| Avascular necrosis of the femoral head (ANFH) is a progressive pathological process that primarily afflicts people20-50years of age. Without timely effective treatment, ANFH causes in situ avascular necrosis, and ultimately deforms the bone. This can severely impair the patients’life quality and work capacity, causing great burden to society and their families. According to the incomplete statistics of the World Health Organization, there are30,000,000individuals with ANFH worldwide,5,000,000-7,500,000in China. There are150,000-200,000people born with ANFH annually, and the incidence rate increases year by year. So far, no single treatment for ANFH achieves consistently good results and this becomes a difficult and urgent problem to be addressed by medical science. Thus, the potential social and economic benefits of a new efficient therapeutic method for treating early ANFH are self-evident.Whether the etiology of ANFH is traumatic or aseptic nontraumatic, the basic cause is a vicious cycle of elevated intraosseous pressure and obstructed blood supply in the femoral head. ANFH is considered irreversible, and any diagnostic or therapeutic strategy for ANFH is best introduced in the early stage. Early intervention will reduce intraosseous pressure and improve the blood supply to the necrotic femoral head. Osseous repair should be performed alongside supplemental interventions. The induction of blood vessel regeneration and the construction of a collateral circulation are the most effective ways to break the vicious pathological cycle. These entail new study directions for ANFH therapy.Angiogenic factors are a group of cytokines that induce the generation of blood vessels. Different angiogenic factors own various biological activities. Consequently, it is critical to choose an appropriate angiogenic factor. Hepatocyte growth factor (HGF) is a multifunctional cytokine produced by mesenchymal cells, which also has a potent angiogenic function. HGF can also inhibit cellular apoptosis and alleviate tissue fibrosis. HGF has shown excellent potential applications in therapies for avascular cardiopathy and peripheral arterial occlusion and is likely to be an excellent angiogenic factor in therapy for ANFH.Bone marrow mesenchymal stem cells (BMSCs) are a kind of stromal stem cell, derived from the bone marrow, and have multiple differentiation potentials. It is easy for BMSCs to be isolated from the bone marrow and expanded. BMSCs secrete large amounts of cell growth factors and angiogenic factors, and are easily transfected with an exogenous gene. Moreover, allogenic transplantation induces no immunological rejection. BMSCs also contain bone progenitor cells and have excellent potential for differentiation into osteoblasts. BMSCs are recognized as the most potentially useful seed cells in bone tissue engineering in the clinical context in the near future. At present, treatment of early ANFH with allogenic BMSCs has shown attractive application prospects.There are significant advantages in the use of HGF transgenic BMSCs over BMSCs without exogenous gene transfer:(1) transgenic BMSCs have the capacity to promote bone generation and at the same time secrete HGF to induce blood vessel generation and improve the blood supply in the femoral head;(2) HGF has chemotactic effects on BMSCs, and locally secreted high concentrations of HGF can sustain BMSCs in the necrotic area and promote their differentiation into osteoblasts;(3) HGF inhibits BMSC apoptosis, and the blood vessels induced by HGF can improve the environment in which BMSCs live, increasing the survival rate of transplanted BMSCs and improving their therapeutic efficacy.Gene therapy, stem cell transplantation, and tissue engineering have been rationally combined in our study. The therapeutic HGF gene was transfected into the target BMSCs with the AdMax adenovirus vector system. The transgenic BMSCs were used as seed cells to transfer into the necrotic area of the femoral head to treat early ANFH. Simultaneously with physical decompression, the HGF secreted by the transplanted cells was released in a sustained way from BMSCs and induced the local regeneration of new blood vessels and the construction of a collateral circulation, thus improving the blood supply in the femoral head. In the local environment, BMSCs are induced to differentiate into osteoblasts to repair the necrotic bone tissue. Our study integrates decompression, vascular regeneration, and bone reconstruction and provides a new therapeutic mode for ANFH.Objects:Basing on determining the patterns and mechanisms of HGF effects on BMSCs proliferation and osteogenic differentiation in vitro, assess the treatment efficies of early stage hormone-or trauma-induced ANFH with HGF transgenic BMSC plantation and preliminary explore the ANFH pathogenesis and the molecular mechanisms of treatment. Methods:1. Isolation, culture and identification rabbit BMSCsBone marrow was aspirated from the bilateral posterior superior iliac spine and placed into low glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) containing50U/ml of heparin sodium and10%fetal bovine serum (FBS). The dissociated cell mixture was agitated then centrifuged at800×g for5min. The cell pellet was resuspended, then cultured in DMEM-HG containing10%FBS,100U/ml penicillin,100mg/ml streptomycin and2mM L-glutamine at37℃. After72h, nonadherent debris was removed and adherent cells were cultured to the second generation.BMSCs were cultured in various differentiation induction medium to differentiate into osteoblasts, chondroblasts and adipocytes. After12and24d, NBT-BCIP staining and alizarin red sulfate (AR-S) staining were used to detect the osteogenic differentiation. After27d, Alcian Blue and Oil Red O were used to detect the chondroblast and adipocyte differentiation, respectively. At the same time, real-time quantitative PCR (qPCR) was used to detect the mRNA expression of the indicated lineage-associated markers. MSCs, osteoblasts, chondroblasts and adipocytes specifically expressed Vimentin, Runx2, SOX9and PPAR-gamma3, respectively.2. Determine the patterns and mechanisms of HGF effects on BMSCs proliferation and osteogenic differentiation.In osteogenic induction medium, BMSCs were treated with20ng/mL or100ng/mL HGF. Cell proliferation and viability were assessed with EdU incorporation and WST-8methods. NBT-BCIP staining was used to determine the alkaline phosphatase (ALP) activity and AR-S staining was to detect the formation of calcium deposition in the extracellular matrix. Western blot and qPCR were used to detect the expression of the HGF receptor---c-Met, cell cycle inhibitor---p27and transcription factors required for osteogenic differentiation---Runx2and Osterix. After pretreatment of BMSCs with the specific signaling pathway inhibitors, PD98059(30μM) or Wortmannin (100nM) for1h, Western blot was used to detect the activation of ERK1/2and Akt signaling pathway and their effects on BMSCs proliferation and osteogenic differentiation were also assayed.3. Transfection of rabbit BMSCs with recombinant Ad-HGF and detection of HGF expression.After PCR identification, recombinant adenovirus vector carrying HGF gene (Ad-HGF) was amplified and purified with cesium chloride gradient centrifugation and its titer was measured by TCID50assay before transfection into the second passage of rabbit BMSCs. The transcription and expression of HGF gene in the transfected BMSCs was detected by RT-PCR, in situ hybridization, and immunohistochemistry.4. Establishment and examination of a rabbit early stage hormone-induced ANFH modelANFH was induced by a combination of hypersensitivity vasculitis caused by injection of horse serum and subsequent administration of a high dose of corticosteroid. The pathological changes were detected with digital radiography (DR), computed tomography (CT), magnetic resonance imaging (MRI), ink artery infusion angiography, hematoxylin-eosin staining, and immunohistochemistry. 5. Treatment of early stage hormone-induced ANFH with HGF transgenic BMSCs.BMSCs were transplanted by core decompression under the guidance of CT. Therapeutic efficacy was evaluated4weeks later by CT, MRI, CT perfusion imaging, ink artery infusion angiography, hematoxylin-eosin staining and immunohistochemical staining. Treatment with simple core decompression was used as the control.To explore the therapeutic mechanisms, transplantations of blank Ad vector-infected BMSCs and uninfected BMSCs were used as the controls. Four weeks later, the therapeutic efficacies were assessed by histological examination with HE staining. p27, Runx2and Osterix mRNA expression was assayed with qPCR, and the expressions of HGF, p-ERK1/2, p-Akt proteins were detected with immunohistochemistry.6. Establishment and examination of a rabbit early stage traumatic ANFH modelAnimals were anaesthetized with an intravenous injection of30mg·kg-1body weight30%pentobarbitone sodium. A posterolateral incision was made in the left hip under aseptic conditions, and a3cm incision was made in the joint capsule to expose the femoral head. All soft tissue attachments, including the annular ligament, were detached, and the femoral neck was severed at the base. The pathological changes were detected with CT, MRI, HE staining, and immunohistochemistry at1d,1week,2week, and3week post trauma.7. Treatment of early stage traumatic ANFH with HGF transgenic BMSCs. One week after trauma, BMSCs were transplanted by core decompression under the guidance of CT. Then the leg was fixed with a small splint. Therapeutic efficacy was evaluated4weeks later by HE staining and immunohistochemical staining. Treatment with transplantations of blank Ad vector-infected BMSCs and uninfected BMSCs were used as the controls.To explore the therapeutic mechanisms, at2d,2weeks, and4weeks after transplantation, the expressions of HGF, p-ERK1/2, p-Akt proteins were detected with immunohistochemistry.8. Statistical analysisAll measurement data were expressed as of x±s. Differences in BMSCs proliferation after treatment with cell signaling pathway inhibitors assayed by EdU incorporation, BMSCs viability assayed by WST-8, ALP activities assayed by NBT-BCIP staining, calcium deposition after treatment with cell signaling pathway inhibitors assayed by AR-S staining, c-Met, Osterix, Runx2and p27mRNA expression assayed by qPCR, the expression of HGF, p-ERK1/2and p-Akt in vivo assayed by immunohistochemistry were determined using a Factorial variance analysis of design information. Differences in BMSCs proliferation assayed by EdU incorporation, calcium deposition assayed by AR-S staining, the richment of bone marrow cells in medullary cavities, the ratio of empty lacunae, the expression of vWF, CD105, PCNA, Col I, OCN and VEGF in vivo assayed by immunohistochemistry were determined using a One-Way analysis of variance (ANOVA). Heterogeneity of variance was corrected with Welch method. Multiple comparisons were performed under the premise of significantly difference using least significant difference (LSD) or Dunnett’s T3multiple comparison tests. All reported P-values were two-sided and P-values<0.05were considered statistically significant. Statistical analyses were performed using the SPSS version16.0for windows statistical package.Results1. Culture of rabbit BMSCs with pluripotencyRabbit BMSCs were successfully cultured through isolation with bone marrow biopsy and purification through adherency. Specific tissue staining and qPCR demonstrated the pluripotency of BMSCs which could differentiated into osteoblasts, chondroblasts and adipocytes.2. Determination of HGF effects on BMSCs proliferation and osteogenic differentiation.①the pattern of HGF effects on BMSCs proliferation and osteogenic differentiationCell proliferation activities were assayed by EdU incorporation and WST-8methods, and the osteogenic differentiation was determined by NBT-BCIP and AR-S staining. The results showed that in the osteogenic induction medium,100ng/mL HGF promoted BMSCs proliferation but inhibits osteogenic differentiation. On the contrary,20ng/mL HGF suppressed the BMSCs proliferation somewhat, while significantly enhances the osteogenic differentiation.②Mechanism study1:Phosphorylation of c-Met is correlated with the concentration of HGF and may affect the osteogenic differentiation of BMSCsTreatment with20ng/mL HGF significantly increased c-Met receptor expression and induced stronger activation. However,100ng/mL HGF partially inhibited the expression, and the activation effects on c-Met was obviously lower than20ng/mL HGF. In turn, these effects may further affect the downstream signaling pathway to diversely regulate osteogenic differentiation. Thus, c-Met expression may control the ability of BMSCs to mobilize after HGF stimulation; high levels of c-Met promote osteogenic differentiation and low levels induce proliferation.③Mechanism study2:Requirement of ERK1/2and Akt pathways for BMSCs proliferation and osteogenic differentiation100ng/mL HGF significantly promoted BMSC proliferation and inhibited osteogenic differentiation through activation of ERK signaling pathway and inhibition of Akt signaling pathway. By contrast,20ng/mL HGF inhibited the ERK pathway but activated Akt pathway to significantly enhance BMSCs osteogenic differentiation, but the promotion effects on cell proliferation was lower than that of100ng/mL HGF. It suggested that various concentration of HGF exerted different effects on BMSCs through activation of different signaling pathway.④Mechanism study3:Effects of HGF concentration on expression of the cell cycle inhibitor, p27and transcription factors required for osteogenic differentiationIn the osteogenic induction medium,20ng/mL HGF increased expression of p27, Runx2and Osterix in BMSCs to enhance osteogenic differentiation. In contrast,100ng/mL HGF exerted inhibition effects on expression of p27, Runx2and Osterix. It suggested that various concentration of HGF exerted different effects on BMSCs through regulation of expressions of p27, Runx2and Osterix.3. High expression level of HGF from Ad-HGF transfected BMSCsThe infection titer of Ad-HGF was2.6×1010TCID50/mL. The expressions of HGF mRNA and protein were confirmed in the transfected BMSCs.4. Establishment of a rabbit early stage hormone-induced ANFH modelThe radiological and pathological changes of the model corresponded to the clinical characteristics of early stage ANFH. DR showed bilaterally increased bone density, an unclear epiphyseal line, and blurred texture of cancellous bone. CT showed spot-like low-density imaging of cancellous bone, thinner cortical bone, osteoporosis, and an unclear epiphyseal line. MRI showed bone marrow edema and spot-like high signals in T2-weighted imaging in cancellous bone. Ink artery infusion angiography showed fewer obstructed blood vessels in the femoral head. HE staining of pathological sections showed fewer trabeculae and thin bone, an increased proportion of empty osteocyte lacunae, decreased hematopoiesis, thrombosis, and fat cell hypertrophy. Immunohistochemistry showed attenuated expression of vascular endothelial growth factor in osteoblasts and chondrocytes, and on the inner membrane of blood vessels. Immunohistochemistry assay of the marker of new blood vessels---CD105and the marker of tissue repair---type I collagen indicated that after modeling, the revascularization of the femoral head was suppressed, the osteogenic activity decreased. There was lack of tissue self-repair and the early stage hormone-induced ANFH was developed4weeks after injection of hormone.5. Treatment efficies of rabbit early stage hormone-induced ANFH with HGF transgenic BMSCs.①Assessment of therapeutic efficacyImaging and histopathological analyses showed that treatment with HGF transgenic BMSCs transplantation significantly enhanced blood vessel regeneration and bone reconstruction in the necrotic area of the femoral head among the three groups. DR showed clear bone texture and the edges of both femoral heads. CT showed the decreased spot-like low-density imaging. MRI showed the unobvious spot-like or line-like high-signal imaging. ink artery infusion angiography showed revascularization, with the recovery of big blood vessels in the metaphysis. HE staining of pathological sections showed a relatively regular arrangement of trabeculae and obvious bone regeneration. The newly generated capillaries were visible on the bone plates of the trabeculae, and the bone marrow was rich in hematopoietic tissue.②In vivo mechanism study:The expression of p27, Runx2and Osterix were highest in the HGF transgenic BMSCs group, the peak level appeared at2weeks after transplantation. The expression of HGF decreased in the ANFH group, but reached the highest level in the HGF transgenic BMSCs group. The peak level of HGF appeared at2d after transplantation, accompanied with increased activation level of ERK signaling pathway. After then, both the levels of HGF and p-ERK gradually decreased. Two weeks later, the level of p-Akt increased gradually, the peak level appeared at4weeks after transplantation. The results demonstrated that changes in HGF expression level in vivo after transplantation of HGF transgenic BMSCs was similar with in vitro after transfection of BMSCs with Ad-HGF. Meanwhile, this sequent changes in HGF concentration mediated by the adenovirus vector could regulate BMSCs proliferation and osteogenic differentiation in vivo to promote tissue repair and regeneration.6. Establishment of a rabbit early stage stage traumatic ANFH modelAn experimental rabbit model of early stage traumatic ONFH was established, validated, and used for an evaluation of therapy. CT and MRI confirmed that this model represents clinical Association Research Circulation Osseous (ARCO) phase I or II ONFH, which was also confirmed by the presence of significant tissue damage in osseous tissue and vasculature. Pathological examination detected obvious self-repair of bone tissue up to2weeks after trauma, as indicated by revascularization (marked by CD105) and expression of collagen type I (Col I), osteocalcin, and proliferating cell nuclear antigen. However, due to the interruption of blood flow, the tissue self-repair could not keep up with the progression of necrosis, and the early stage traumatic ANFH developed3weeks after operation.7. Treatment efficies of rabbit early stage traumatic ANFH with HGF transgenic BMSCs.Histopathological examinations showed that treatment with HGF transgenic BMSCs transplantation significantly promoted tissue recovery from injury. The expression pattern of Col I assayed by immunohistochemistry in treatment group was reverse to that in untreated group, suggesting the increase in the activity of osteoblasts. Meanwhile, the expressions of VEGF and CD105were up-regulated, suggesting the occurrence of revascularization. These results confirmed the efficacies of transplantation of BMSCs. HGF regulated the activities of BMSCs through the mechanisms similar to that in early stage hormone-induced ANFH to promote tissue repair.Conclusions1. HGF transgenic BMSCs were achieved and provided the treatment basis of early stage ANFH.2. Rabbit early stage hormone-induced or traumatic ANFH models were established. These works provided the basis to further study the pathogenesis of early stage ANFH and to develop new, effective therapeutic method, and provided an appropriate platform for efficacy assessment of new therapies.3. Combination of HGF transgenic BMSCs and core decompression can effectively promote revascularization and bone matrix reconstruction in the necrotic region, and is hopeful to be a new treatment method for early stage ANFH. It is worthy to further explore the mechanisms how changes in HGF concentration regulate BMSCs activities and promote bone regeneration for promotion of therapeutic efficacies, thus provide the basis for future clinical applications.4. The tissue self-repair in early stage traumatic ANFH provides a therapeutic window to perform therapeutic intervention, which will cooperate with tissue self-repair to promote therapeutic efficacies. |
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