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Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation.

ABSTRACT: Bone extracellular matrix (ECM) is a natural composite made of collagen and mineral hydroxyapatite (HA). Dynamic cell-ECM interactions play a critical role in regulating cell differentiation and function. Understanding the principal ECM cues promoting osteogenic differentiation would be pivotal for both bone tissue engineering and regenerative medicine. Altering the mineral content generally modifies the stiffness as well as other physicochemical cues provided by composite materials, complicating the "cause-effect" analysis of resultant cell behaviour. To isolate the contribution of mechanical cues from other HA-derived signals, we developed and characterised composite HA/gelatin scaffolds with different mineral contents along with a set of stiffness-matched HA-free gelatin scaffolds. Samples were seeded with human periosteal derived progenitor cells (PDPCs) and cultured over 7 days, analysing their resultant morphology and gene expression. Our results show that both stiffness and HA contribute to directing PDPC osteogenic differentiation, highlighting the role of stiffness in triggering the expression of osteogenic genes and of HA in accelerating the process, particularly at high concentrations.

SUBMITTER: Mattei G

PROVIDER: S-EPMC4451686 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation.

Mattei Giorgio G Ferretti Concetta C Tirella Annalisa A Ahluwalia Arti A Mattioli-Belmonte Monica M

Scientific reports 20150602

Bone extracellular matrix (ECM) is a natural composite made of collagen and mineral hydroxyapatite (HA). Dynamic cell-ECM interactions play a critical role in regulating cell differentiation and function. Understanding the principal ECM cues promoting osteogenic differentiation would be pivotal for both bone tissue engineering and regenerative medicine. Altering the mineral content generally modifies the stiffness as well as other physicochemical cues provided by composite materials, complicatin ...[more]

PMID: 26035412

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Project description:BackgroundTendon-derived stem cells (TDSCs) are key factors associated with regeneration and healing in tendinopathy. The aim of this study was to investigate the effects of mechanical stiffness and topographic signals on the differentiation of TDSCs depending on age and pathological conditions.Materials and methodsWe compared TDSCs extracted from normal tendon tissues with TDSCs from tendinopathic Achilles tendon tissues of Sprague Dawley rats in vitro and TDSCs cultured on nanotopographic cues and substrate stiffness to determine how to control the TDSCs. The tendinopathy model was created using a chemical induction method, and the tendon injury model was created via an injury-and-overuse method. Norland Optical Adhesive 86 (NOA86) substrate with 2.48 GPa stiffness with and without 800 nm-wide nanogrooves and a polyurethane substrate with 800 nm-wide nanogrooves were used.ResultsTDSCs from 5-week-old normal tendon showed high expression of type III collagen on the flat NOA86 substrate. In the 15-week normal tendon model, expression of type III collagen was high in TDSCs cultured on the 800 nm NOA86 substrates. However, in the 15-week tendon injury model, expression of type III collagen was similar irrespective of nanotopographic cues or substrate stiffness. The expression of type I collagen was also independent of nanotopographic cues and substrate stiffness in the 15-week normal and tendon injury models. Gene expression of scleraxis was increased in TDSCs cultured on the flat NOA86 substrate in the 5-week normal tendon model (P=0.001). In the 15-week normal tendon model, scleraxis was highly expressed in TDSCs cultured on the 800 nm and flat NOA86 substrate (P=0.043). However, this gene expression was not significantly different between the substrates in the 5-week tendinopathy and 15-week tendon injury models.ConclusionDevelopment and maturation of tendon are enhanced when TDSCs from normal tendons were cultured on stiff surface, but not when the TDSCs came from pathologic models. Therapeutic applications of TDSCs need to be flexible based on tendon age and tendinopathy.

Project description:Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE.

Project description:BackgroundPeriosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs.MethodsWe detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16.ResultsWnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity.ConclusionTogether, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

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Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation.

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Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation.

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