Yongchang Yao

The control of anchorage-dependent cell behavior within a hydrogel/ microcarrier system in an osteogenic model

The use of injectable hydrogels for tissue engineering purposes such as bone regeneration has been hampered by the mass depletion of cells after encapsulation, due to the lack of a proper interface between hydrogel matrices and osteo-progenitor cells. Efforts to graft bioactive molecules as cell attachment moieties have achieved limited success. In this study, we devised a solution to promote cellular focal adhesion within hydrogels, and elicit the mechanism behind cellular survival/death therein. We found that the fulfillment of ligation between cellular integrins and extracellular ligands, instead of the expression of integrins per se, is essential to avoid apoptosis in gel-encapsulated anchorage-dependent cells (ADCs). Absence of such ligation brought about mass cell death in our osteogenic model with osteoblasts (as representative of ADCs) and failure of osteogenic commitment of mesenchymal stem cells (as representative of anchorage-dependent progenitors). We have designed a gel-based composite system that works as a suspension of injectable cell-laden microcarriers in hydrogel, as compared to the conventional cell-suspended hydrogels. Injectable microscopic anchors (microcarriers) not only provide platforms for cellular focal adhesion but also facilitate the cells to overcome gel enlacement and fully spread out into their natural morphology. Further in vitro and in vivo osteogenic investigations show the composites to be a competent potential injectable vehicle for the conveyance of ADCs and regenerations of bone and other tissues.

Antisense makes sense in engineered regenerative medicine.

The use of antisense strategies such as ribozymes, oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) in gene therapy, in conjunction with the use of stem cells and tissue engineering, has opened up possibilities in curing degenerative diseases and injuries to non-regenerating organs and tissues. With their unique ability to down-regulate or silence gene expression, antisense oligonucleotides are uniquely suited in turning down the production of pathogenic or undesirable proteins and cytokines. Here, we review the antisense strategies and their applications in regenerative medicine with a focus on their efficacies in promoting cell viability, regulating cell functionalities as well as shaping an optimal microenvironment for therapeutic purposes.

Gene Transfer and Living Release of Transforming Growth Factor-β3 for Cartilage Tissue Engineering Applications

Cartilage restoration continues to present a tremendous clinical challenge due to its nonvascular nature. Many studies have demonstrated that chondrogenesis of progenitor cells can be achieved in vitro by manual dose of growth factors; however, it remains a vital difficulty in feeding growth factors to implanted therapeutic cells in vivo. Herein, we constructed recombinant adenovirus encoding human transforming growth factor-beta3 (hTGF-beta3) and practiced it in rat bone marrow-derived mesenchymal stromal cells and articular chondrocytes for cartilage regeneration. Optimal viable transduction and transgenic hTGF-beta3 production were achieved; consequently, positive expression of cartilage marker-collagen type II was enabled in the infected progenitors. We thus conclude that recombinant adenovirus encoding TGF-beta3 gene has been successfully established and validated for cartilage tissue engineering applications.

A dual-functioning adenoviral vector encoding both transforming growth factor-β3 and shRNA silencing type I collagen: Construction and controlled release for chondrogenesis

Hyaline articular cartilage degeneration is a common clinical syndrome globally, whereas cell-based therapy has proved to be a good solution to such problems. Given that transforming growth factor (TGF-beta3) is helpful in maintaining chondrocytic phenotype or stimulating chondrogenic differentiation of stem cells, vectors containing TGF-beta3 expression cassette can be delivered to therapeutic cells. One problem involved in the application of therapeutic cells in chondrogenesis is the undesirable production of type I collagen in such cells as chondrocytes and synovial mesenchymal stem cells during ex vivo culture, which undermines the mechanical strength of engineered cartilage. RNA interference (RNAi) strategy can be used to knock down its expression to allow better biological and mechanical functions in artificial tissues. In this study, for the first time we report the construction of an adenoviral vector that can express both TGF-beta3 to promote chondrogenesis and short hairpin RNA (shRNA) targeting type I collagen to block its production. This dual-functioning vector (Ad-dual) was found to function well in three model cell types: human fibroblast, osteoblast and porcine chondrocyte in terms of the release of TGF-beta3 protein and down-regulation of type I collagen production. Besides, we also tested its efficacy in porcine synovial mesenchymal stem cells, highlighting its potential applications in cell-based therapy for the treatment of articular cartilage degeneration.

Effects of combinational adenoviral vector‐mediated TGFβ3 transgene and shRNA silencing type I collagen on articular chondrogenesis of synovium‐derived mesenchymal stem cells

In this study, transgenic effects of combination of transforming growth factor (TGF) β3 and shRNA silencing type I collagen (Col I) on chondrogenesis of synovium‐derived mesenchymal stem cells (SMSCs) were evaluated. SMSCs were infected with recombinant adenoviruses encoding TGFβ3 (Ad‐TGFβ3) and/or anti‐Col I shRNA (Ad‐shRNA) separately, simultaneously (Ad‐combination), or conjugately (Ad‐double, mediated by one vector encoding both). The transduced SMSCs were encapsulated in alginate hydrogel and cultured for 30 days in chondrogenic medium. The expression of cartilaginous extracellular matrix components was investigated by quantitative real‐time RT‐PCR (qRT‐PCR) and histological staining. qRT‐PCR showed an up‐regulation in chondrocytes marker genes such as type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP) in Ad‐TGFβ3, Ad‐double, and Ad‐combination groups on day 30. Whereas, Ad‐TGFβ3 treatment induced significant elevation in Col I, which could be largely resisted by anti‐Col I shRNA functionality. Histological and immunohistochemical staining results were consistent with our qRT‐PCR data. These results demonstrate that the application of combinational adenoviral vector‐mediated transgenic TGFβ3 and shRNA targeting Col I possesses the potential in promoting the chondrogenic differentiation of SMSCs as well as inhibiting the formation of fibrocartilage. Biotechnol. Bioeng. 2010;106: 818–828.

Optimal Construction and Delivery of Dual-Functioning Lentiviral Vectors for Type I Collagen-Suppressed Chondrogenesis in Synovium-Derived Mesenchymal Stem Cells

ABSTRACTPurposeThis study aims to deliver both transforming growth factor β3 (TGF-β3) and shRNA targeting type I collagen (Col I) by optimal construction and application of various dual-functioning lentiviral vectors to induce Col I-suppressed chondrogenesis in synovium-derived mesenchymal stem cells (SMSCs).MethodsWe constructed four lentiviral vectors (LV-1, LV-2, LV-3 and LV-4) with various arrangements of the two expression cassettes in different positions and orientations. Col I inhibition efficiency and chondrogenic markers were assessed with qPCR, ELISA and staining techniques. Among the four vectors, LV-1 has two distant and reversely oriented cassettes, LV-2 has two distant and same-oriented cassettes, LV-3 has two proximal and reversely oriented cassettes, and LV-4 has two proximal and same-oriented cassettes. Col I and chondrogenic markers, including type II collagen (Col II), aggrecan and glycosaminoglycan (GAG), were examined in SMSCs cultured in 3-D alginate hydrogel.ResultsAll of the four vectors showed distinct effects in Col I level as well as diverse inductive efficiencies in upregulation of the cartilaginous markers. Based on real-time PCR results, LV-1 was optimal towards Col I-suppressed chondrogenesis.ConclusionLV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.

Co-transduction of lentiviral and adenoviral vectors for co-delivery of growth factor and shRNA genes in mesenchymal stem cells-based chondrogenic system

Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co‐transduced in a synovial mesenchymal stem cells (SMSCs)‐based articular chondrogenic system by which two transgenes were co‐delivered – the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co‐delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three‐dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral‐mediated TGFβ3 release and adenoviral‐mediated shRNA expression (LV‐T + Ad‐sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs. Copyright

In vitro study of chondrocyte redifferentiation with lentiviral vector-mediated transgenic TGF-β3 and shRNA suppressing type I collagen in three-dimensional culture.

Chondrocytes are the primary candidate therapeutic cells to cure cartilaginous lesions. Ideally, for transplantation, autologous chondrocytes are isolated from the patient, amplified in vitro, seeded in a scaffold and implanted back. However, significant concerns arise with chondrocyte dedifferentiation during monolayer amplification, whereby cells lose their chondrocytic phenotype by rapidly downregulating the expression of cartilage markers such as type II collagen (Col II) and aggrecan. The accompanying upregulation in type I collagen (Col I) is also problematic, as it leads to unexpected fibrosis and causes such engineered cartilage to lack the desired mechanical strength to make up joint lesions. Transforming growth factor‐β3 (TGF‐β3) has been proved effective in maintaining chondrocytic morphology and promoting total collagen production. In this study, we aimed to deliver the TGF‐β3 gene into dedifferentiated chondrocytes with recombinant lentiviral vectors; by transgenic expression of TGF‐β3, chondrocytic redifferentiation is catalysed. Simultaneously, shRNA targeting Col I was also incorporated into the vector to suppress Col I production. The results indicated that chondrocytes underwent dedifferentiation in monolayer culture in the presence or absence of transgenic TGF‐β3. In three‐dimensional culture, effective redifferentiation was managed in the dedifferentiated chondrocytes that were transduced with transgenic TGF‐β3. The incorporation and expression of Col I‐targeting shRNA were also effective in reducing Col I production in a post‐transcriptional manner. Copyright

Redifferentiation of Dedifferentiated Chondrocytes by Adenoviral Vector-Mediated TGF-β3 and Collagen-1 Silencing shRNA in 3D Culture

Autologous chondrocytes remain one of the most preferable candidates among various therapeutic cell species because of their high efficacy, despite remarkable progress in discovery and development of therapeutic cells for cartilage regenerative medicine to date. However, the essential process of cell expansion via repeated monolayer sub-cultures inevitably induces chondrocytic dedifferentiation. In this study, we aimed to achieve and enhance redifferentiation of dedifferentiated chondrocytes with dual genes of transforming growth factor (TGF)-β3 and short hairpin RNA (shRNA) that restore chondrocytic phenotype and silence fibrous collagen type I (Col I), respectively. It was hypothesized that gene delivery of the two targets would promote chondrogenesis in chondrocytes, and meanwhile inhibit the expression of the undesired Col I. Three types of recombinant adenoviruses were constructed. Two of them were of single-function vectors with the ability to express either TGF-β3 (Ad-TGFβ3) or shRNA (specific for Col I, Ad-shRNA); the third type was of double-function vectors that encode both TGF-β3 and anti-Col I shRNA (Ad-double). We infected the dedifferentiated chondrocytes with Ad-double, or co-transduced them with Ad-TGFβ3 and Ad-shRNA at the same time (designated as Ad-combination). Data from real-time RT-PCR and histological staining suggested a restoration in the expression of cartilage-specific genes including aggrecan, type II collagen, and cartilage oligomeric matrix protein (COMP); while a significant down-regulation of Col I expression was observed in groups treated with Ad-double and Ad-combination compared to other control groups. These results demonstrated that, by genetic modification, dedifferentiated chondrocytes managed to redifferentiate back to chondrocytic phenotype, which may greatly facilitate cartilage regenerative medicine by providing sufficient number of competent therapeutic cells.

Continuous supply of TGFβ3 via adenoviral vector promotes type I collagen and viability of fibroblasts in alginate hydrogel

In recent years, transforming growth factor‐β3 (TGFβ3) has interested more and more researchers with its competence in engineered histogenesis. In the present study we employed recombinant adenoviral vectors to deliver the constitutively active TGFβ3 gene to human dermal fibroblasts, which could maintain the continuous secretion of TGFβ3 from the cells. The expression of type I collagen in the Ad‐TGFβ3 group increased significantly in comparison with other three groups: Neg (cells without treatment of the adenovirus), Ad‐null (cells with treatment of the adenovirus, without the inserted gene) and Ad‐shRNA (cells with treatment of the adenovirus encoding shRNA specific for type I collagen). Additionally, we demonstrated that TGFβ3 enhanced the expression of Smad4 while inhibiting that of MMP‐9, thus promoting the collagen transcription via the Smad signal transduction pathway and restraining collagen degradation by MMP‐9, which contributed to the increasing type I collagen expression level. As type I collagen mediates cell–material interactions by providing anchorage, the viability of encapsulated fibroblasts in Ad‐TGFβ3 group was significantly higher than that in other three groups. Accordingly, this approach forms an effective way to improve the compatibility of non‐adhesive hydrogels containing anchorage‐dependent cells. Copyright