissociated cell mixture was agitated then centrifuged at injected into the abdomen twice a week for 2 weeks. At 800 g for 5 min. The cell pellet was resuspended, then the time of injecting the hormone, 200 000 U of penicillin cultured in high-glucose Dulbecco’s modified Eagle’s was injected into the buttock of each animal. At the end medium containing 10% fetal calf serum, 100 U mlÀ1 of week 5 after the hormone injection, the animals were penicillin, 100 mg mlÀ1 streptomycin and 2 mML-gluta- examined with several imaging techniques (described mine (Invitrogen, Carlsbad, CA, USA) at 37 1C. After below) to establish baseline of disease. During the 72 h, nonadherent debris was removed and adherent establishment of ANFH model, four rabbits died of cells were cultured to the second generation. infection. Bone marrow stromal stem cells were infected with Ad-GFP within a multiplicity of infection range of Transplantation of autologous BMSCs to ANFH model 50–400. The transfection efficiency was determined by Preparation of transfected BMSCs and medical FG. flow cytometry (FACSCalibur, Becton Dickinson and Co., Fibrin glue (FG) solution contained two components. The Franklin Lakes, NJ, USA). first is solution I: 2000 kIU mlÀ1 aprotinin (Lanzhou Dadeli Biochemical Pharmaceutical Co. Ltd, Lanzhou, Monitoring of BMSC HGF expression. Both HGF Gansu, China) was prepared at 37 1C in sterile water mRNA and protein expression were monitored in and used to dissolve freeze-dried human fibrinogen BMSCs. The HGF mRNA expression was assayed by (Shanghai RAAS Blood Products Co. Ltd., Shanghai,

Gene Therapy Treating ANFH with HGF-transgenic BMSCs QWenet al 1533 China) to a concentration of 80 mg mlÀ1. The second is Conventional CT examination À1 solution II: 40 mmol l CaCl2 (Guangzhou Jinhuada Computer tomography examination was performed Chemical Reagent Co. Ltd, Guangzhou, Guangdong, with a Lightspeed 16 spiral CT scanner (GE Company, China) was prepared in sterile water and used to New York, USA). The animals were anesthetized and dissolve thrombin (Jin Kang Pharmaceutical Co. Ltd., fixed in the supine position on the operating board, and Nanjing, Jiangsu, China) to a concentration of hip were positioned symmetrically and as laterally 400 IU mlÀ1. At 48 h after transfection, the Ad-HGF- as possible. Transect scanning of the entire hip joint transfected autologous BMSCs were collected and their was performed, including the upper and lower edges. concentration was adjusted to 107 mlÀ1. One milliliter of The scanning parameters were 100 kV and 220 mA. The the cell suspension was centrifuged at 800 g for 5 min combination detector was 16 Â 0.625 mm, the screw pitch and the cell pellet was resuspended in 900 ml of solution was 0.625:1, the bed speed was 5.62 mm/r, the recon- I. This preparation of solution I with transfected BMSCs struction layer was 1.25 mm thick and the diameter of and FG and solution II would be mixed fully and was field of view was 9.6 cm. hereafter termed BMSC–FG. MRI examination The examination was conducted using the Magnetom Core decompression and transplantation of BMSCs to Vision PLUS 1.5 T superconducting MRI machine (Sie- the femoral head. All animals receiving core decom- mens). An orthogonal head coil was placed on the pression were anesthetized. A mixture of 1.5 ml of anesthetized rabbit, and the center of the coil was located veterinary sumianxin II and 1 ml of atropine sulfate on the hip joint. Fast spin echo was used. T2WI fat- was injected into the buttock. The first dose was 0.7– suppression sequence (FS-T2WI) was collected twice in À1 0.8 ml kg and a half-dose was injected 30–40 min later. the coronal position. The layer thickness was 3 mm and Core decompression was then conducted under CT the field of view was 100 Â 100 mm. control with Somatom PLUS 4 CT scanner (Siemens, Erlangen, Germany). A custom-made trocar was applied CT perfusion imaging to puncture the femoral head and conduct core decom- Cross sections of the hip joints were first scanned pression, as well as to deliver BMSCs or BMSC–FG routinely. Layers that both included the femoral head mixtures. A disposable dispensing needle (1.2 Â 38 mm) and were thinner than 1 cm were chosen for rescan in the was used as the puncture trocar sheath, with the needle axial plane. The thickness of the layers were 2.5 Â 4mm matching the 18G OptiMed Vitesse biopsy gun (diameter and the layers were scanned once every 2 s. Four 1.2 mm) as the trocar needle. Before use, the needle was milliliters of contrast reagent Iohexol (300 mg mlÀ1; shaped by a grinding wheel and sandpaper to neatly Yangtze River Pharmacy Group, Taizhou, Jiangsu, match the top-inclined plane of the trocar sheath with China) was injected into an ear vein at a rate of optimal smoothness and sharpness. In addition, punc- 1.5 ml sÀ1, and the same layers were subjected to ture trocars were sterilized by soaking for 2 h in dynamic enhanced scanning 45 times in 90 s. potentiated glutaraldehyde solution immediately before the operation. The femoral head was punctured once. For transgenic BMSC group, 106 cells in 100 ml high-glucose Ink artery infusion angiography and analysis Dulbecco’s modified Eagle’s medium containing no fetal The animals were anesthetized as described in the core calf serum were transferred into the necrotic femoral decompression section above and the abdominal aorta head. For transgenic BMSC-FG groups, an aliquot (90 ml) was exposed. After the blood was washed away with of solution I containing suspended cells and 10 mlof saline, a 7:3 v/v mixture of ink (Shanghai Hero Group solution II were transferred with a duplex syringe, Co. Ltd, Shanghai, China) and dextran (Shanghai Fu Min through the tunnel of the core decompression. The Pharmaceutical Factory, Shanghai, China) was injected animals were examined at the end of 4 weeks after into the aorta, until the skin of the bilateral crura and 1 decompression surgery. nails were uniformly black. The body was kept at 4 C overnight, and the next day, the thighbones were removed and fixed in 4% paraformaldehyde (Tianjin Evaluation of treatment. All animals received CT Baishi Chemical Industry Co. Ltd, Tianjin, China) for 3 perfusions and MRI examination. The treated animals days. Then these were decalcified with 10% were divided into scanning groups organized by hip ethylenediamine tetraacetic acid disodium salt (Na2- treatment identity. Ink Artery Infusion Angiography was EDTA, Jinhuada) in posphate-buffered saline (GIBCO performed on the following subsets: four hips from the BRL). The samples were dehydrated stepwise and normal group (two animals), four hips from the ANFH turned transparent by exposure to dimethylbenzene group (two animals), four hips from the core decom- (Jinhuada) and sodium salicylate (Jinhuada). Samples pression-alone group, four hips from transgenic BMSC were then sectioned into 50-mm-thick layers and ob- group and eight hips from the transgenic BMSC–FG served under a stereomicroscope. Configuration of the group. Histopathology and immunolabeling with ERK1/ femoral head, blood distribution and growth condition 2 was performed on the following sets: six hips from the were observed. The ratio of the area of ink artery to that normal group (3 animals), six hips from the ANFH of the whole femoral head was counted using at least five group, four hips from the core decompression-alone randomly selected fields per section. group, four from the transgenic BMSC group, and eight hips from the transgenic BMSC–FG group. The scans Histopathology and immunohistochemistry were analyzed by two different observers, each blind to After euthanization by air injection through ear vein, treatment groups. both femoral heads including the metaphyses and

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Nature 1989; 342: 440–443. the bone marrow hematopoietic cells were counted at a 10 Nakamura Y, Morishita R, Higaki J, Kida I, Aoki M, Moriguchi A  400 magnification, by at least five randomly selected et al. Hepatocyte growth factor (HGF) is a novel member of endothelium-specific growth factors: additive stimulatory effect fields per section. For immunolabeling of ERK1/2, the of HGF with basic fibroblast growth factor, but not vascular slides were incubated with mouse monoclonal anti- endothelial growth factor. J Hypertens 1996; 14: 1067–1072. ERK1/2 (1:250) (sc-7383; Santa Cruz Biotechonology Inc., 11 Nakamura Y, Morishita R, Nakamura S, Aoki M, Moriguchi A, Santa Cruz, CA, USA) for 1 h, followed by incubation Matsumoto K et al. A vascular modulator, hepatocyte growth with biotinylated goat anti-mouse IgG working solution factor, is associated with systolic pressure. Hypertension 1996; 28: (BA1001, BOSTER bioengineering Co. Ltd, Wuhan, 409–413. Hubei, China). 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