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 (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

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 ß-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. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.

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. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability.

Mechanical regulation of glycogen synthase kinase 3ß (GSK3ß) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein.

Mechanical signals can inactivate glycogen synthase kinase 3ß (GSK3ß), resulting in stabilization of ß-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 GSK3ß in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3ß at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3ß at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3ß, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3ß inactivation was prevented, whereas insulin inhibition of GSK3ß was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3ß. Thus, mechanical regulation of GSK3ß downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.

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. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSCs expressing insulin-like growth factor-I (MSC(IGF)), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and nonunions. MSC(IGF) or traceable MSC(IGF)-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knockout (Irs1(-/-)) mice with a stabilized tibia fracture. Healing was assessed using biomechanical testing, microcomputed tomography (µCT), and histological analyses. We found that systemically transplanted MSC(IGF) through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSC(IGF) to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced osteoglastogenesis in MSCs was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1(-/-) mice as a nonunion fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap. A callus that was basically absent in Irs1(-/-) left untransplanted or transplanted with MSCs. We provided evidence of effects and mechanisms for transplanted MSC(IGF) in fracture repair and potentially to treat nonunions.

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 3ß (GSK3ß) with ß-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 GSK3ß, such that total cycle number can be reduced while still inhibiting adipogenesis. Amplification of Akt signaling is facilitated by a dynamic restructuring of the cell in response to mechanical signals, as evidenced by a transient increase in focal adhesion (FA) number and increased RhoA activity. Preventing FA assembly or development of tension blocks activation of Akt by mechanical signals, but not by insulin. This indicates that the FA infrastructure is essential to the physical, but not necessarily the chemical, sensitivity, and responsiveness of the cell. Exploiting the transient nature of cytoskeletal remodeling may represent a process to enhance cell responsiveness to mechanical input and ultimately define the fate of MSCs with a minimal input.

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. Currently, a successful clinical therapy has not been identified. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and their three-dimensional aggregation can further enhance therapeutic efficacy. In this study, MSC aggregation into spheroids was optimized in vitro based on cellular viability, spheroid size, and trophic factor secretion. The regenerative potential of the optimized MSC spheroid therapy was then investigated in a murine model of VML injury. Experimental groups included an untreated VML injury control, intramuscular injection of MSC spheroids, and MSC spheroids encapsulated in a fibrin-laminin hydrogel. Compared to the untreated VML group, the spheroid encapsulating hydrogel group enhanced myogenic marker (i.e., MyoD and myogenin) protein expression, improved muscle mass, increased presence of centrally nucleated myofibers as well as small fibers (<500 µm2 ), modulated pro- and anti-inflammatory macrophage marker expression (i.e., iNOS and Arginase), and increased the presence of CD146+ pericytes and CD31+ endothelial cells in the VML injured muscles. Future studies will evaluate the extent of functional recovery with the spheroid encapsulating hydrogel therapy.

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. Small interfering RNA targeting GSK3beta prevented both strain-induced induction of beta-catenin and an increase in COX2, a factor associated with increased osteoprogenitor phenotype. Small interfering RNA knockdown of beta-catenin blocked mechanical reduction of peroxisome proliferator-activated receptor gamma and adiponectin, implicating beta-catenin in strain inhibition of adipogenesis. In contrast, the effect of both mechanical and pharmacologic inhibition of GSK3beta on the putative beta-catenin target, COX2, was unaffected by beta-catenin knockdown. GSK3beta inhibition caused accumulation of nuclear NFATc1; mechanical strain increased nuclear NFATc1, independent of beta-catenin. NFATc1 knockdown prevented mechanical stimulation of COX2, implicating NFATc1 signaling. Finally, inhibition of GSK3beta caused association of RNA polymerase II with the COX2 gene, suggesting transcription initiation. These results demonstrate that mechanical inhibition of GSK3beta induces activation of both beta-catenin and NFATc1 signaling, limiting adipogenesis via the former and promoting osteoblastic differentiation via NFATc1/COX2. Our novel findings suggest that mechanical loading regulates mesenchymal stem cell differentiation through inhibition of GSK3beta, which in turn regulates multiple downstream effectors.

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. beta-Catenin actions are less well understood in mature osteoblasts: while beta-catenin contributes to control of osteoclastic bone resorption via alteration of the osteoprotegerin/RANKL ratio, a specific regulatory role during osteoblast bone synthesis has not yet been determined. The proven ability of mechanical factors to prevent beta-catenin degradation and induce nuclear translocation through Lrp-independent mechanisms suggests processes by which exercise might modulate bone mass via control of lineage allocation, in particular, by preventing precursor distribution into the adipocyte pool. Effects resulting from mechanical activation of beta-catenin in mature osteoblasts and osteocytes likely modulate bone resorption, but whether beta-catenin is involved in osteoblast synthetic function remains to be proven for both mechanical and soluble mediators. As beta-catenin appears to support the downstream effects of multiple osteogenic factors, studies clarifying when and where beta-catenin effects occur will be relevant for translational approaches aimed at preventing bone loss and terminal adipogenic conversion.

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. Indomethacin stimulated adipogenesis (increased aP2, adiponectin and lipid droplets) of CH310T1/2 stem cells as well as marrow-derived MSCs to a degree similar to the PPARγ2 ligand, rosiglitazone. Unlike rosiglitazone, indomethacin significantly upregulated PPARγ2 expression. Indomethacin and the COX2 specific inhibitor celecoxib suppressed PGE2 production, but celecoxib did not induce adipogenesis. As well, addition of PGE2 failed to reverse indomethacin induced adipogenesis, indicating that indomethacin's effects were prostaglandin independent. In MSCs over-expressing PPARγ2 and RXRα, indomethacin did not increase PPAR-induced transcription, while rosiglitazone and 15d-PGJ2 did (1.7- and 1.3-fold, respectively, P < 0.001). We considered whether indomethacin might directly affect C/EBPß proximally to PPARγ2 induction. Indomethacin significantly increased C/EBPß expression and protein within 24 h of addition. These results indicate that indomethacin promotes adipogenesis by increasing C/EBPß and PPARγ2 expression in a prostaglandin-independent fashion. This effect of indomethacin is pertinent to potential deleterious effects of this commonly used anti-inflammatory drug on bone remodeling and tissue healing.

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. Frequency domain simulations showed that the spatially-averaged pressure at the dish surface alternated between a minimum and maximum level as the distance between the dish and transducer increased. The ratio of the maximum to minimum level was 6.5-fold when the air interface was present, and this ratio dropped to 1.8-fold with replacement of the air interface. However, radial pressure variability was present with or without the air interface in the bioreactor model. Time-dependent simulations showed that the increase in acoustic pressure to a maximum level after US signal activation and the pressure drop after signal cessation were faster when the water-coupled non-reflective layer was used to replace the air layer below the dish, generating a pressure pattern that more closely followed the applied pulsed ultrasound signal due to reduced wave reflection and interference. Overall, this work showed that having water rather than air in contact with the lower dish surface when paired with an acoustic absorbent layer resulted in a less variable pressure field, providing an improved bioreactor design for in vitro experiments.

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. This highlights a need for a delivery vehicle for a sustained release of PRP to improve its therapeutic effect. In this study, we used microfluidics to fabricate biodegradable PRP-loaded polyethylene glycol (PEG) microspheres. PRP was incorporated into the microspheres as a lyophilized PRP powder either as is (powder PRP) or first solubilized and pre-clotted to remove clots (liquid PRP). A high PRP loading of 10% w/v was achieved for both PRP preparations. We characterized the properties of the resulting PRP-loaded PEG microspheres including swelling, modulus, degradation, and protein release as a function of PRP loading and preparation. Overall, loading powder PRP into the PEG microspheres significantly affected the properties of microspheres, with the most pronounced effect noted in degradation. We further determined that microsphere degradation in the presence of powder PRP was affected by platelet aggregation and clotting. Platelet aggregation did not prevent but prolonged sustained PRP release from the microspheres. The delivery system developed and characterized herein could be useful for the loading and releasing of PRP to promote tissue regeneration and wound healing or to suppress tissue degeneration in osteoarthritis, and intervertebral disc degeneration.

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. In this study, we evaluated the therapeutic value of PRP released from a polyethylene glycol (PEG) hydrogel on articular chondrocytes/cartilage explants derived from OA patients. Lyophilized PRP (PRGF) was encapsulated in PEG hydrogels at 10% w/v and hydrogel swelling, storage modulus and degradation and PRGF release kinetics were determined. PRGF releasate from the hydrogels was collected on day 1, 4, and 11. Encapsulation of PRGF at 10% w/v in PEG hydrogels had minimal effect on hydrogel properties. PRGF was released with an initial burst followed by sustained release until complete hydrogel degradation. Effect of PRGF releasates and bolus PRGF (1% w/v PRGF) on patient-derived cartilage explants or chondrocytes was assessed by chondrocyte proliferation (pico-green assay), gene expression for COL1A1, COL2A1, MMP13, COX2, and NFKB1 (real-time polymerase chain reaction), and measurement of nitric oxide concentration (Griess' assay). Compared to bolus PRGF, PRGF releasates enhanced chondrocyte proliferation, suppressed the expression of genes like MMP13, NFKB1, COL1A1, and COL2A1 and reduced levels of nitric oxide. Taken together, these results indicate that release of PRGF from PEG hydrogels may improve the therapeutic efficacy of PRP and merits further investigation in an animal model of OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2401-2410, 2019.

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. A decrease in active and total beta-catenin levels during adipogenic differentiation was entirely prevented by daily application of mechanical strain; furthermore, strain induced beta-catenin nuclear translocation. Inhibition of glycogen synthase kinase-3beta by lithium chloride or SB415286 also prevented adipogenesis, suggesting that preservation of beta-catenin levels was important to strain inhibition of adipogenesis. Indeed, mechanical strain inactivated glycogen synthase kinase-3beta, which was preceded by Akt activation, indicating that strain transmits antiadipogenic signals through this pathway. Cells grown under adipogenic conditions showed no increase in osteogenic markers runt-related transcription factor (Runx) 2 and osterix (Osx); subsequent addition of bone morphogenetic protein 2 for 2 d increased Runx2 but not Osx expression in unstrained cultures. When cultures were strained for 5 d before bone morphogenetic protein 2 addition, Runx2 mRNA increased more than in unstrained cultures, and Osx expression more than doubled. As such, mechanical strain enhanced MSC potential to enter the osteoblast lineage despite exposure to adipogenic conditions. Our results indicate that MSC commitment to adipogenesis can be suppressed by mechanical signals, allowing other signals to promote osteoblastogenesis. These data suggest that positive effects of exercise on both fat and bone may occur during mesenchymal lineage selection.

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. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(-/-) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(-/-) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(-/-) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(-/-) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.

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. Cell studies showed that cav-1 deficiency leads to increased osteoblast differentiation. These results suggest that cav-1 helps to maintain osteoblast progenitors in a less differentiated state. INTRODUCTION: The absence of caveolin-1 in cellular membranes causes dysregulated signaling. To understand the role of the caveolar microdomain in bone homeostasis, we examined the skeletal phenotype of 5- and 8-wk-old cav-1(-/-) mice. MATERIALS AND METHODS: High-resolution microCT imaging showed a region-specific effect of cav-1 deficiency on the skeleton. At 5 wk, cav-1(-/-) mice had increased epiphyseal bone volume (+58.4%, p = 0.05); at 8 wk, metaphyseal bone volume was increased by 77.4% (p = 0.008). Cortical bone at the femoral mid-diaphysis showed that the periosteal area of cav-1(-/-) mice significantly exceeded that of cav-1(+/+) mice by 23.9% and 16.3% at 5 and 8 wk, respectively, resulting in increased mechanical properties (I(max): +38.2%, p = 0.003 and I(mi): +23.7%, p = 0.03). RESULTS: Histomorphometry complemented microCT results showing increased bone formation rate (BFR) at trabecular and cortical sites at 5 wk, which supported findings of increased bone at 8 wk in cav-1(-/-) mice. Formal mechanical testing of the femoral diaphysis confirmed increased bone structure: stiffness increased 33% and postyield deflection decreased 33%. Stromal cells from cav-1(-/-) marrow showed a 23% increase in von Kossa-positive nodules; osteoclastogenesis was also modestly increased in cav-1-deficient marrow. Knockdown of cav-1 with siRNA in wildtype stromal cells increased alkaline phosphatase protein and expression of osterix and Runx2, consistent with osteoblast differentiation. CONCLUSIONS: These data suggest that cav-1 helps to maintain a less differentiated state of osteoblast progenitor cells, and the absence of cav-1 causes bone to mature more rapidly. Caveolin-1 may thus be a target for altering skeletal homeostasis.

Hydrogel effects on bone marrow stromal cell response to chondrogenic growth factors.

The aim of this study was to investigate the effects of alginate and agarose on the response of bone marrow stromal cells (BMSCs) to chondrogenic stimuli. Rat BMSCs were expanded in monolayer culture with or without FGF-2 supplementation. Cells were then seeded in 2% alginate and agarose gels and cultured in media with or without TGF-beta1 or dexamethasone (Dex). Sulfated glycosaminoglycans (sGAGs), collagen type II, and aggrecan were expressed in all groups that received TGF-beta1 treatment during hydrogel culture. Expansion of rat BMSCs in the presence of FGF-2 increased production of sGAG in TGF-beta1-treated groups over those cultures that were treated with TGF-beta1 alone in alginate cultures. However, in agarose, cells exposed to FGF-2 during expansion produced less sGAG within TGF-beta1-supplemented groups over those cultures treated with TGF-beta1 alone. Dex was required for optimal matrix synthesis in both hydrogels, but was found to decrease cell viability in agarose constructs. These results indicate that the response of BMSCs to a chondrogenic growth factor regimen is scaffold dependent.

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. Thus, it is beneficial to provide a primary source of hydroxyapatite within the scaffold, expediting the process of mineralization during bone regeneration. In this study, chitosan-gelatin (CG) cryogels were incorporated with various forms of hydroxyapatite to evaluate effects on the standard characteristics of cryogels, as well as the potential for increased mineralization. Testing included the comparison of porosity, swelling, mechanical integrity, cellular infiltration, and mineralization potential between all types of cryogels. The addition of bone char to CG cryogels produced scaffolds with appropriate porosity and interconnectivity. Additionally, the bone char cryogels exhibited an adequate swelling potential, suitable mechanical properties, excellent cell attachment, and increased mineralization. These properties support this cryogel for such an application in tissue engineering.

Bone formation on tissue-engineered cartilage constructs in vivo: effects of chondrocyte viability and mechanical loading.

Interactions between bone and cartilage formation are critical during growth and fracture healing and may influence the functional integration of osteochondral repair constructs. In this study, the ability of tissue-engineered cartilage constructs to support bone formation under controlled mechanical loading conditions was evaluated using a lapine hydraulic bone chamber model. Articular chondrocytes were seeded onto polymer disks, cultured for 4 weeks in vitro, and then transferred to empty bone chambers previously implanted into rabbit femoral metaphyses. The effects of chondrocyte viability within the implanted constructs and in vivo mechanical loading on bone formation were tested in separate experiments. After 4 weeks in vivo, biopsies from the chambers consisted of a complex composite of bone, cartilage, and fibrous tissue, with bone forming in direct apposition to the cartilage constructs. Microcomputed tomography imaging of the chamber biopsies revealed that the implantation of viable constructs nearly doubled the bone volume fraction of the chamber tissue from 0.9 to 1.6% as compared with the implantation of devitalized constructs in contralateral control chambers. The application of an intermittent cyclic mechanical load was found to increase the bone volume fraction of the chamber tissue from 0.4 to 3.6% as compared with no-load control biopsies. The results of these experiments demonstrate that tissue-engineered cartilage constructs implanted into a well-vascularized bone defect will support direct appositional bone formation and that bone formation is significantly influenced by the viability of chondrocytes within the constructs and the local mechanical environment in vivo.

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. We asked if mTORC2 played a role in mechanical adaptation of the cytoskeleton. We found that during bi-axial strain-induced cytoskeletal restructuring, mTORC2 and Akt colocalize with newly assembled focal adhesions (FA). Disrupting the function of mTORC2, or that of its downstream substrate Akt, prevented mechanically induced F-actin stress fiber development. mTORC2 becomes associated with vinculin during strain, and knockdown of vinculin prevents mTORC2 activation. In contrast, mTORC2 is not recruited to the FA complex during its activation by insulin, nor does insulin alter cytoskeletal structure. Further, when rictor was knocked down, the ability of mesenchymal stem cells (MSC) to enter the osteoblastic lineage was reduced, and when cultured in adipogenic medium, rictor-deficient MSC showed accelerated adipogenesis. This indicated that cytoskeletal remodeling promotes osteogenesis over adipogenesis. In sum, our data show that mTORC2 is involved in stem cell responses to biophysical stimuli, regulating both signaling and cytoskeletal reorganization. As such, mechanical activation of mTORC2 signaling participates in mesenchymal stem cell lineage selection, preventing adipogenesis by preserving ß-catenin and stimulating osteogenesis by generating a stiffer cytoskeleton.