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Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds.

ABSTRACT: Tooth formation during embryogenesis is controlled through a complex interplay between mechanical and chemical cues. We have previously shown that physical cell compaction of dental mesenchyme cells during mesenchymal condensation is responsible for triggering odontogenic differentiation during embryogenesis, and that expression of Collagen VI stabilizes this induction. In addition, we have shown that synthetic polymer scaffolds that artificially induce cell compaction can induce embryonic mandible mesenchymal cells to initiate tooth differentiation both in vitro and in vivo. As embryonic cells would be difficult to use for regenerative medicine applications, here we explored whether compressive scaffolds coated with Collagen VI can be used to induce adult bone marrow stromal cells (BMSCs) to undergo an odontogenic lineage switch. These studies revealed that when mouse BMSCs are compressed using these scaffolds they increase expression of critical markers of tooth differentiation in vitro, including the key transcription factors Pax9 and Msx1. Implantation under the kidney capsule of contracting scaffolds bearing these cells in mice also resulted in local mineralization, calcification and production of dentin-like tissue. These findings show that these chemically-primed compressive scaffolds can be used to induce adult BMSCs to undergo a lineage switch and begin to form dentin-like tissue, thus raising the possibility of using adult BMSCs for future tooth regeneration applications.

SUBMITTER: Hashmi B

PROVIDER: S-EPMC5796538 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds.

Hashmi Basma B Mammoto Tadanori T Weaver James J Ferrante Thomas T Jiang Amanda A Jiang Elisabeth E Feliz Juani J Ingber Donald E DE

Stem cell research 20170817

Tooth formation during embryogenesis is controlled through a complex interplay between mechanical and chemical cues. We have previously shown that physical cell compaction of dental mesenchyme cells during mesenchymal condensation is responsible for triggering odontogenic differentiation during embryogenesis, and that expression of Collagen VI stabilizes this induction. In addition, we have shown that synthetic polymer scaffolds that artificially induce cell compaction can induce embryonic mandi ...[more]

PMID: 28841424

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Project description:Interfaces between soft tissue and bone are characterized by transitional gradients in composition and structure that mediate substantial changes in mechanical properties. For interfacial tissue engineering, scaffolds with mineral gradients have shown promise in controlling osteogenic behavior of seeded bone marrow stromal cells (bMSCs). Previously, we have demonstrated a 'top-down' method for creating monolithic bone-derived scaffolds with patterned mineral distributions similar to native tissue. In the present work, we evaluated the ability of these scaffolds to pattern osteogenic behavior in bMSCs in basic, osteogenic, and chondrogenic biochemical environments. Immunohistochemical (IHC) and histological stains were used to characterize cellular behavior as a function of local mineral content. Alkaline phosphatase, an early marker of osteogenesis, and osteocalcin, a late marker of osteogenesis, were positively correlated with mineral content in basic, osteogenic, and chondrogenic media. The difference in bMSC behavior between the mineralized and demineralized regions was most pronounced in an basic biochemical environment. In the mineralized regions of the scaffold, osteogenic markers were clearly present as early as 4 days in culture. In osteogenic media, osteogenic behavior was observed across the entire scaffold, whereas in chondrogenic media, there was an overall reduction in osteogenic biomarkers. Overall, these results indicate local mineral content of the scaffold plays a key role in spatially patterning bMSC behavior. Our results can be utilized for the development of interfacial tissue engineered scaffolds and understanding the role of local environment in determining bMSC behavior. STATEMENT OF SIGNIFICANCE: Soft tissue-to-bone interfaces, such as tendon-bone, ligament-bone, and cartilage-bone, are ubiquitous in mammalian musculoskeletal systems. These interfacial tissues have distinct, hierarchically-structured gradients of cellular, biochemical, and materials components. Given the complexity of the biological structures, interfacial tissues present unique challenges for tissue engineering. Here, we demonstrate that material-derived cues can spatially pattern osteogenic behavior in bone marrow stromal cells (bMSCs). Specifically, we observed that when the bMSCs are cultured on bone-derived scaffolds with mineral gradients, cells in contact with higher mineral content display osteogenic behavior at earlier times than those on the unmineralized substrate. The ability to pattern the cellular complexity found in native interfaces while maintaining biologically relevant structures is a key step towards creating engineered tissue interfaces.

Project description:Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl Biomater, 2016. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 989-1001, 2017.

Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

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Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds.

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Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds.

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