Carrageenan-Based Crowding and Confinement Combination Approach to Increase Collagen Deposition for In Vitro Tissue Development.

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body's environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo.

Co-delivery of fibrin-laminin hydrogel with mesenchymal stem cell spheroids supports skeletal muscle regeneration following trauma.

Volumetric muscle loss (VML) is traumatic or surgical loss of skeletal muscle with resultant functional impairment. Skeletal muscle's innate capacity for regeneration is lost with VML due to a critical loss of stem cells, extracellular matrix, and neuromuscular junctions. Consequences of VML include permanent disability or delayed amputations of the affected limb.

Micro-Clotting of Platelet-Rich Plasma Upon Loading in Hydrogel Microspheres Leads to Prolonged Protein Release and Slower Microsphere Degradation.

Platelet-rich plasma (PRP) is an autologous blood product that contains a variety of growth factors (GFs) that are released upon platelet activation. Despite some therapeutic potential of PRP in vitro, in vivo data are not convincing. Bolus injection of PRP is cleared rapidly from the body diminishing its therapeutic efficacy.

Computational model to evaluate modulation of the acoustic field in an ultrasonic bioreactor by incorporation of a water layer bounded by an acoustic absorbent boundary layer.

Ultrasonic bioreactors have been used for in vitro experimentation to study cellular responses to low-intensity pulsed ultrasound. The presence of an air interface in these bioreactors contributes to variability in the acoustic pressure field, reducing experimental reproducibility. A multiphysics finite element model was developed to simulate the acoustic field in an in-dish ultrasonic bioreactor, where the transducer is immersed in culture medium above the dish surface, and the effects of replacing air below the dish in the bioreactor with a water layer bounded by an acoustic absorbent layer were evaluated.

Platelet-Rich Plasma Released From Polyethylene Glycol Hydrogels Exerts Beneficial Effects on Human Chondrocytes.

Osteoarthritis (OA) is a debilitating joint disease resulting from chronic joint inflammation and erosion of articular cartilage. A promising biological treatment for OA is intra-articular administration of platelet-rich plasma (PRP). However, immediate bolus release of growth factors limits beneficial therapeutic effects of PRP, thus necessitating the demand for sustained release platforms.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca-permeable channel, may regulate this signaling.

The calcification potential of cryogel scaffolds incorporated with various forms of hydroxyapatite for bone regeneration.

Cryogels are advantageous scaffolds for bone regeneration applications due to their high mechanical stability and macroporous structure. Anatomically, bone is composed of collagen and hydroxyapatite and during remodeling, these structural components are replaced. However, early forms of mineralization include calcium salts which take up to months to be converted to the desired hydroxyapatite form.

Bone marrow fat accumulation accelerated by high fat diet is suppressed by exercise.

Marrow adipose tissue (MAT), associated with skeletal fragility and hematologic insufficiency, remains poorly understood and difficult to quantify. We tested the response of MAT to high fat diet (HFD) and exercise using a novel volumetric analysis, and compared it to measures of bone quantity. We hypothesized that HFD would increase MAT and diminish bone quantity, while exercise would slow MAT acquisition and promote bone formation.

Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells.

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of β-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement.

mTORC2 regulates mechanically induced cytoskeletal reorganization and lineage selection in marrow-derived mesenchymal stem cells.

The cell cytoskeleton interprets and responds to physical cues from the microenvironment. Applying mechanical force to mesenchymal stem cells induces formation of a stiffer cytoskeleton, which biases against adipogenic differentiation and toward osteoblastogenesis. mTORC2, the mTOR complex defined by its binding partner rictor, is implicated in resting cytoskeletal architecture and is activated by mechanical force.

Mechanical regulation of chondrogenesis.

Mechanical factors play a crucial role in the development of articular cartilage in vivo. In this regard, tissue engineers have sought to leverage native mechanotransduction pathways to enhance in vitro stem cell-based cartilage repair strategies. However, a thorough understanding of how individual mechanical factors influence stem cell fate is needed to predictably and effectively utilize this strategy of mechanically-induced chondrogenesis.

Mechanical strain downregulates C/EBPβ in MSC and decreases endoplasmic reticulum stress.

Exercise prevents marrow mesenchymal stem cell (MSC) adipogenesis, reversing trends that accompany aging and osteoporosis. Mechanical input, the in-vitro analogue to exercise, limits PPARγ expression and adipogenesis in MSC. We considered whether C/EBPβ might be mechanoresponsive as it is upstream to PPARγ, and also is known to upregulate endoplasmic reticulum (ER) stress.

Mechanical input restrains PPARγ2 expression and action to preserve mesenchymal stem cell multipotentiality.

Exercise-generated signals are pro-osteogenic and anti-adipogenic within the marrow. In vitro studies indicate that mechanical signals directly block adipogenic differentiation through activation of β-catenin and by limiting PPARγ2 expression. Whether mechanically generated β-catenin can inhibit adipogenesis during PPARγ transactivation is unknown.

Mechanical regulation of glycogen synthase kinase 3β (GSK3β) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein.

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC.

Mechanically induced focal adhesion assembly amplifies anti-adipogenic pathways in mesenchymal stem cells.

The fate of pluripotent mesenchymal stem cells (MSC) is determined through integration of chemical, spatial, and physical signals. The suppression of MSC adipogenesis by mechanical stimuli, which requires Akt-induced inhibition of glycogen synthase kinase 3β (GSK3β) with β-catenin activation, can be enhanced by repetitive dosing within a single day. Here, we demonstrate that reapplication of cyclic strain within a 24-hour period leads to amplification of both Akt activation and its subsequent inhibition of GSK3β, such that total cycle number can be reduced while still inhibiting adipogenesis.

Mesenchymal stem cells expressing insulin-like growth factor-I (MSCIGF) promote fracture healing and restore new bone formation in Irs1 knockout mice: analyses of MSCIGF autocrine and paracrine regenerative effects.

Failures of fracture repair (nonunions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe, and feasible. The contributions of MSC to the reparative process can occur through autocrine and paracrine effects.

Mechanical signal influence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen.

Mechanical signals of both low and high intensity are inhibitory to fat and anabolic to bone in vivo, and have been shown to directly affect mesenchymal stem cell pools from which fat and bone precursors emerge. To identify an idealized mechanical regimen which can regulate MSC fate, low intensity vibration (LIV; <10 microstrain, 90 Hz) and high magnitude strain (HMS; 20,000 microstrain, 0.17 Hz) were examined in MSC undergoing adipogenesis.

Mechanical activation of β-catenin regulates phenotype in adult murine marrow-derived mesenchymal stem cells.

Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow-derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells.

Indomethacin promotes adipogenesis of mesenchymal stem cells through a cyclooxygenase independent mechanism.

Regulation of mesenchymal stem cell (MSC) lineage selection is important for the generation of bone mass. Inhibition of cyclooxygenase-2 (COX2) may increase adipogenesis at the cost of decreasing osteoprogenitor output. Here we investigated the role of COX2 and its products during MSC differentiation.

Beta-catenin--a supporting role in the skeleton.

In the last 5 years a role for beta-catenin in the skeleton has been cemented. Beginning with mutations in the Lrp5 receptor that control beta-catenin canonical downstream signals, and progressing to transgenic models with bone-specific alteration of beta-catenin, research has shown that beta-catenin is required for normal bone development. A cell critical to bone in which beta-catenin activity determines function is the marrow-derived mesenchymal stem cell (MSC), where sustained beta-catenin prevents its distribution into adipogenic lineage.

Mechanical loading regulates NFATc1 and beta-catenin signaling through a GSK3beta control node.

Mechanical stimulation can prevent adipogenic and improve osteogenic lineage allocation of mesenchymal stem cells (MSC), an effect associated with the preservation of beta-catenin levels. We asked whether mechanical up-regulation of beta-catenin was critical to reduction in adipogenesis as well as other mechanical events inducing alternate MSC lineage selection. In MSC cultured under strong adipogenic conditions, mechanical load (3600 cycles/day, 2% strain) inactivated GSK3beta in a Wnt-independent fashion.

Beta-catenin levels influence rapid mechanical responses in osteoblasts.

Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/beta-catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce beta-catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on beta-catenin signaling.

Mechanical strain inhibits adipogenesis in mesenchymal stem cells by stimulating a durable beta-catenin signal.

The ability of exercise to decrease fat mass and increase bone mass may occur through mechanical biasing of mesenchymal stem cells (MSCs) away from adipogenesis and toward osteoblastogenesis. C3H10T1/2 MSCs cultured in highly adipogenic medium express peroxisome proliferator-activated receptor gamma and adiponectin mRNA and protein, and accumulate intracellular lipid. Mechanical strain applied for 6 h daily inhibited expression of peroxisome proliferator-activated receptor gamma and adiponectin mRNA by up to 35 and 50%, respectively, after 5 d.

The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells.

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis.

Caveolin-1 knockout mice have increased bone size and stiffness.

Unlabelled: The skeletal phenotype of the cav-1(-/-) mouse, which lacks caveolae, was examined. muCT and histology showed increased trabecular and cortical bone caused by the gene deletion. Structural changes were accompanied by increased mechanical properties.