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The release of an anti-angiogenic agent, such as type XVIII/endostatin, from an implantable scaffold may be of benefit in the repair of articular cartilage. The objectives of this study are to develop an injectable mesenchymal stem cell (MSC)-incorporating collagen-based hydrogel capable of undergoing covalent cross-linking in vivo and overexpressing endostatin using nonviral transfection, and to investigate methods for the retention of the endostatin protein within the scaffolds. The effects of different cross-linking agents (genipin, transglutaminase-2, and microbial transglutaminase) and different binding molecules for endostatin retention (heparin, heparan sulfate, and chondroitin sulfate) are evaluated. Cartilaginous constructs that overexpress endostatin for 3 weeks are successfully engineered. Most of the endostatin is released into the surrounding media and is not retained within the constructs. The presence of two common basement membrane molecules, laminin and type IV collagen, which have been reported in developing and mature articular cartilage and are generally associated with type XVIII collagen in vivo, is also observed in the engineered cartilaginous constructs. Endostatin-producing cartilaginous constructs can be formulated by growing nonvirally transfected mesenchymal stem cells in collagen gels covalently cross-linked using genipin, transglutaminase-2, and microbial transglutaminase. These constructs warrant further investigation for cartilage repair procedures. The novel finding of laminin and type IV collagen in the engineered cartilage constructs may be of importance for future work toward understanding the role of basement membrane molecules in chondrogenesis and in the physiology and pathology of articular cartilage., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Although there is widespread recognition of the importance of angiogenesis in tissue repair, there is little work on the inhibition of angiogenesis in the context of tissue engineering of naturally avascular tissues, like articular cartilage. The objective was to engineer a collagen-scaffold-based cartilaginous construct overexpressing a potent antiangiogenic factor, endostatin, using nonviral transfection. Endostatin-plasmid-supplemented collagen scaffolds were seeded with mesenchymal stem cells and chondrocytes and cultured for 20–22 days. The effects of the following variables on endostatin expression and chondrogenesis were examined: collagen scaffold material, method of nonviral vector incorporation, plasmid load, culture medium, and oxygen tension. An increase and peak of endostatin protein was observed during the first week of culture, followed by a decrease to low levels, suggesting that overexpression of endostatin could be sustained for several days using the nonviral vector. The amount of endostatin produced was tunable with the external factors. Chondrogenesis was observed in the engineered constructs cultured in chondrogenic medium at the 3-week time point, demonstrating that endostatin did not inhibit the chondrogenic potential of mesenchymal stem cells or the general viability of the cells. The ability to engineer endostatin-expressing cartilaginous constructs will be of value for future work exercising regulatory control of angiogenesis in cartilage repair.

To directly examine the role of vascular endothelial growth factor (VEGFA) in cartilage development, we conditionally knocked out Vegfa in chondrocytes, using the Col2a1 promoter to drive expression of Cre recombinase. Our study of Vegfa conditional knockout (CKO) mice provides new in-vivo evidence for two important functions of VEGFA in bone formation. First, VEGFA plays a significant role in both early and late stages of cartilage vascularization, since Vegfa CKO mice showed delayed invasion of blood vessels into primary ossification centers and delayed removal of terminal hypertrophic chondrocytes. Second, VEGFA is crucial for chondrocyte survival, since massive cell death was seen in joint and epiphyseal regions of Vegfa CKO endochondral bones. Chondrocytes in these regions were found to upregulate expression of Vegfa in wild-type mice at the time when massive cell death occurred in the Vegfa CKO mice. The expression of the VEGFA receptors Npr1 and Npr2 in epiphyseal chondrocytes and lack of blood vessel reduction in the vicinity of the cartilaginous elements in the Vegfa CKO mice raise the possibility that the observed cell death is the result of a direct involvement of VEGFA in chondrocyte survival. Interestingly, the extensive cell death seen in Vegfa CKO null bones had a striking similarity to the cell death phenotype observed when hypoxia-inducible factor 1 alpha (Hif1a) expression was abolished in developing cartilage. This similarity of cell death phenotypes and the deficient VEGFA production in Hif1a null epiphyseal chondrocytes demonstrate that HIF1 alpha and VEGFA are components of a key pathway to support chondrocyte survival during embryonic bone development.

Replacement of nonvascular cartilage by bone and bone marrow is a critical step in bone development. In a recent issue of Developmental Cell, report that a distinct population of immature precursors of bone-forming cells migrate into the cartilage in intimate association with invading blood vessels. [ABSTRACT FROM AUTHOR]

Abstract: Mechanical stress is an important epigenetic factor for regulating skeletal remodeling, and application of force can lead to remodeling of both bone and cartilage. Chondrocytes, osteoblasts and osteoclasts all participate and interact with each other in this remodeling process. To study cellular responses to mechanical stimuli in a system that can be genetically manipulated, we used mouse midpalatal suture expansion in vivo. Six-week-old male C57BL/6 mice were subjected to palatal suture expansion by opening loops with an initial force of 0.56 N for the periods of 1, 3, 5, 7, 14 or 28 days. Periosteal cells in expanding sutures showed increased proliferation, with Ki67-positive cells representing 1.8±0.1% to 4.5±0.4% of total suture cells in control groups and 12.0±2.6% to 19.9±1.2% in experimental/expansion groups (p <0.05). Starting at day 1, cells expressing alkaline phosphatase and type I collagen were seen. New cartilage and bone formation was observed at the oral edges of the palatal bones at day 7; at the nasal edges only bone formation without cartilage appeared to occur. An increase in osteoclast numbers suggested increased bone remodeling, ranging from 60 to 160% throughout the experimental period. Decreased Saffranin O staining after day 3 suggested decreased proteoglycan content in the secondary cartilage. Micro-CT showed a significant increase in maxillary width at days 14 and 28 (from 2334±4 μm to 2485±3 μm at day 14 and from 2383±5 μm to 2574±7 μm at day 28, p <0.001). The suture width was increased at days 14 and 28, except in the oral third region at day 28 (from 48±5 μm to 36±4 μm, p <0.05). Bone volume/total volume was significantly reduced at days 14 and 28 (50.2±0.7% vs. 68.0±3.7% and 56.5±1.0% vs. 60.9±1.3%, respectively, p <0.05), indicative of increased bone marrow space. These findings demonstrate that expansion forces across the midpalatal suture promote bone resorption through activation of osteoclasts and bone and cartilage formation via increased proliferation and differentiation of periosteal cells. Mouse midpalatal suture expansion would be useful in further studies of the ability of mineralized tissues to respond to mechanical stimulation. [Copyright &y& Elsevier]

Runx2–Cbfa1, a Runt transcription factor, plays important roles during skeletal development. It is required for differentiation and function of osteoblasts. In its absence, chondrocyte hypertrophy is severely impaired and there is no vascularization of cartilage templates during skeletal development. These tissue-specific functions of Runx2 are likely to be dependent on its interaction with other proteins. We have therefore searched for proteins that may modulate the activity of Runx2. The yeast two-hybrid system was used to identify a groucho homologue, Grg5, as a Runx2-interacting protein. Grg5 enhances Runx2 activity in a cell culture-based assay and by analyses of postnatal growth in mice we demonstrate that Grg5 and Runx2 interact genetically. We also show that Runx2 haploinsufficiency in the absence of Grg5 results in a more severe delay in ossification of cranial sutures and fontanels than occurs with Runx2 haploinsufficiency on a wild-type background. Finally, we find shortening of the proliferative and hypertrophic zones, and expansion of the resting zone in the growth plates of Runx2+/- Grg5-/- mice that are associated with reduced Ihh expression and Indian hedgehog (Ihh) signaling. We therefore conclude that Grg5 enhances Runx2 activity in vivo. [Copyright &y& Elsevier]