Dataset Information

Collagen type XV and the 'osteogenic status'.

ABSTRACT: We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo-induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo-induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as 'mineralized MSCs', during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium 'per se', negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early-phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.

SUBMITTER: Lisignoli G

PROVIDER: S-EPMC5571525 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Collagen type XV and the 'osteogenic status'.

Lisignoli Gina G Lambertini Elisabetta E Manferdini Cristina C Gabusi Elena E Penolazzi Letizia L Paolella Francesca F Angelozzi Marco M Casagranda Veronica V Piva Roberta R

Journal of cellular and molecular medicine 20170322 9

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different ost ...[more]

PMID: 28332281

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Project description:The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss- and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development.Significance statementIn addition to the archetypal axon guidance cues, the extracellular matrix provides local information that guides motor axons from the spinal cord to their muscle targets. Many of the proteins involved are unknown. Using the zebrafish model, we identified an unexpected role of the extracellular matrix collagen XV in motor axon pathfinding. We show that the synthesis of collagen XV-B by slow muscle precursors and its deposition in the common motor path are dependent on a novel two-step mechanism that determines axon decisions at a choice point during motor axonogenesis. Zebrafish and humans use common molecular cues and regulatory mechanisms for the neuromuscular system development. And as such, our study reveals COL15A1 as a candidate gene for orphan neuromuscular disorders.

Project description:Biomineralized collagen with intrafibrillar calcium phosphate mineral provides an excellent mimic of the composition and structure of the extracellular matrix of bone, from nano- to micro-scale. Scaffolds prepared from this material have the potential to become the next-generation of synthetic bone graft substitutes, as their unique properties make them closer to the native tissue than synthetic alternatives currently available to clinicians. To understand the interaction between biomineralized collagen and cells that are relevant in the context of bone regeneration, we studied the growth and osteogenic differentiation of bone marrow derived human mesenchymal stromal cells (hMSCs) cultured on biomineralized collagen membranes, and compared it to the cell behavior on collagen membranes without mineral. Cells proliferated normally on both biomimetic membranes, and were more triggered to differentiate toward the osteogenic lineage by the biomineralized collagen. This was shown by the elevated mRNA levels of RUNX2, SPP1, ENPP1, and OCN after 3 days of culture, and COL1A1 after 14 days of culture on mineralized collagen. The mRNA levels of the tested markers of osteogenesis were lower on collagen membranes without mineral, with the exception of OCN, which was more highly expressed on collagen than on biomineralized collagen membranes. Expression by hMSCs of OPG, a gene involved in inhibition of osteoclastogenesis, was higher on biomineralized collagen at day 3, while M-CSF, involved in osteoblast-osteoclast communication, was upregulated on both membranes at day 3 and 14 of culture. Alkaline phosphatase activity of hMSCs was high on both biomimetic membranes when compared with cells cultured on tissue culture plastic. Cell-induced mineralization was observed on collagen membranes, while the high mineral content of the biomineralized membranes prohibited a reliable analysis of cell-induced mineralization on these membranes. In conclusion, we have identified that both collagen and biomineralized collagen support proliferation, osteogenic differentiation and mineralization of hMSCs, with biomineralized membranes having a more pronounced positive effect. These findings support the existing evidence that biomineralized collagen is a promising material in the field of bone regeneration.

Project description:Cationic liposomes composed of a novel lipid (N-{6-amino-1-[N-(9Z) -octadec9-enylamino] -1-oxohexan-(2S) -2-yl} -N'- {2- [N, N-bis(2-aminoethyl) amino] ethyl} -2-hexadecylpropandiamide) (OO4) and dioleoylphosphatidylethanolamine (DOPE) possess high amounts of amino groups and are promising systems for lipofection. Moreover, these cationic liposomes can also be used as a polycationic entity in multilayer formation using layer-by-layer technique (LbL), which is a method to fabricate surface coatings by alternating adsorption of polyanions and polycations. Since liposomes are suitable for endocytosis by or fusion with cells, controlled release of their cargo on site is possible. Here, a polyelectrolyte multilayer (PEM) system was designed of chondroitin sulfate (CS) and collagen type I (Col I) by LbL technique with OO4/DOPE liposomes embedded in the terminal layers to create an osteogenic microenvironment. Both, the composition of PEM and cargo of the liposomes were used to promote osteogenic differentiation of C2C12 myoblasts as in vitro model. The internalization of cargo-loaded liposomes from the PEM into C2C12 cells was studied using lipophilic (Rhodamine-DOPE conjugate) and hydrophilic (Texas Red-labeled dextran) model compounds. Besides, the use of Col I and CS should mimic the extracellular matrix of bone for future applications such as bone replacement therapies. Physicochemical studies of PEM were done to characterize the layer growth, thickness, and topography. The adhesion of myoblast cells was also evaluated whereby the benefit of a cover layer of CS and finally Col I above the liposome layer was demonstrated. As proof of concept, OO4/DOPE liposomes were loaded with dexamethasone, a compound that can induce osteogenic differentiation. A successful induction of osteogenic differentiation of C2C12 cells with the novel designed liposome-loaded PEM system was shown. These findings indicate that designed OH4/DOPE loaded PEMs have a high potential to be used as drug delivery or transfection system for implant coating in the field of bone regeneration and other applications.

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Collagen type XV and the 'osteogenic status'.

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Collagen type XV and the 'osteogenic status'.

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