Hepatocyte Growth Factor Gene-modified Mesenchymal Stem Cells For The Treatment Of Osteonecrosis Of The Femoral Head

As a common refractory disease, osteonecrosis of the femoral head (ONFH) is a chronic disabling clinical entity that usually leads to destruction of the hip joint and loss of work ability. Most cases of ONFH occur between 30 and 60 years of age. Osteonecrosis is a disease of unknown pathogenesis and various causes. The therapy is so troublesome that no ideal treatment protocols have been accepted as yet. The essential pathology of ONFH involves ischemic events followed by death of bone and marrow elements. And secondary osteoarthritis and destruction of the hip joint would ineluctably develop after femur head collapses.Mesenchymal stem cells (MSCs) have a strong osteogenic potential both in vitro and in vivo, which are considered to be one of seed cells that occupy the greatest potential for bone tissue engineering. Several clinical trials based on the ultilization of MSCs on the bone repair have been approved into investigation. Hepatocyte growth factor (HGF) has the powerful angiogenic activity and anti-apoptosis function. In the present study, series of basic and clinical experiments had been carried out to observe the feasibility and effectiveness of MSCs modified with HGF gene in the treatment of ONFH, providing supportive data for clinical use of adult stem cells modified with certain genes to manage bone injury and osteonecrosis diseases.The work described here include four parts that will be detailed below: (1) investigations with an experimental animal model of ONFH; (2) establishment of a method to isolate and culture-expand human bone marrow MSCs with animal serum-free medium; (3) safty evaluation on MSCs-HGF; and (4) treatment of ONFH with HGF gene-modified MSCs: a case report.In the first part of the present study, the therapeutic effects of hepatocyte growth factor gene-modified mesenchymal stem cells on rabbit femoral head osteonecrosis are described. Bone marrow MSCs from New Zealand rabbits were obtained by means of gradient centrifugation and subsequent early adhesion separation. The adherent cells exhibited a uniform morphology and differentiation abilities into osteoblasts and adipoblasts. The osteonecrosis of the femoral head was developed surgically and MSCs- or MSCs-HGF-coated biomaterials or materials alone were implanted into the osteonecrotic zone. Three months after operation, pathological analysis and histological scores were conducted. The resluts revealed that the bone defects from both control and MSCs-treated femoral heads were filled with fibrous tissues, though blood vessels were evident in MSCs group, whereas new bony tissues were obvious in MSCs-HGF-treated defects. The result was further confirmed by Lane-Sandhu scaling, which indicated that new bone formation was more evident in MSCs-HGF compared with MSCs or control group (P<0.0001). The results definitely suggested that in comparison with MSCs, MSCs-HGF display greater osteoneneic capacity at least in this model.To investigate the underlying mechanisms responsible for the observations above, MSCs or MSCs-HGF were labeled with carboxyfluorescein diacetate succinmidyl ester dye (CFSE) , a cell-tracking marker, and incubated with the hypoxia-mimicking agent, cobalt chloride. FCM analysis showed that when cells had been pre-treated with coblat chloride at a concentration of 150μmol/L for 72 hours, the proportions of MSCs and MSCs-HGF that had experienced divisions were 85.1±4.0% and 94.5±3.1% respectively. When the concentration increased to 300μmol/L, the proportions decreased to 81.6±3.5% and 94.0±2.8%, respectively. Statistical analysis proved the significance of the differences between two groups (P=0.0049) and suggested that HGF modification provide protective effects on MSCs from hypoxic injury.Prevoius data have confirmed that intercellularization of proteins from calf serum into cultured MSCs take place, which is a limiting factor for the clinical ultization of MSCs culture-expanded in the standard medium containing calf sera. Therefore, a novel methodology had been investigated to expand human MSCs with a animal serum-free medium. In the second part of our studies, human platelet lysates (PL) were prepared by freezing and thawing the pooled platelet-rich plasma. Human MSCs were cultured in media containing various concentrations of PL and their proliferation status was evaluated by MTT assay and the CFSE-labeling technique. The results showed that MSCs cultured in PL at a concentration of 1.25% expanded at an efficiency comparable to those cultured in medium containing FCS at a concentration of 10%. Furthermore, fibroblastoid colony-forming unit and primary culture growth curve assays validated the results above, confirming the efficiency of MSCs proliferation in 1.25%PL.To investigate if MSCs cultured in PL occupied similar phenotypic and functional characteristics compared to their counterparts obtained in culture of 10%FCS, a series of experiments were performed to observe the morphological, immunotypical and differentiation features. The results revealed that MSCs cultured in PL were fibroblast-like in morphology, homogenously expressed CD29, CD73, CD105, CD166 and HLA-ABC and were negative for CD14, CD31, CD34, CD45 and HLA-DR. Further, the cells had the abilities of in vitro osteogenesis and adipogenesis.To observe the potential differences of gene expression styles in MSCs cultured in these two media, a series of cytokine mRNA expression levels were analyzed with a microarray assay. The results showed that compared to MSCs cultured in standard medium, six genes including BF, BGLAP, CCL7, FST, PLAU and TNFβ-, were up-regulated in MSCs collected from PL culture, while three were down-regulated inclusive of CKTSF1B1, IGFBP3, and JAK2. Furthermore, with cell passaging, PL seemed inclined to maintain the expression levels of BGLAP and FST in MSCs, whereas decreased the levels of CKTSF1B1 and JAK2 genes. All the results above had been confirmed by means of realtime PCR. Thus, the novel protocol described here would be relatively safe and applicable for MSCs culture-expansion in the process of MSCs-based cell therapy. The data here might also provide highlight on the mechanisms underlying the chemotactism, migration, proliferation, differentiation and immune regulation of MSCs.In the third part of our studies, the biological properties and the safty issues of MSCs-HGF were concerned. MSCs were infected with adenovirus vectors encoding GFP or HGF gene (Ad-GFP or Ad-HGF). The efficiency of infection and expression of HGF were detected by FCM and ELISA assay, respectively. It showed that almost all of the cells expressed GFP when transfected at an MOI of 150. HGF secretion from MSCs-HGF reached the highest level of 147±18ng/ml 48 hours after infection and could last for more than one week. Further, it was found that the HGF gene modification would not change the cell phenotype and differetiation capacity. However, this modification could promote the MSCs proliferation in a hypoxia environment. G-band analysis showed that after continuous culture till passage 11th, the chromosomal amounts and structure maintained normal in MSCs-HGF. Moreover, MSCs-HGF were proved to be of no oncogenicity as assessed by the soft agar colony formation assay and subcutaneous inoculation of cells into the BALB/c nude mice. When MSCs-HGF were intravenously administrated into BALB/c mice at a dose of 1.0×107 (4.0×105 cells/gram body weight), no any adverse events were observed. During the two-week observation period, the increase of body weight of MSCs-HGF- and sham-treated mice was comparable. Additionaly, histological examination on the sections of the hearts, lung, liver, spleen and kindney found no evidence of pathological changes. Based on the results above, it is suggested that MSCs-HGF produced herein might fit the requirements of clinical application, as described in the"Main issues of standarization on clinical trials with human somatic cell and gene therapy"that released by China's State Food and Drug Administration (SFDA).Based on the data above, a patient with steroid-induced ONFH (ARCO Grade IIIB) was enrolled and treated with MSCs-HGF. Heparinized bone marrow was harvested; MSCs were isolated, culture-expanded, and infected with Ad-HGF. MSCs-HGF cells (an aliquot of 1.0×107) were collected 48 hr after infection and surgically implanted into the necrotic zone of the affected femoral head. During the nine-month follow-up, the clinical symptoms and imaging change were evaluated. The results showed that the therapeutic operation was performed smoothly and no any severe complications were observed. After nine months, there was a significant reduction in hip joint pain measured with the WOMAC index. The Harris scores of hip joint were obviously improved, which indicated respectively for clinical symptoms (63 versus 76) and for joint range of motion (2.5 versus 4.2). Computer Tomography displayed reconstructive repair manifestation, which exhibited as decreased low-intensity region accompanied with new bone formation. The classification of disease severity was improved from stageⅢ- B to stage-ⅢA, according to the system of the Association Research Circulation Osseous. Therefore, the preliminary clinical result implies that implantation of autologous MSCs modified by HGF gene appears to be a safe and effective treatment for osteonecrosis of the femoral head. Our results here appeal a further large-scale clinical trial.The data described here provide support on the conclusions detailed as below:(1) MSCs modified by HGF gene exhibit therapeutic effects in the rabbit ONFH model, and this modification might endow MSCs with the resistance to hypoxic injury. Further, the enhanced angiogenesis elicited by MSCs-HGF could be attributed to the effectiveness of bone repair.(2) A methodology to culture human MSCs with animal serum-free medium has been successfully established, and the cells occupy the biological properties similar to those harvested from culture with standard FCS-containing medium.(3) Microarray and subsequent fluorescent real-time PCR analysis proved that compared to MSCs cultured in FCS, the expression levels of several cytokines in MSCs cultured in PL were different. More detailed investigations are needed to clarify the underlying mechanisms.(4) MSCs could be infected effectively by Ad-HGF and HGF gene modification had little effect on the cellular phenotypic and differentiaiton features. However, overexpression of HGF in MSCs protected cells from hypoxic injury.(5) In vitro experiments and this case report might have provided helpful information on the safty of MSCs-HGF in the management of the necrosis of the femoral head.