Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.     

Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro

Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity.     

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.     

New recombinant serotypes of AAV vectors

AAV based vectors can achieve stable gene transfer with minimal vector related toxicities. AAV serotype 2 (AAV2) is the first AAV that was vectored for gene transfer applications. However, the restricted tissue tropism of AAV and its low transduction efficiency have limited its further development as vector. Recent studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5 and 6 have shown improved potency and broadened tropism of the AAV vector by packaging the same vector genome with different AAV capsids. In an attempt to search for potent AAV vectors with enhanced performance profiles, molecular techniques were employed for the detection and isolation of endogenous AAVs from a variety of human and non-human primate (NHP) tissues. A family of novel primate AAVs consisting of 110 non-redundant species of proviral sequences was discovered and turned to be prevalent in 18-19% of the tissues evaluated. Phylogenetic and functional analyses revealed that primate AAVs are segregated into clades based on phylogenetic relatedness. The members within a clade share functional and serological properties. Initial evaluation in mouse models of vectors based on these novel AAVs for tissue tropism and gene transfer potency led to the identification of some vector with improved gene transfer to different target tissues. Gene therapy treatment of several mouse and canine models with novel AAV vectors achieved long term phenotypic corrections. Vectors based on new primate AAVs could become the next generation of efficient gene transfer vehicles for various gene therapy applications.     

Preliminary study of primarily cultured C57 articular cartilage transfected with plasmid IDO-EGFP by lipofectamine]

AIM: To determine the transfection efficiency and transient expression of pIDO-EGFP gene in primarily cultured C57 articular cartilage of mice, and to establish a transfection method of the primarily cultured articular cartilage in mice. METHODS: Plasmid IDO-EGFP was amplified in Escherichia coli. The primarily cultured mouse chondrocytes which were initially obtained from articular cartilage were cultured in vitro and transfected with pIDO-EGFP by lipofectamine2000 reagent under optimized condition. Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscopy (LSCM), and transfection efficiency was determined by flow cytometry. RESULTS: There was obvious expression of EGFP at 24 h after transfection. The transfection efficiency of pIDO-EGFP into primarily cultured mouse chondrocytes reached 36.43% at 48 hours and the transfection did not affect the process of cell adherence. CONCLUSION: IDO gene has been successfully transfected into primarily cultured chondrocytes by means of lipofectamine2000 reagent and the chondrocytes can survive in vitro. Satisfactory efficiency of transient transfection can be reached under optimized condition, which will provide a basis for gene introduction and modification of tissue engineered cartilage.     

Akt与ERK1/2在人骨关节炎软骨细胞中的表达

目的: 观察蛋白激酶B(Akt)与细胞外信号调节激酶1/2(ERK1/2)在正常和骨关节炎(OA)软骨细胞中的表达,探讨Akt与ERK1/2在OA病程中的意义。方法: 手术中取5例正常和18例OA人膝关节软骨组织,包埋制备切片,免疫组织化学技术观察 p-Akt及p-ERK1/2在正常和OA 软骨组织中的表达;培养人软骨细胞,甲苯胺蓝染色、免疫组化鉴定并观察聚集蛋白聚糖及Ⅱ型胶原在正常和OA软骨细胞中的表达; Western blotting技术检测Akt、p-Akt、ERK1/2、p-ERK1/2、磷酸化70 kD核糖体蛋白S6激酶(p-p70S6K)及增殖细胞核抗原(PCNA)蛋白在正常和OA软骨细胞中表达水平; 实时荧光定量PCR技术检测聚集蛋白聚糖及Ⅱ型胶原在正常和OA软骨细胞中mRNA表达水平。结果: 与正常软骨细胞比较,OA软骨细胞内p-Akt和p-p70S6K蛋白表达明显降低(P<0.05),且聚集蛋白聚糖和Ⅱ型胶原mRNA和蛋白在OA软骨细胞中的表达水平降低(P<0.05),而 p-ERK1/2和PCNA蛋白表达明显提高(P<0.05)。结论: Akt可能通过p-p70S6K来调控OA软骨细胞外基质聚集蛋白聚糖及II型胶原的合成,ERK1/2可能通过PCNA来调控OA软骨细胞增殖;Akt与ERK1/2可能参与了OA的病理过程。     

AAV-6 mediated efficient transduction of mouse lower airways

AAV1 and AAV6 are two closely related AAV serotypes. In the present study, we found AAV6 was more efficient in transducing mouse lower airway epithelia in vitro and in vivo than AAV1. To further explore the mechanism of this difference, we found that significantly more AAV1 bound to mouse airway epithelia than AAV6, yet transduction by AAV6 was far superior. Lectin competition assays demonstrated that both AAV1 and AAV6 similarly utilize α-2, 3-, and to a lesser extend α-2, 6- linked sialic acids as the receptors for transduction. Furthermore, the rates of AAV endocytosis could not account for the transduction differences of AAV1 and AAV6. Finally, it was revealed that AAV6 was less susceptible to ubiquitin/proteasome-mediated blocks than AAV1 when transducing mouse airway epithelia. Thus compared with AAV1, AAV6 has a unique ability to escape proteasome-mediated degradation, which is likely responsible for its higher transduction efficiency in mouse airway epithelium.     

脂质体介导pIDO-EGFP转染原代培养软骨细胞的初步研究

目的：检测脂质体介导pIDO-EGFP转染原代培养的C57小鼠关节软骨细胞的瞬时表达及转染效率，建立原代培养的小鼠关节软骨细胞转染方法。方法：大肠杆菌中扩增pIDO-EGFP质粒，在最优化条件下通过lipofectamine2000^TM转染试剂将pIDO-EGFP质粒转入原代培养的小鼠关节软骨细胞。应用荧光显微镜和激光共聚焦显微镜观察其转染过程及瞬时表达情况，流式细胞术检测其转染效率。结果：质粒携带的增强型绿色荧光蛋白在转染后24h得到了明显表达，48h后流式细胞术检测其转染效率为36．43％，未影响软骨细胞贴壁过程。结论：经绿色荧光蛋白检测表明，脂质体成功地将IDO基因转染进入原代培养的软骨细胞。转染后的软骨细胞在体外仍能存活，在最优化的条件下能达到良好的瞬时转染效率，为组织工程化软骨细胞基因导入和基因修饰提供了思路。     

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain

Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.     

In vivo cultivation of human articular chondrocytes in a nude mouse-based contained defect organ culture model

The nude mouse model is an established method to cultivate and investigate tissue engineered cartilage analogues under in vivo conditions. One limitation of this common approach is the lack of appropriate surrounding articular tissues. Thus the bonding capacity of cartilage repair tissue cannot be evaluated. Widely applied surgical techniques in cartilage repair such as conventional and three-dimensional autologous chondrocyte implantation (ACI) based on a collagen gel matrix cannot be included into nude mouse studies, since their application require a contained defect. The aim of this study is to apply an organ culture defect model for the in vivo cultivation of different cell-matrix-constructs.Cartilage defects were created on osteochondral specimens which had been harvested from 10 human knee joints during total knee replacement. Autologous chondrocytes were isolated from the cartilage samples and cultivated in monolayer until passage 2. On each osteochondral block defects were treated either by conventional ACI or a collagen gel seeded with autologous chondrocytes, including a defect left empty as a control. The samples were implanted into the subcutaneous pouches of nude mice and cultivated for six weeks. After retrieval, the specimens were examined histologically, immunohistochemically and by cell morphology quantification.In both, ACI and collagen gel based defect treatment, a repair tissue was formed, which filled the defect and bonded to the adjacent tissues. The repair tissue was immature with low production of collagen type II. In both groups redifferentiation of chondrocytes remained incomplete. Different appearances of interface zones between the repair tissue and the adjacent cartilage were found.The presented contained defect organ culture model offers the possibility to directly compare different types of clinically applied biologic cartilage repair techniques using human articular tissues in a nude mouse model.     

The bovine patella as a model of early osteoarthritis

The bovine patella model has been used extensively for studying important structure–function aspects of articular cartilage, including its degeneration. However, the degeneration seen in this model has, to our knowledge, never been adequately compared with human osteoarthritis (OA). In this study, bovine patellae displaying normal to severely degenerate states were compared with human tissue displaying intact cartilage to severe OA. Comparisons of normal and OA features were made with histological scoring, morphometric measurements, and qualitative observations. Differential interference contrast microscopy was used to image early OA changes in the articular cartilage matrix and to investigate whether this method provided comparable quality of visualisation of key structural features with standard histology. The intact bovine cartilage was found to be similar to healthy human cartilage and the degenerate bovine cartilage resembled the human OA tissues with regard to structural disruption, cellularity changes, and staining loss. The extent of degeneration in the bovine tissues matched the mild to moderate range of human OA tissues; however, no bovine samples exhibited late‐stage OA. Additionally, in both bovine and human tissues, cartilage degeneration was accompanied by calcified cartilage thickening, tidemark duplication, and the advancement of the cement line by protrusions of bony spicules into the calcified cartilage. This comparison of degeneration in the bovine and human tissues suggests a common pathway for the progression of OA and thus the bovine patella is proposed to be an appropriate model for investigating the structural changes associated with early OA.     

Immunolocalization of water channel aquaporins in human knee articular cartilage with intact and early degenerative regions

Aquaporins (AQPs), a family of water channel proteins expressed in various cells and tissues, serve as physiological pathways of water and small solute transport. Articular cartilage is avascular tissue with unique biomechanical structure, a major component of which is “water”. Our objective is to investigate the immunolocalization and expression pattern changes of AQPs in articular cartilage with normal and early degenerative regions in the human knee joint, which is the joint most commonly involved in osteoarthritis (OA). Two isoforms (AQPs 1 and 3) of AQPs were examined by immunohistochemical analyses using isoform-specific antibodies with cartilage samples from OA patients undergoing total knee arthroplasty. AQP 1 and AQP 3 were expressed in human knee articular cartilage and were localized in chondrocytes, both in the intact and early degenerative cartilage regions. Compared to the intact cartilage, both AQP 1 and AQP 3 immunopositive cells were observed at the damaged surface area in the degenerative region. These findings suggest that these AQPs play roles in metabolic water regulation in articular cartilage of load bearing joints and that they are responsible for OA onset.     

Adeno-associated virus mediated gene transfer into lung cancer cells promoting CD40 ligand-based immunotherapy

Expression of the CD40 ligand (CD40L) on tumors can activate host immune systems and produce antitumor effects against the tumors. To deliver the CD40L gene efficiently, we evaluated the efficiency of transduction of different serotypes of adeno-associated virus (AAV) vectors in lung cancer A549 cells and compared the transduction efficiency of a conventional AAV vector with that of self-complementary AAV (scAAV) vectors as well. We determined that serotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. And the transduction efficiency of scAAV2/5 was significantly higher than conventional AAV2/5. Furthermore, pre-treatment with carboplatin, which is a chemotherapeutic agent used in lung cancer chemotherapy, substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L were used to tranduce CD40L into A549 cells, which were then co-cultivated with immature human dendritic cells (DCs). Interleukin 12 (IL-12) that was released was measured in the culture supernatant. Specificity of the immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L. The in vivo antitumor activity was evaluated in BALB/c nude mice bearing A549 lung cancer. scAAV2/5-CD40L showed significant inhibitory effects on the growth of transplanted tumor cells as compared with control group. These studies suggest that recombinant AAV2/5-CD40L may provide an effective form of therapy for lung cancer.     

增强型绿色荧光蛋白基因转染对原代培养的人软骨细胞细胞周期的影响

目的:观察转染增强型绿色荧光蛋白 (EGFP) 基因后原代培养的人鼻中隔软骨细胞的细胞周期的变化,建立原代培养的人鼻中隔软骨细胞的示踪方法。方法:在大肠杆菌中扩增pEGFP-N1 质粒,通过Amaxa细胞核转染仪将pEGFP-N1质粒转入原代培养的人鼻中隔软骨细胞,应用激光共聚焦显微镜观察其转染过程及瞬时表达情况,流式细胞仪检测其转染效率和细胞周期的变化。结果:增强型绿色荧光蛋白基因在转染24 h后得到了明显表达,48 h后流式细胞仪检测其表达率为35.37％,细胞周期没有明显的改变,且未影响软骨细胞的贴壁过程。结论:经pEGFP-N1质粒转染的原代培养的人鼻中隔软骨细胞仍能在体外存活,对软骨细胞的生长没有明显的影响,pEGFP-N1是转染原代培养的人鼻中隔软骨细胞较为理想的瞬时表达载体,也是组织工程化软骨形成过程的良好示踪剂。     

Enhanced apoptotic and reduced protective response in chondrocytes following endoplasmic reticulum stress in osteoarthritic cartilage

Endoplasmic reticulum (ER) stress has been shown to participate in many disease pathologies. Although recent reports have demonstrated that ER stress in chondrocytes is present in human osteoarthritis (OA), its role in the pathology of cartilage degeneration, such as chondrocyte apoptosis, remains unclear. In the present study, we investigated the expression of phosphorylated PERK (pPERK), ubiquitin (Ub), GRP78, CHOP, phosphorylated JNK (pJNK) and cleaved caspase-3 (C-CASP3) and the mRNA splicing of XBP1 (XBP1 splicing) in human OA cartilage by immunohistochemistry and RT-PCR. Additionally, human chondrocytes were treated with several concentrations of tunicamycin, an ER stress inducer, to assess the impact of ER stress on the mRNA expression of CHOP, XBP1 splicing and apoptosis, as determined by real-time PCR, RT-PCR and ELISA analyses respectively. In human OA cartilage, the number of chondrocytes expressing pPERK, Ub, CHOP and pJNK positively correlated with cartilage degeneration and the number of C-CASP3-positive chondrocytes. XBP1 splicing and GRP78 expression in severe OA containing the greatest number of C-CASP3-positive chondrocytes were similar to the levels in mild OA, however, XBP1 splicing was higher in moderate OA than in mild and severe OA. Tunicamycin dose dependently increased CHOP expression and apoptosis of cultured chondrocytes. Although tunicamycin upregulated XBP1 splicing in cultured chondrocytes, its impact on XBP1 splicing was weakened at higher concentrations. In conclusion, the present results indicate that ER stress may contribute to chondrocyte apoptosis along with OA progression, which was closely associated with an enhanced apoptotic response and a reduced protective response by the cells.     

GFP和RFP报道基因在人体不同细胞中转导率的比较研究

目的 比较慢病毒载体介导的绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）报道基因在人体不同细胞中的转导率,为再生医学和组织工程学研究中报道基因的选择提供依据.方法 将构建的GFP和RFP慢病毒载体分别转染人骨髓间充质干细胞（hMSC）、人脐静脉内皮细胞株（Eahy926）和人肺泡上皮细胞株（A549）.4d后,用流式细胞仪检测GFP和RFP的转导率,并利用激光共聚焦显微镜观察GFP和RFP的表达特征.结果 GFP和RFP在hMSC、Eahy926和A549三种细胞间的转导率均有显著性差异（P﹤0.01）;在hMSC或Eahy926或A549的同种细胞中GFP和RFP的转导率也有显著性差异（P﹤0.01）;RFP在部分Eahy926和A549细胞局部高表达.结论 慢病毒载体介导的GFP和RFP报道基因在人体不同细胞中的转导率明显不同.     

β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold

Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker.     

DCC is expressed in a CD166-positive subpopulation of chondrocytes in human osteoarthritic cartilage and modulates CRE activity

Objective: In a recent study we determined a strong differential expression of DCC in OA compared to normal chondrocytes and a strong impact of the DCC receptor on cellular mobility triggered by its ligand Netrin-1. Migration of chondrocytes or their progenitor cells may play a role in remodeling of cartilage and pathological conditions. The purpose of this study is to identify subsets of chondrocytes expressing DCC and to understand signaling pathways used by DCC in chondrocytes. Methods: Immunofluorescent histology of human cartilage was used to determine the expression pattern of CD166, DCC and p-CREB. Cell culture of chondrocytes and SW1353, transient transfection, siRNA transfection, EMSA, luciferase assay, quantitative RT-PCR, ELISA, and Western Blotting were used to study signaling down-stream of DCC. Results: DCC expressing chondrocytes are mainly located in the surface layers of OA cartilage. These also express CD166 indicating that DCC expressing chondrocytes are progenitor cells. Interestingly, expression of DCC reduces cAMP levels, CREB DNA-binding activity and CRE activity in chondrocytes, whereas down-regulation of DCC results in induction of CRE signaling. Conclusion: In summary, DCC is up-regulated in CD166-positive chondrogenic progenitor cells in OA and induces down-regulation of CREB. These findings indicate that migration of CD166 positive progenitor cells to sites of cartilage damage may be directed by regulation of DCC signaling.