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Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods.

ABSTRACT: Key aspects of native endochondral bone development and fracture healing can be mimicked in mesenchymal stem cells (MSCs) through standard in vitro chondrogenic induction. Exploiting this phenomenon has recently emerged as an attractive technique to engineer bone tissue, however, relatively little is known about the best conditions for doing so. The objective of the present study was to compare the bone-forming capacity and angiogenic induction of hypertrophic cell constructs containing human adipose-derived stem cells (hASCs) primed for chondrogenesis in two different culture systems: high-density pellets and alginate bead hydrogels. The hASC constructs were subjected to 4 weeks of identical chondrogenic induction in vitro, encapsulated in an agarose carrier, and then implanted subcutaneously in immune-compromised mice for 8 weeks to evaluate their endochondral potential. At the time of implantation, both pellets and beads expressed aggrecan and type II collagen, as well as alkaline phosphatase (ALP) and type X collagen. Interestingly, ASCs in pellets formed a matrix containing higher glycosaminoglycan and collagen contents than that in beads, and ALP activity per cell was higher in pellets. However, after 8 weeks in vivo, pellets and beads induced an equivalent volume of mineralized tissue and a comparable level of vascularization. Although osteocalcin and osteopontin-positive osteogenic tissue and new vascular growth was found within both types of constructs, all appeared to be better distributed throughout the hydrogel beads. The results of this ectopic model indicate that hydrogel culture may be an attractive alternative to cell pellets for bone tissue engineering via the endochondral pathway. Copyright © 2016 John Wiley & Sons, Ltd.

SUBMITTER: Weiss-Bilka HE

PROVIDER: S-EPMC5773408 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods.

Weiss-Bilka Holly E HE McGann Megan E ME Meagher Matthew J MJ Roeder Ryan K RK Wagner Diane R DR

Journal of tissue engineering and regenerative medicine 20170409 1

Key aspects of native endochondral bone development and fracture healing can be mimicked in mesenchymal stem cells (MSCs) through standard in vitro chondrogenic induction. Exploiting this phenomenon has recently emerged as an attractive technique to engineer bone tissue, however, relatively little is known about the best conditions for doing so. The objective of the present study was to compare the bone-forming capacity and angiogenic induction of hypertrophic cell constructs containing human ad ...[more]

PMID: 27690279

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Project description:The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 - a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

Project description:Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of ?-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14?days-of-age (n?=?5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9?days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14?days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14?days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/?-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.

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Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods.

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Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods.

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