In vitro evaluation of a double-stranded self-complementary adeno-associated virus type2 vector in bone marrow stromal cells for bone healing

Background: Both adenoviral and lentiviral vectors have been successfully used to induce bone repair by over-expression of human bone morphogenetic protein 2 (BMP-2) in primary rat bone marrow stromal cells in pre-clinical models of ex vivo regional gene therapy. Despite being a very efficient means of gene delivery, there are potential safety concerns that may limit the adaptation of these viral vectors for clinical use in humans. Recombinant adeno-associated viral (rAAV) vector is a promising viral vector without known pathogenicity in humans and has the potential to be an effective gene delivery vehicle to enhance bone repair. In this study, we investigated gene transfer in rat and human bone marrow stromal cells in order to evaluate the effectiveness of the self-complementary AAV vector (scAAV) system, which has higher efficiency than the single-stranded AAV vector (ssAAV) due to its unique viral genome that bypasses the rate-limiting conversion step necessary in ssAAV.Methods: Self-complementaryAAV2 encoding GFP and BMP-2 (scAAV2-GFP and scAAV2-BMP-2) were used to transduce human and rat bone marrow stromal cells in vitro, and subsequently the levels of GFP and BMP-2 expression were assessed 48 hours after treatment. In parallel experiments, adenoviral and lentiviral vector mediated over-expression of GFP and BMP-2 were used for comparison.Results: Our results demonstrate that the scAAV2 is not capable of inducing significant transgene expression in human and rat bone marrow stromal cells, which may be associated with its unique tropism.Conclusions: In developing ex vivo gene therapy regimens, the ability of a vector to induce the appropriate level of transgene expression needs to be evaluated for each cell type and vector used. © 2011 Alaee et al; licensee BioMed Central Ltd

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity.

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration

The enthesis: a review of the tendon-to-bone insertion

The integration of tendon into bone occurs at a specialized interface known as the enthesis. The fibrous tendon to bone enthesis is established through a structurally continuous gradient from uncalcified tendon to calcified bone. The enthesis exhibits gradients in tissue organization classified into four distinct zones with varying cellular compositions, mechanical properties, and functions in order to facilitate joint movement. Damage to tendinous insertions is common in the field of orthopaedic medicine and often involves surgical intervention that requires the attempted recreation of the natural organization of tendon into bone. The difficulty associated with recreating the distinct organization may account for the surgical challenges associated with reconstruction of damaged insertion sites. These procedures are often associated with high failure rates and consequently require revision procedures. Management of tendinous injuries and reconstruction of the insertion site is becoming a popular topic in the field of orthopaedic medicine

Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing

<p><i>Purpose of this study</i>: To elucidate the origin of cell populations that contribute to rotator cuff healing, we developed a mouse surgical model where a full-thickness, central detachment is created in the supraspinatus. <i>Materials and methods</i>: Three different inducible Cre transgenic mice with Ai9-tdTomato reporter expression (PRG4-9, αSMA-9, and AGC-9) were used to label different cell populations in the shoulder. The defect was created surgically in the supraspinatus. The mice were injected with tamoxifen at surgery to label the cells and sacrificed at 1, 2, and 5 weeks postoperatively. Frozen sections were fluorescently imaged then stained with Toluidine Blue and re-imaged. <i>Results</i>: Three notable changes were apparent postoperatively. (1) A long thin layer of tissue formed on the bursal side overlying the supraspinatus tendon. (2) The tendon proximal to the defect initially became hypercellular and disorganized. (3) The distal stump at the insertion underwent minimal remodeling. In the uninjured shoulder, tdTomato expression was seen in the tendon midsubstance and paratenon cell on the bursal side in PRG4-9, in paratenon, blood vessels, and periosteum of acromion in SMA-9, and in articular cartilage, unmineralized fibrocartilage of supraspinatus enthesis, and acromioclavicular joint in AGC-9 mice. In the injured PRG4-9 and SMA-9 mice, the healing tissues contained an abundant number of tdTomato+ cells, while minimal contribution of tdTomato+ cells was seen in AGC-9 mice. <i>Conclusions</i>: The study supports the importance of the bursal side of the tendon to rotator cuff healing and PRG4 and αSMA may be markers for these progenitor cells.</p

Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing

<p><i>Purpose of this study</i>: To elucidate the origin of cell populations that contribute to rotator cuff healing, we developed a mouse surgical model where a full-thickness, central detachment is created in the supraspinatus. <i>Materials and methods</i>: Three different inducible Cre transgenic mice with Ai9-tdTomato reporter expression (PRG4-9, αSMA-9, and AGC-9) were used to label different cell populations in the shoulder. The defect was created surgically in the supraspinatus. The mice were injected with tamoxifen at surgery to label the cells and sacrificed at 1, 2, and 5 weeks postoperatively. Frozen sections were fluorescently imaged then stained with Toluidine Blue and re-imaged. <i>Results</i>: Three notable changes were apparent postoperatively. (1) A long thin layer of tissue formed on the bursal side overlying the supraspinatus tendon. (2) The tendon proximal to the defect initially became hypercellular and disorganized. (3) The distal stump at the insertion underwent minimal remodeling. In the uninjured shoulder, tdTomato expression was seen in the tendon midsubstance and paratenon cell on the bursal side in PRG4-9, in paratenon, blood vessels, and periosteum of acromion in SMA-9, and in articular cartilage, unmineralized fibrocartilage of supraspinatus enthesis, and acromioclavicular joint in AGC-9 mice. In the injured PRG4-9 and SMA-9 mice, the healing tissues contained an abundant number of tdTomato+ cells, while minimal contribution of tdTomato+ cells was seen in AGC-9 mice. <i>Conclusions</i>: The study supports the importance of the bursal side of the tendon to rotator cuff healing and PRG4 and αSMA may be markers for these progenitor cells.</p

Skeletal Muscle Contractile Gene (TNNT3, MYH3, TPM2) Mutations Not Found in Vertical Talus or Clubfoot

Arthrogryposis presents with lower limb contractures that resemble clubfoot and/or vertical talus. Recently, mutations in skeletal muscle contractile genes MYH3 (myosin heavy chain 3), TNNT3 (troponin T3), and TPM2 (tropomyosin 2) were identified in patients with distal arthrogryposis DA2A (Freeman-Sheldon syndrome) or DA2B (Sheldon-Hall syndrome). We asked whether the contractile genes responsible for distal arthrogryposis are also responsible for cases of familial clubfoot or vertical talus. We determined the frequency of MYH3, TNNT3, and TPM2 mutations in patients with idiopathic clubfoot, vertical talus, and distal arthrogryposis type 1 (DA1). We resequenced the coding exons of the MYH3, TNNT3, and TPM2 genes in 31 patients (five with familial vertical talus, 20 with familial clubfoot, and six with DA1). Variants were evaluated for segregation with disease in additional family members, and the frequency of identified variants was determined in a control population. In one individual with DA1, we identified a de novo TNNT3 mutation (R63H) previously identified in an individual with DA2B. No other causative mutations were identified, though we found several previously undescribed single-nucleotide polymorphisms of unknown importance. Although mutations in MYH3, TNNT3, and TPM2 are frequently associated with distal arthrogryposis syndromes, they were not present in patients with familial vertical talus or clubfoot. The TNNT3 R63H recurrent mutation identified in two unrelated individuals may be associated with either DA1 or DA2B