Understanding tissue-engineered endochondral ossification; towards improved bone formation

Endochondral ossification (EO) is the process by which the long bones of the body form and has proven to be a promising method in tissue engineering for achieving cell-mediated bone formation. The present review centred on state-of-the-art research pertaining to mesenchymal stem cells (MSCs)-mediated endochondral bone formation, focusing on the role of donor cells, extracellular matrix and host immune cells during tissue-engineered bone formation. Possible research avenues to improve graft outcome and bone output were highlighted, as well as emerging research that, when applied to tissue-engineered bone grafts, offers new promise for improving the likelihood of such grafts transition from bench to bedside

Long-term expansion, enhanced chondrogenic potential, and suppression of endochondral ossification of adult human MSCs via WNT signaling mo

Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair

Silencing of anti-chondrogenic microRNA-221 in human mesenchymal stem cells promotes cartilage repair in vivo

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation towards the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the anti-chondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs towards chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a pro-chondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. This article is protected by copyright. All rights reserved

Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derivedfrom the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary

Recellularization of auricular cartilage via elastase-generated channels

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tiss

Activin receptor-like kinase receptors ALK5 and ALK1 are both required for TGFβ-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor beta; (TGFbeta;) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFbeta; receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFbeta; receptors did not clearly associate with chondrogenic induction. TGFbeta; increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFbeta;. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFbeta;-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFbeta;-induced chondrogenic differentiation of BMSCs, and TGFbeta; not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors

Emerging Infectious Disease leads to Rapid Population Decline of Common British Birds

Emerging infectious diseases are increasingly cited as threats to wildlife, livestock and humans alike. They can threaten geographically isolated or critically endangered wildlife populations; however, relatively few studies have clearly demonstrated the extent to which emerging diseases can impact populations of common wildlife species. Here, we report the impact of an emerging protozoal disease on British populations of greenfinch Carduelis chloris and chaffinch Fringilla coelebs, two of the most common birds in Britain. Morphological and molecular analyses showed this to be due to Trichomonas gallinae. Trichomonosis emerged as a novel fatal disease of finches in Britain in 2005 and rapidly became epidemic within greenfinch, and to a lesser extent chaffinch, populations in 2006. By 2007, breeding populations of greenfinches and chaffinches in the geographic region of highest disease incidence had decreased by 35% and 21% respectively, representing mortality in excess of half a million birds. In contrast, declines were less pronounced or absent in these species in regions where the disease was found in intermediate or low incidence. Also, populations of dunnock Prunella modularis, which similarly feeds in gardens, but in which T. gallinae was rarely recorded, did not decline. This is the first trichomonosis epidemic reported in the scientific literature to negatively impact populations of free-ranging non-columbiform species, and such levels of mortality and decline due to an emerging infectious disease are unprecedented in British wild bird populations. This disease emergence event demonstrates the potential for a protozoan parasite to jump avian host taxonomic groups with dramatic effect over a short time period

Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derivedfrom the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary

Enhanced Chondrogenic Capacity of Mesenchymal Stem Cells After TNFα Pre-treatment

Mesenchymal stem cells (MSCs) are promising cells to treat cartilage defects due to their chondrogenic differentiation potential. However, an inflammatory environment during differentiation, such as the presence of the cytokine TNFα, inhibits chondrogenesis and limits the clinical use of MSCs. On the other hand, it has been reported that exposure to TNFα during in vitro expansion can increase proliferation, migration, and the osteogenic capacity of MSCs and therefore can be beneficial for tissue regeneration. This indicates that the role of TNFα on MSCs may be dependent on the differentiation stage. To improve the chondrogenic capacity of MSCs in the presence of an inflamed environment, we aimed to determine the effect of TNFα on the chondrogenic differentiation capacity of MSCs. Here, we report that TNFα exposure during MSC expansion increased the chondrogenic differentiation capacity regardless of the presence of TNFα during chondrogenesis and that this effect of TNFα during expansion was reversed upon TNFα withdrawal. Interestingly, pre-treatment with another pro-inflammatory cytokine, IL-1β, did not increase the chondrogenic capacity of MSCs indicating that the pro-chondrogenic effect is specific for TNFα. Finally, we show that TNFα pre-treatment increased the levels of SOX11 and active β-catenin suggesting that these intracellular effectors may be useful targets to improve MSC-based cartilage repair. Overall, these results suggest that TNFα pre-treatment, by modulating SOX11 levels and WNT/β-catenin signaling, could be used as a strategy to improve MSC-based cartilage repair