HGF基因修饰BMSCs治疗早期股骨头缺血性坏死的研究
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摘要
股骨头缺血性坏死(avascular necrosis of the femoral head, ANFH)是临床多发病和常见病,致残率极高,多见于20-50岁青壮年,严重影响患者的生活质量和劳动能力,给社会和家庭造成巨大负担。据世界卫生组织不完全统计,全世界约有3000万人患有此病；我国患者约500-750万,每年新发病例15-20万,发病率呈逐年上升趋势。ANFH目前尚无特效疗法,已成为世界医学界亟待攻克的难题。寻找新的有效的早期治疗方法、保存自身股骨头显得尤为重要,其潜在的社会和经济效益不言而喻。不论何种病因引起的ANFH,其根本原因是存在骨内压升高→血供障碍这一恶性循环。一般认为本病是一不可逆过程,因此,ANFH的治疗策略应该是早期治疗,降低骨内压、改善股骨头血供,打破恶性循环,同时进行坏死区骨质修复。利用血管生长因子诱导新血管生成和侧枝循环建立,是打破ANFH病理恶性循环最有效的手段,这也为ANFH治疗提供了新的研究方向。血管生长因子是一组可以诱导血管生成的细胞因子。不同的血管生成因子具有不完全相同的生物学活性,因此,选择何种血管生长因子至关重要。肝细胞生长因子(hepatocyte growth factor, HGF)由问质细胞产生,具有很强的促血管内皮细胞(vascular endothelial cells, VECs)增殖和促血管生成作用,并可抑制细胞凋亡和减轻组织纤维化,已在治疗缺血性心脏病和肢体动脉闭塞等疾病中展现出良好的应用前景,有望成为治疗ANFH理想的血管生长因子。
骨髓基质干细胞(bone marrow mesenchymal stem cells, BMSCs)是一种由骨髓中分离获得的具有多种分化潜能的间质干细胞,是体内参与组织再生和修复的重要干细胞成分之一,取材方便,易于被外源基因转染,同时还能分泌大量的促细胞和血管生长因子,其成分中含有骨祖细胞,具有良好的向成骨细胞分化的潜能。此外,BMSCs不具免疫原性,因此同种异体移植无免疫排斥反应,被认为是骨组织工程近阶段最有希望应用于临床的种子细胞。目前,用BMSCs治疗早期ANFH已显示出诱人的应用前景。
将基因治疗和干细胞移植治疗相结合,用HGF基因修饰BMSCs治疗ANFH,比单用BMSCs具有明显的优势,表现在：(1)BMSCs在发挥其成骨能力的同时分泌HGF,诱导新血管生成,改善股骨头血供；(2)HGF对BMSCs有趋化作用,局部高分泌的HGF可使BMSCs'‘停留”在坏死区局部,利于其向成骨细胞分化：(3)HGF能抑制BMSCs凋亡,其诱生的血管可改善移植细胞的生存环境,提高BMSCs移植存活率,进而提高治疗效果。
本课题将基因治疗、干细胞移植、组织工程技术有机结合,以HGF为目的基因、以BMSCs为靶细胞,借助AdMax腺病毒载体系统,将HGF基因转入BMSCs;再以其为种子细胞,经髓芯减压隧道移植到股骨头坏死区,用于治疗早期ANFH。在减压的同时,移植细胞分泌的HGF诱导局部新血管生成和侧枝循环建立,改善股骨头血供；BMSCs在局部微环境诱导下向成骨细胞分化,修复坏死区骨质,这种集减压、血管生成和骨质重建一体化的治疗技术,为ANFH治疗提供了一种全新的模式。
目的
在体外探讨HGF调节BMSCs增殖与成骨分化活性的方式与机制的基础上,评价HGF基因修饰BMSCs治疗早期激素性和创伤性ANFH的疗效,并初步探讨发病与治疗的分子机理。
方法：
1、分离培养与鉴定兔BMSCs
取4-8周龄健康新西兰大白兔,麻醉后,于双侧髂后上棘行穿刺法抽吸骨髓,抗凝处理后低速离心去上清,以10%FBS-低糖DMEM培养液重悬培养,3d时换液去除未贴壁细胞,其后每3d换液1次；待细胞汇合成单层后消化,传代细胞培养至第二代备用。
采用诱导培养基体外诱导BMSCs向成骨细胞、成软骨、脂肪细胞分化。培养12和24天后,磷酸苯二钠基质(NBT-BCIP)法染色和茜素红(AR-S)染色以检测成骨分化水平；培养27天后,阿利新蓝染色以检测成软骨分化水平,油红O染色以检测成脂分化水平；同时以qPCR法检测BMSCs标志基因Vimentin、成骨细胞标志基因Runx2、成软骨细胞标志基因SOX9与脂肪细胞标志基因PPAR-y3mRNA的表达水平。
2、体外检测HGF调节BMSCs增殖与分化活性的方式与机制
在成骨诱导环境中,以低浓度(20ng/mL)和高浓度(100ng/mL)的HGF处理BMSCs,EdU掺入法检测细胞增殖,WST-8法检测细胞活性；NBT-BCIP法行碱性磷酸酶(Alkaline phosphatase, ALP)染色,AR-S法检测钙结节的形成。Western和qPCR检测HGF受体c-Met、细胞分化开关分子——细胞周期抑制分子p27和成骨分化相关转录因子Runx2与Osterix的表达；以ERK信号通路特异性抑制剂PD98059(30lμM)或Akt信号通路特异性抑制剂Wortmannin (100nM)预处理兔BMSCs1h, Western检测信号通路活化水平及其对BMSCs增殖与分化的影响。
3、重组腺病毒Ad-HGF转染兔BMSCs,检测HGF的表达和活性
重组腺病毒Ad-HGF在293细胞中扩增、氯化铯法超离纯化,TCID50法测定感染性滴度,然后感染BMSCs, RT-PCR、原位杂交、免疫组化检测转染后细胞中HGF的转录和表达。
4、兔早期激素性ANFH造模与检测
采用马血清注射造成兔过敏性血管炎,再应用大剂量肾上腺糖皮质激素(醋酸泼尼松龙),诱导兔激素性ANFH。4周后利用DR、CT、MRI、动脉墨汁灌注造影、病理切片HE染色、免疫组化等多种方法,观察ANFH的影像及病理变化。
5、HGF基因修饰BMSCs治疗兔早期激素性ANFH的研究
在CT引导下行髓芯减压术,将HGF基因修饰BMSCs经减压隧道移植入兔早期激素性ANFH模型坏死区。治疗后4周,利用DR、CT、MRI、动脉墨汁灌注造影、病理切片HE染色、免疫组化等多种检测手段评价疗效,同时设置髓芯减压组为对照。
为探讨其机理,以腺病毒空载体转染BMSCs和未转染BMSCs为对照,术后4周,病理切片HE染色评价疗效；术后2天、2周、4周取材,qPCR检测p27、Runx2与Osterix mRNA的表达,免疫组化检测HGF、p-ERK1/2、p-Akt的表达。
6、兔早期创伤性ANFH造模与检测
采用截断股骨颈的方法造模：逐层切开表皮与肌肉,打开关节囊,切断股骨头周围、含圆韧带在内的血管与韧带,截断股骨颈基底部,使股骨头断端分离3-5min后复位,逐层缝合。术后3天、1周、2周、3周,利用CT、MRI、病理切片HE染色、免疫组化等多种方法,观察ANFH的影像及病理变化。
7、HGF基因修饰BMSCs治疗兔早期创伤性ANFH的研究
创伤后1周,在CT引导下行髓芯减压术,将HGF基因修饰BMSCs经减压隧道移植入兔早期创伤性ANFH模型坏死区,然后用小夹板固定截断股骨颈。术后4周,利用病理切片HE染色、免疫组化等手段评价疗效,同时设置腺病毒空载体转染BMSCs和未转染BMSCs为对照。
为探讨其机理,术后2天、2周、4周取材,免疫组化检测HGF、p-ERK1/2、p-Akt的表达。
8、统计学分析
所有计量资料结果用x±s表示。EdU法检测信号通路抑制剂处理后BMSCs增殖、WST-8法检测BMSCs活性、NBT-BCIP法检测ALP活性、AR-S染色法检测信号通路抑制剂处理后BMSCs钙结节形成、qPCR检测成骨诱导环境中不同剂量HGF对c-Met、Osterix、Runx2与p27mRNA表达水平的长期影响、IHC检测体内HGF、p-ERK1/2与p-Akt表达水平采用析因设计资料的方差分析；EdU法检测未施信号通路抑制剂处理的BMSCs增殖、AR-S染色法检测钙结节形成、骨髓细胞与股骨头骨髓的面积比、空骨陷窝率、IHC检测体内vWF、CD105、PCNA、Col I、OCN与VEGF的表达应用单因素方差分析(One-Way ANOVA),方差不齐时用Welch校正,在差异显著的前提下进行多重比较,方差齐时采用LSD法,方差不齐时采用Dunnett's T3法。检验水准a=0.05,双侧检验。采用SPSS16.0for windows统计软件包进行数据分析。
结果
1、成功培养出具有多向分化潜能的兔BMSCs
骨髓穿刺法分离、贴壁法纯化成功培养出兔BMSCs,染色法与qPCR检测证实兔BMSCs具有多向分化潜能,可分化为成骨细胞、成软骨细胞与成脂细胞。
2、体外探讨HGF调节BMSCs增殖与分化活性的方式与机制
①HGF体外调节BMSCs增殖与分化的活性
EdU掺入法与WST-8法检测细胞增殖活性,NBT-BCIP法与AR-S法检测细胞成骨分化。结果表明,100ng/mL HGF在成骨诱导环境中促进BMSCs增殖而抑制其向成骨细胞分化,而20ng/mL HGF对BMSCs增殖有所抑制,但显著促进其向成骨细胞分化。
②机制研究1：不同剂量HGF对其受体c-Met的表达调控
20ng/mL HGF显著提高c-Met的表达与活化水平,而100ng/mL HGF则部分抑制c-Met的表达,且对c-Met的活化效应明显低于20ng/mL HGF。提示不同剂量HGF通过诱导其受体的不同表达与活化水平而产生不同的效应。
③机制研究2：不同剂量HGF对ERK与Akt信号通路活化水平的影响
100ng/mL HGF通过活化ERK通路、抑制Akt通路活化以显著促进BMSCs的增殖而抑制其成骨分化；20ng/mL HGF则抑制ERK通路、活化Akt通路,从而显著促进BMSCs的成骨分化,但对细胞增殖的促进效应低于100ng/mLHGF的作用效果。提示不同剂量HGF通过活化不同信号通路而产生不同效应。
④机制研究3：不同剂量HGF对分化相关转录因子的表达调控
在成骨诱导环境中,20ng/mL HGF显著促进BMSCs中p27的表达,并通过提高Osterix与Runx2的表达以促进BMSCs的成骨分化；而100ng/mL HGF对p27的表达具有抑制效应,成骨分化相关转录因子的表达也受到抑制。提示不同剂量HGF通过调控p27、Osterix与Runx2的表达而产生不同的效应。
3、Ad-HGF转染的BMSCs表达高水平HGF
Ad-HGF感染性滴度为2.6x1010TCID50/mL。转染后的BMSCs在mnRNA和蛋白水平均有HGF表达。
4、成功建立兔早期激素性ANFH模型
DR、CT、MRI、动脉墨汁灌注造影、病理切片HE染色检测结果表明,激素注射后4周,成功制备出兔早期激素性ANFH模型。免疫组化检测新生血管指标--CDl05和组织修复指标--Ⅰ型胶原(ColⅠ),结果提示：激素造模后,股骨头血管生成受抑,成骨反应逐渐减弱,无自身修复现象,4周发展为早期激素性ANFH。
5、HGF基因修饰BMSCs治疗兔早期激素性ANFH的研究
①疗效评价：
影像学和病理组织学检查结果表明,治疗组可显著促进坏死区血管再生和骨质重建,DR显示治疗侧股骨头骨纹理较清楚；CT示点状低密度影数量减少；MRI示点线状高信号影不明显；动脉墨汁灌注造影显示软骨下血管重建,干骺端大血管恢复；病理切片HE染色结果显示骨小梁排列基本规则,骨基质量增多,空骨陷窝少,骨小梁骨板上可见新生毛细血管,边缘有大量成骨细胞,骨髓中造血组织基本恢复。
②体内实验机理研究：
HGF基因修饰BMSCs治疗组p27、Runx2与Osterix的表达水平最高,表达高峰在移植后2周。HGF的表达水平在ANFH模型组中显著降低,但在HGF基因修饰BMSCs治疗组中达到最高峰,表达高峰出现在移植后2天,ERK1/2信号通路活化水平随之增加,之后二者表达水平逐渐降低。2周后p-Akt的水平逐渐增高,表达峰值出现在移植后4周。上述结果表明,HGF基因修饰BMSCs的HGF表达水平具有与体外相同的变化趋势,并通过与体外相同的机制,即由腺病毒载体介导的HGF体内浓度的有序改变来调控BMSCs增殖与分化,发挥促组织修复与再生的功能。
6、成功建立兔早期创伤性ANFH模型
影像学与病理组织学检查结果表明,术后3周,成功制备出处于临床ARCOI-II期的兔早期创伤性ANFH模型。病理组织学检查可见骨组织与血管系统发生明显的坏死病变,同时,创伤后2周内可见明显的组织自我修复。CD105、Col I、OCN和PCNA免疫组化检测结果显示,手术截断股骨颈后,股骨头血管生成受抑,组织损伤显著,术后3天有一定程度的自身修复,之后由于血运阻断,修复失败,3周发展为早期创伤性ANFH。
7、HGF基因修饰BMSCs治疗兔早期创伤性ANFH的研究
病理组织学检查结果表明,HGF基因修饰BMSCs可显著促进创伤组织的恢复,免疫组化检测到ColⅠ的表达模式与未治疗组相反,提示成骨细胞活性增加,同时在治疗组动物中VEGF与CD105表达上调,提示新生血管的发生,进一步证实了BMSCs的疗效。HGF在其中以类似于在激素性ANFH中的机制对BMSCs舌性进行调节,从而促进其修复创伤组织的功能。
结论
1、获得高表达人HGF基因的BMSCs,为其用于早期ANFH的治疗奠定基础。
2、成功诱导了兔早期激素性和创伤性ANFH模型,为进一步研究其发病机理、发展新的有效的早期治疗方法提供了基础,为将来评估新疗法的疗效提供了适宜的平台。
3、HGF基因修饰BMSCs联合隧芯减压能有效促进坏死区血管再生和骨质重建,有望成为一种新的ANFH早期治疗手段。可进一步深入探讨HGF浓度改变对BMSCs活性调节的机制,与促进骨再生、提高ANFH疗效,为将来临床应用提供基础。
4、创伤性ANFH具有自我修复现象,在适当时机进行干预治疗,与组织自身修复相协同,将有助于提高疗效。
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 BMSCs
Bone 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 model
ANFH 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 model
Animals 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 analysis
All 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.
Results
1. Culture of rabbit BMSCs with pluripotency
Rabbit 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 differentiation
Cell 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 BMSCs
Treatment 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 differentiation
100ng/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 differentiation
In 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 BMSCs
The 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 model
The 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 efficacy
Imaging 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 model
An 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.
Conclusions
1. 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.
骨髓基质干细胞(bone marrow mesenchymal stem cells, BMSCs)是一种由骨髓中分离获得的具有多种分化潜能的间质干细胞,是体内参与组织再生和修复的重要干细胞成分之一,取材方便,易于被外源基因转染,同时还能分泌大量的促细胞和血管生长因子,其成分中含有骨祖细胞,具有良好的向成骨细胞分化的潜能。此外,BMSCs不具免疫原性,因此同种异体移植无免疫排斥反应,被认为是骨组织工程近阶段最有希望应用于临床的种子细胞。目前,用BMSCs治疗早期ANFH已显示出诱人的应用前景。
将基因治疗和干细胞移植治疗相结合,用HGF基因修饰BMSCs治疗ANFH,比单用BMSCs具有明显的优势,表现在：(1)BMSCs在发挥其成骨能力的同时分泌HGF,诱导新血管生成,改善股骨头血供；(2)HGF对BMSCs有趋化作用,局部高分泌的HGF可使BMSCs'‘停留”在坏死区局部,利于其向成骨细胞分化：(3)HGF能抑制BMSCs凋亡,其诱生的血管可改善移植细胞的生存环境,提高BMSCs移植存活率,进而提高治疗效果。
本课题将基因治疗、干细胞移植、组织工程技术有机结合,以HGF为目的基因、以BMSCs为靶细胞,借助AdMax腺病毒载体系统,将HGF基因转入BMSCs;再以其为种子细胞,经髓芯减压隧道移植到股骨头坏死区,用于治疗早期ANFH。在减压的同时,移植细胞分泌的HGF诱导局部新血管生成和侧枝循环建立,改善股骨头血供；BMSCs在局部微环境诱导下向成骨细胞分化,修复坏死区骨质,这种集减压、血管生成和骨质重建一体化的治疗技术,为ANFH治疗提供了一种全新的模式。
目的
在体外探讨HGF调节BMSCs增殖与成骨分化活性的方式与机制的基础上,评价HGF基因修饰BMSCs治疗早期激素性和创伤性ANFH的疗效,并初步探讨发病与治疗的分子机理。
方法：
1、分离培养与鉴定兔BMSCs
取4-8周龄健康新西兰大白兔,麻醉后,于双侧髂后上棘行穿刺法抽吸骨髓,抗凝处理后低速离心去上清,以10%FBS-低糖DMEM培养液重悬培养,3d时换液去除未贴壁细胞,其后每3d换液1次；待细胞汇合成单层后消化,传代细胞培养至第二代备用。
采用诱导培养基体外诱导BMSCs向成骨细胞、成软骨、脂肪细胞分化。培养12和24天后,磷酸苯二钠基质(NBT-BCIP)法染色和茜素红(AR-S)染色以检测成骨分化水平；培养27天后,阿利新蓝染色以检测成软骨分化水平,油红O染色以检测成脂分化水平；同时以qPCR法检测BMSCs标志基因Vimentin、成骨细胞标志基因Runx2、成软骨细胞标志基因SOX9与脂肪细胞标志基因PPAR-y3mRNA的表达水平。
2、体外检测HGF调节BMSCs增殖与分化活性的方式与机制
在成骨诱导环境中,以低浓度(20ng/mL)和高浓度(100ng/mL)的HGF处理BMSCs,EdU掺入法检测细胞增殖,WST-8法检测细胞活性；NBT-BCIP法行碱性磷酸酶(Alkaline phosphatase, ALP)染色,AR-S法检测钙结节的形成。Western和qPCR检测HGF受体c-Met、细胞分化开关分子——细胞周期抑制分子p27和成骨分化相关转录因子Runx2与Osterix的表达；以ERK信号通路特异性抑制剂PD98059(30lμM)或Akt信号通路特异性抑制剂Wortmannin (100nM)预处理兔BMSCs1h, Western检测信号通路活化水平及其对BMSCs增殖与分化的影响。
3、重组腺病毒Ad-HGF转染兔BMSCs,检测HGF的表达和活性
重组腺病毒Ad-HGF在293细胞中扩增、氯化铯法超离纯化,TCID50法测定感染性滴度,然后感染BMSCs, RT-PCR、原位杂交、免疫组化检测转染后细胞中HGF的转录和表达。
4、兔早期激素性ANFH造模与检测
采用马血清注射造成兔过敏性血管炎,再应用大剂量肾上腺糖皮质激素(醋酸泼尼松龙),诱导兔激素性ANFH。4周后利用DR、CT、MRI、动脉墨汁灌注造影、病理切片HE染色、免疫组化等多种方法,观察ANFH的影像及病理变化。
5、HGF基因修饰BMSCs治疗兔早期激素性ANFH的研究
在CT引导下行髓芯减压术,将HGF基因修饰BMSCs经减压隧道移植入兔早期激素性ANFH模型坏死区。治疗后4周,利用DR、CT、MRI、动脉墨汁灌注造影、病理切片HE染色、免疫组化等多种检测手段评价疗效,同时设置髓芯减压组为对照。
为探讨其机理,以腺病毒空载体转染BMSCs和未转染BMSCs为对照,术后4周,病理切片HE染色评价疗效；术后2天、2周、4周取材,qPCR检测p27、Runx2与Osterix mRNA的表达,免疫组化检测HGF、p-ERK1/2、p-Akt的表达。
6、兔早期创伤性ANFH造模与检测
采用截断股骨颈的方法造模：逐层切开表皮与肌肉,打开关节囊,切断股骨头周围、含圆韧带在内的血管与韧带,截断股骨颈基底部,使股骨头断端分离3-5min后复位,逐层缝合。术后3天、1周、2周、3周,利用CT、MRI、病理切片HE染色、免疫组化等多种方法,观察ANFH的影像及病理变化。
7、HGF基因修饰BMSCs治疗兔早期创伤性ANFH的研究
创伤后1周,在CT引导下行髓芯减压术,将HGF基因修饰BMSCs经减压隧道移植入兔早期创伤性ANFH模型坏死区,然后用小夹板固定截断股骨颈。术后4周,利用病理切片HE染色、免疫组化等手段评价疗效,同时设置腺病毒空载体转染BMSCs和未转染BMSCs为对照。
为探讨其机理,术后2天、2周、4周取材,免疫组化检测HGF、p-ERK1/2、p-Akt的表达。
8、统计学分析
所有计量资料结果用x±s表示。EdU法检测信号通路抑制剂处理后BMSCs增殖、WST-8法检测BMSCs活性、NBT-BCIP法检测ALP活性、AR-S染色法检测信号通路抑制剂处理后BMSCs钙结节形成、qPCR检测成骨诱导环境中不同剂量HGF对c-Met、Osterix、Runx2与p27mRNA表达水平的长期影响、IHC检测体内HGF、p-ERK1/2与p-Akt表达水平采用析因设计资料的方差分析；EdU法检测未施信号通路抑制剂处理的BMSCs增殖、AR-S染色法检测钙结节形成、骨髓细胞与股骨头骨髓的面积比、空骨陷窝率、IHC检测体内vWF、CD105、PCNA、Col I、OCN与VEGF的表达应用单因素方差分析(One-Way ANOVA),方差不齐时用Welch校正,在差异显著的前提下进行多重比较,方差齐时采用LSD法,方差不齐时采用Dunnett's T3法。检验水准a=0.05,双侧检验。采用SPSS16.0for windows统计软件包进行数据分析。
结果
1、成功培养出具有多向分化潜能的兔BMSCs
骨髓穿刺法分离、贴壁法纯化成功培养出兔BMSCs,染色法与qPCR检测证实兔BMSCs具有多向分化潜能,可分化为成骨细胞、成软骨细胞与成脂细胞。
2、体外探讨HGF调节BMSCs增殖与分化活性的方式与机制
①HGF体外调节BMSCs增殖与分化的活性
EdU掺入法与WST-8法检测细胞增殖活性,NBT-BCIP法与AR-S法检测细胞成骨分化。结果表明,100ng/mL HGF在成骨诱导环境中促进BMSCs增殖而抑制其向成骨细胞分化,而20ng/mL HGF对BMSCs增殖有所抑制,但显著促进其向成骨细胞分化。
②机制研究1：不同剂量HGF对其受体c-Met的表达调控
20ng/mL HGF显著提高c-Met的表达与活化水平,而100ng/mL HGF则部分抑制c-Met的表达,且对c-Met的活化效应明显低于20ng/mL HGF。提示不同剂量HGF通过诱导其受体的不同表达与活化水平而产生不同的效应。
③机制研究2：不同剂量HGF对ERK与Akt信号通路活化水平的影响
100ng/mL HGF通过活化ERK通路、抑制Akt通路活化以显著促进BMSCs的增殖而抑制其成骨分化；20ng/mL HGF则抑制ERK通路、活化Akt通路,从而显著促进BMSCs的成骨分化,但对细胞增殖的促进效应低于100ng/mLHGF的作用效果。提示不同剂量HGF通过活化不同信号通路而产生不同效应。
④机制研究3：不同剂量HGF对分化相关转录因子的表达调控
在成骨诱导环境中,20ng/mL HGF显著促进BMSCs中p27的表达,并通过提高Osterix与Runx2的表达以促进BMSCs的成骨分化；而100ng/mL HGF对p27的表达具有抑制效应,成骨分化相关转录因子的表达也受到抑制。提示不同剂量HGF通过调控p27、Osterix与Runx2的表达而产生不同的效应。
3、Ad-HGF转染的BMSCs表达高水平HGF
Ad-HGF感染性滴度为2.6x1010TCID50/mL。转染后的BMSCs在mnRNA和蛋白水平均有HGF表达。
4、成功建立兔早期激素性ANFH模型
DR、CT、MRI、动脉墨汁灌注造影、病理切片HE染色检测结果表明,激素注射后4周,成功制备出兔早期激素性ANFH模型。免疫组化检测新生血管指标--CDl05和组织修复指标--Ⅰ型胶原(ColⅠ),结果提示：激素造模后,股骨头血管生成受抑,成骨反应逐渐减弱,无自身修复现象,4周发展为早期激素性ANFH。
5、HGF基因修饰BMSCs治疗兔早期激素性ANFH的研究
①疗效评价：
影像学和病理组织学检查结果表明,治疗组可显著促进坏死区血管再生和骨质重建,DR显示治疗侧股骨头骨纹理较清楚；CT示点状低密度影数量减少；MRI示点线状高信号影不明显；动脉墨汁灌注造影显示软骨下血管重建,干骺端大血管恢复；病理切片HE染色结果显示骨小梁排列基本规则,骨基质量增多,空骨陷窝少,骨小梁骨板上可见新生毛细血管,边缘有大量成骨细胞,骨髓中造血组织基本恢复。
②体内实验机理研究：
HGF基因修饰BMSCs治疗组p27、Runx2与Osterix的表达水平最高,表达高峰在移植后2周。HGF的表达水平在ANFH模型组中显著降低,但在HGF基因修饰BMSCs治疗组中达到最高峰,表达高峰出现在移植后2天,ERK1/2信号通路活化水平随之增加,之后二者表达水平逐渐降低。2周后p-Akt的水平逐渐增高,表达峰值出现在移植后4周。上述结果表明,HGF基因修饰BMSCs的HGF表达水平具有与体外相同的变化趋势,并通过与体外相同的机制,即由腺病毒载体介导的HGF体内浓度的有序改变来调控BMSCs增殖与分化,发挥促组织修复与再生的功能。
6、成功建立兔早期创伤性ANFH模型
影像学与病理组织学检查结果表明,术后3周,成功制备出处于临床ARCOI-II期的兔早期创伤性ANFH模型。病理组织学检查可见骨组织与血管系统发生明显的坏死病变,同时,创伤后2周内可见明显的组织自我修复。CD105、Col I、OCN和PCNA免疫组化检测结果显示,手术截断股骨颈后,股骨头血管生成受抑,组织损伤显著,术后3天有一定程度的自身修复,之后由于血运阻断,修复失败,3周发展为早期创伤性ANFH。
7、HGF基因修饰BMSCs治疗兔早期创伤性ANFH的研究
病理组织学检查结果表明,HGF基因修饰BMSCs可显著促进创伤组织的恢复,免疫组化检测到ColⅠ的表达模式与未治疗组相反,提示成骨细胞活性增加,同时在治疗组动物中VEGF与CD105表达上调,提示新生血管的发生,进一步证实了BMSCs的疗效。HGF在其中以类似于在激素性ANFH中的机制对BMSCs舌性进行调节,从而促进其修复创伤组织的功能。
结论
1、获得高表达人HGF基因的BMSCs,为其用于早期ANFH的治疗奠定基础。
2、成功诱导了兔早期激素性和创伤性ANFH模型,为进一步研究其发病机理、发展新的有效的早期治疗方法提供了基础,为将来评估新疗法的疗效提供了适宜的平台。
3、HGF基因修饰BMSCs联合隧芯减压能有效促进坏死区血管再生和骨质重建,有望成为一种新的ANFH早期治疗手段。可进一步深入探讨HGF浓度改变对BMSCs活性调节的机制,与促进骨再生、提高ANFH疗效,为将来临床应用提供基础。
4、创伤性ANFH具有自我修复现象,在适当时机进行干预治疗,与组织自身修复相协同,将有助于提高疗效。
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 BMSCs
Bone 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 model
ANFH 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 model
Animals 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 analysis
All 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.
Results
1. Culture of rabbit BMSCs with pluripotency
Rabbit 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 differentiation
Cell 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 BMSCs
Treatment 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 differentiation
100ng/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 differentiation
In 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 BMSCs
The 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 model
The 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 efficacy
Imaging 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 model
An 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.
Conclusions
1. 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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