Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

AbstractThe severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV

TGF-β Type II Receptor/MCP-5 Axis: At the Crossroad between Joint and Growth Plate Development

Despite its clinical significance, the mechanisms of joint morphogenesis are still elusive. Here, we show by combining laser-capture microdissection for RNA sampling with microarray analysis, that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by down-regulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in-vivo, ex-vivo and in-vitro approaches, we showed that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2Prx1KO), which lack joint development and fail chondrocyte hypertrophy, showed up-regulation of interzone-MCP-5. In-vivo and ex-vivo blockade of the sole MCP-5 receptor, CCR2, in Tgfbr2Prx1KO led to rescue of joint formation and growth plate maturation; while in control mice determined an acceleration of endochondral growth plate mineralization. Taken together, we characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth

Use of glycol chitosan modified by 5β-cholanic acid nanoparticles for the sustained release of proteins during murine embryonic limb skeletogenesis

Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-Agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol-chitosan–5β-cholanic-acid-conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission-electron-microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% of released after 48-hours. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age (E)13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC-nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology

Mesenchymal stem cells at the intersection of cell and gene therapy

Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secret bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases

Joint TGF-β Type II Receptor-Expressing Cells: Ontogeny and Characterization as Joint Progenitors

TGF-β type II receptor (Tgfbr2) signaling plays an essential role in joint-element development. The Tgfbr2PRX-1KO mouse, in which the Tgfbr2 is conditionally inactivated in developing limbs, lacks interphalangeal joints and tendons. In this study, we used the Tgfbr2-β-Gal-GFP-BAC mouse as a LacZ/green fluorescent protein (GFP)-based read-out to determine: the spatial and temporally regulated expression pattern of Tgfbr2-expressing cells within joint elements; their expression profile; and their slow-cycling labeling with bromodeoxyuridine (BrdU). Tgfbr2-β-Gal activity was first detected at embryonic day (E) 13.5 within the interphalangeal joint interzone. By E16.5, and throughout adulthood, Tgfbr2-expressing cells clustered in a contiguous niche that comprises the groove of Ranvier and the synovio-entheseal complex including part of the perichondrium, the synovium, the articular cartilage superficial layer, and the tendon's entheses. Tgfbr2-expressing cells were found in the synovio-entheseal complex niche with similar temporal pattern in the knee, where they were also detected in meniscal surface, ligaments, and the synovial lining of the infrapatellar fat pad. Tgfbr2-β-Gal-positive cells were positive for phospho-Smad2, signifying that the Tgfbr2 reporter was accurate. Developmental-stage studies showed that Tgfbr2 expression was in synchrony with expression of joint-morphogenic genes such as Noggin, GDF5, Notch1, and Jagged1. Prenatal and postnatal BrdU-incorporation studies showed that within this synovio-entheseal-articular-cartilage niche most of the Tgfbr2-expressing cells labeled as slow-proliferating cells, namely, stem/progenitor cells. Tgfbr2-positive cells, isolated from embryonic limb mesenchyme, expressed joint progenitor markers in a time- and TGF-β-dependent manner. Our studies provide evidence that joint Tgfbr2-expressing cells have anatomical, ontogenic, slow-cycling trait and in-vivo and ex-vivo expression profiles of progenitor joint cells

Synovial Joints: from Development to Homeostasis

Synovial joint morphogenesis occurs through the condensation of mesenchymal cells into a non-cartilaginous region known as interzone, and the specification of progenitor cells that commit to the articular fate. Although several signaling molecules are expressed by the interzone, the mechanism is poorly understood. For treatments of cartilage injuries, it is critical to discover the presence of joint progenitor cells in adult tissues and their expression gene pattern. Potential stem cells niches have been found in different joint regions, such as the surface zone of articular cartilage, synovium and groove of Ranvier. Inherited joint malformation as well as joint degenerating conditions are often associated with other skeletal defects, and may be seen as the failure of morphogenic factors to establish the correct microenvironment in cartilage and bone. Therefore, exploring how joints form can help us understand how cartilage and bone are damaged and to develop drugs to reactivate this developing mechanism

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I + MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I + MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I + MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I + MSC could be a novel therapeutic approach in patients who have inadequate fracture repair

Mesenchymal Stem Cells Expressing Insulin-like Growth Factor-I (MSCIGF) Promote Fracture Healing and Restore New Bone Formation in Irs1 Knockout Mice: Analyses of MSCIGF Autocrine and Paracrine Regenerative Effects

Failures of fracture repair (non-unions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe and feasible. The contributions of MSC to the reparative process can occur through autocrine as well as paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSC expressing insulin-like growth factor-I (MSCIGF), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and non-unions. MSCIGF or traceable MSCIGF-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knock-out (Irs1−/−) mice with a stabilized tibia fracture. Healing was assed using biomechanical testing, micro-computed-tomography (µCT) and histological analyses. We found that systemically transplanted MSCIGF through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSCIGF to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced induced osteoglastogenesis in MSC was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1−/− mice as a non-union fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap; a callus that basically absent in Irs1−/− left untransplanted or transplanted with MSC. We provided evidence of effects and mechanisms for transplanted MSCIGF in fracture repair and potentially to treat non-unions

3D printing of multilayered scaffolds for rotator cuff tendon regeneration

Repairing massive rotator cuff tendon defects remains a challenge due to the high retear rate after surgical intervention. 3D printing has emerged as a promising technique that enables the fabrication of engineered tissues with heterogeneous structures and mechanical properties, as well as controllable microenvironments for tendon regeneration. In this study, we developed a new strategy for rotator cuff tendon repair by combining a 3D printed scaffold of polylactic-co-glycolic acid (PLGA) with cell-laden collagen-fibrin hydrogels. We designed and fabricated two types of scaffolds: one featuring a separate layer-by-layer structure and another with a tri-layered structure as a whole. Uniaxial tensile tests showed that both types of scaffolds had improved mechanical properties compared to single-layered PLGA scaffolds. The printed scaffold with collagen-fibrin hydrogels effectively supported the growth, proliferation, and tenogenic differentiation of human adipose-derived me-senchymal stem cells. Subcutaneous implantation of the multilayered scaffolds demonstrated their excellent in vivo biocompatibility. This study demonstrates the feasibility of 3D printing multilayered scaffolds for application in rotator cuff tendon regeneration

Tissue engineering a tendon-bone junction with biodegradable braided scaffolds

Abstract Background Tendons play an important role in transferring stress between muscles and bones and in maintaining the stability of joints. Tendon tears are difficult to heal and are associated with high recurrence rates. So, the objective of this study was to develop a biodegradable scaffold for tendon-bone junction regeneration. Methods Two types of polylactic acid (PLA) yarns, having fibers with round and four deep grooved cross-sections, were braided into tubular scaffolds and cultured with murine Transforming growth factor beta type II receptor (Tgfbr2)-expressing joint progenitor cells. The scaffolds were designed to mimic the mechanical, immuno-chemical and biological properties of natural mouse tendon-bone junctions. Three different tubular scaffolds measuring 2 mm in diameter were braided on a Steeger 16-spindle braiding machine and biological and mechanical performance of the three scaffolds were evaluated. Results The mechanical test results indicated that three different braided scaffold structures provided a wide range of mechanical properties that mimic the components of tendon bone junction and results of the biological tests confirmed cell viability, active cell attachment and proliferation throughout all three scaffolds. Conclusions This study has identified that the three proposed types of braided scaffolds with some improvement in their structures have the potential to be used as scaffolds for the regeneration of a tendon bone tissue junction