Engineering zonal cartilaginous tissue by modulating oxygen levels and mechanical cues through the depth of infrapatellar fat pad stem cell laden hydrogels.

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2 mm or 4 mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2 mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2 mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96 MPa to 1.45 MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4 mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.European Research Council Starter Grant. Grant Number: 25846

Betacellulin inhibits osteogenic differentiation and stimulates proliferation through HIF-1α

Cellular signaling via epidermal growth factor (EGF) and EGF-like ligands can determine cell fate and behavior. Osteoblasts, which are responsible for forming and mineralizing osteoid, express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation. Transgenic mice over-expressing the EGF-like ligand betacellulin (BTC) exhibit increased cortical bone deposition; however, because the transgene is ubiquitously expressed in these mice, the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown. We have therefore examined the influence of BTC upon mesenchymal stem cell (MSC) and pre-osteoblast differentiation and proliferation. BTC decreases the expression of osteogenic markers in both MSCs and pre-osteoblasts; interestingly, increases in proliferation require hypoxia-inducible factor-alpha (HIF-α), as an HIF antagonist prevents BTC-driven proliferation. Both MSCs and pre-osteoblasts express EGF receptors ErbB1, ErbB2, and ErbB3, with no change in expression under osteogenic differentiation. These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIF-α in BTC-mediated proliferation

Modulating gradients in regulatory signals within mesenchymal stem cell seeded hydrogels: a novel strategy to engineer zonal articular cartilage.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Engineering organs and tissues with the spatial composition and organisation of their native equivalents remains a major challenge. One approach to engineer such spatial complexity is to recapitulate the gradients in regulatory signals that during development and maturation are believed to drive spatial changes in stem cell differentiation. Mesenchymal stem cell (MSC) differentiation is known to be influenced by both soluble factors and mechanical cues present in the local microenvironment. The objective of this study was to engineer a cartilaginous tissue with a native zonal composition by modulating both the oxygen tension and mechanical environment thorough the depth of MSC seeded hydrogels. To this end, constructs were radially confined to half their thickness and subjected to dynamic compression (DC). Confinement reduced oxygen levels in the bottom of the construct and with the application of DC, increased strains across the top of the construct. These spatial changes correlated with increased glycosaminoglycan accumulation in the bottom of constructs, increased collagen accumulation in the top of constructs, and a suppression of hypertrophy and calcification throughout the construct. Matrix accumulation increased for higher hydrogel cell seeding densities; with DC further enhancing both glycosaminoglycan accumulation and construct stiffness. The combination of spatial confinement and DC was also found to increase proteoglycan-4 (lubricin) deposition toward the top surface of these tissues. In conclusion, by modulating the environment through the depth of developing constructs, it is possible to suppress MSC endochondral progression and to engineer tissues with zonal gradients mimicking certain aspects of articular cartilage.Funding was provided by Science Foundation Ireland (President of Ireland Young Researcher Award: 08/Y15/B1336) and the European Research Council (StemRepair – Project number 258463)

Designing microenvironments for optimal outcomes in tissue engineering and regenerative medicine: From biopolymers to culturing conditions

Bone marrow mesenchymal stem cells have been extensively used for tissue engineering and regenerative medicine applications due to their ease of isolation and expansion and their ability to differentiate towards various lineages of mesodermal origin. Despite these properties, their clinical potential is often hampered by the simplicity of the in vitro environment and its inability to resemble the complex in vivo niche. Herein, different microenvironmental cues (e.g. surface topography, substrate stiffness, mechanical stimulation, oxygen tension and co-culture systems) that have been utilised to enhance the therapeutic efficacy of bone marrow mesenchymal stem cells are discussed.The authors would like to acknowledge the following entities for financial support: H2020, Marie Skłodowska-Curie Actions, Innovative Training Networks 2015 Tendon Therapy Train project (Grant No. 676338); Science Foundation Ireland (SFI) / European Regional Development Fund (Grant Number 13/RC/2073); and SFI Career Development Award (Grant Number 15/CDA/3629)

The impact of hypoxia and cannabinoids on the differentiation of adult rat mesenchymal stem cells in bone and cartilage tissue engineering

THESIS 8708Adult mesenchymal stem cells (MSCs) have the potential to self-renew and differentiate into cartilage, bone, muscle and fat. This subset of bone marrow stromal cells therefore holds great potential for tissue engineering purposes. To-date no successful treatment is available for many cartilage ailments such as rheumatoid arthritis and bone disorders such as osteoporosis. The overall purpose of this research project was to identify the factors that stimulate rat MSC differentiation along the chondrogenic and osteogenic lineages in order to improve in vitro tissue engineering strategies. The impact of a low oxygen environment (hypoxia) was investigated in the regulation of MSC chondrogenesis. MSCs maintained in the presence of TGF-P and dexamethasone in a normoxic environment displayed increased collagen II expression and proteoglycan deposition which concluded that rat MSCs undergo chondrogenic differentiation. Exposure of MSCs to chondrogenic factors for 14 days in normoxia, followed by 7 days in hypoxia (2% oxygen) showed a significantly enhanced chondrogenesis, which verified that a reduced oxygen tension supports MSC differentiation along the chondrogenic route, fhe reduced oxygen environment augmented HIP-la nuclear accumulation, in addition, transactivation of HlF-la was in an AKT and p38 MAPK-dependent mode. A role for HIF-la in the chondrogenic differentiation of MSCs was demonstrated by the prevention of hypoxia-mediated induction of chondrogenesis by siRNA-mediated knockdown of HiF-1 a

Tissue Engineering of Cartilage; Can Cannabinoids Help?

This review discusses the role of the cannabinoid system in cartilage tissue and endeavors to establish if targeting the cannabinoid system has potential in mesenchymal stem cell based tissue-engineered cartilage repair strategies. The review discusses the potential of cannabinoids to protect against the degradation of cartilage in inflamed arthritic joints and the influence of cannabinoids on the chondrocyte precursors, mesenchymal stem cells (MSCs). We provide experimental evidence to show that activation of the cannabinoid system enhances the survival, migration and chondrogenic differentiation of MSCs, which are three major tenets behind the success of a cell-based tissue-engineered cartilage repair strategy. These findings highlight the potential for cannabinoids to provide a dual function by acting as anti-inflammatory agents as well as regulators of MSC biology in order to enhance tissue engineering strategies aimed at cartilage repair