17 research outputs found
Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts.
Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.
Detection of Pentosidine Cross-Links in Cell-Secreted Decellularized Matrices Using Time Resolved Fluorescence Spectroscopy.
Cell-secreted matrices perpetuate the bone-forming phenotype of differentiated mesenchymal stem cells
Prior to transplantation, mesenchymal stem/stromal cells (MSCs) can be induced toward the osteoblastic phenotype using a cocktail of soluble supplements. However, there is little evidence of differentiated MSCs directly participating in bone formation, suggesting that MSCs may either die or revert in phenotype upon transplantation. Cell-secreted decellularized extracellular matrices (DMs) are a promising platform to confer bioactivity and direct cell fate through the presentation of a complex and physiologically relevant milieu. Therefore, we examined the capacity of biomimetic DMs to preserve the mineral-producing phenotype upon withdrawal of the induction stimulus. Regardless of induction duration, ranging up to 6 weeks, MSCs exhibited up to a 5-fold reduction in osteogenic markers within 24 h following stimulus withdrawal. We show that seeding osteogenically induced MSCs on DMs yields up to 2-fold more calcium deposition than tissue culture plastic, and this improvement is at least partially mediated by increasing actin cytoskeletal tension via the ROCK II pathway. MSCs on DMs also secreted 25% more vascular endothelial growth factor (VEGF), a crucial endogenous proangiogenic factor that is abrogated during MSC osteogenic differentiation. The deployment of DMs into a subcutaneous ectopic site enhanced the persistence of MSCs 5-fold, vessel density 3-fold, and bone formation 2-fold more than MSCs delivered without DMs. These results underscore the need for deploying MSCs using biomaterial platforms such as DMs to preserve the in vitro-acquired mineral-producing phenotype and accelerate the process of bone repair
Detection of Pentosidine Cross-Links in Cell-Secreted Decellularized Matrices Using Time Resolved Fluorescence Spectroscopy
Hyperglycemia-mediated, nonenzymatic collagen cross-links such as pentosidine (PENT) can have deleterious effects on cellular interactions with the extracellular matrix (ECM). Present techniques to quantify PENT are limited, motivating the need for improved methods to study the accumulation and contribution of PENT toward diabetic clinical challenges such as impaired bone healing. Current methods for studying PENT are destructive, laborious, and frequently employ oversimplified collagen films that lack the complexity of the native ECM. The primary goal of this study was to evaluate the capacity of time-resolved fluorescence spectroscopy (TRFS) to detect PENT in cell-secreted ECMs possessing enhanced compositional complexity. To demonstrate an application of this method, we assessed the response of human mesenchymal stem cells (MSCs) to cross-linked substrates to explore the role of detected PENT on osteogenic differentiation. We exposed MSC-secreted decellularized matrices (DMs) to 0.66 M ribose for 2 weeks and used TRFS to detect the accumulation of PENT. Ribose treatment resulted in a 30 nm blue shift in peak fluorescence emission and a significant decrease in average lifetime compared to that of control DMs (4.4 ± 0.3 ns vs 3.5 ± 0.09 ns). Evaluation of samples with high performance liquid chromatography (HPLC) confirmed that changes in observed fluorescence were due to PENT accumulation. A strong correlation was found between TRFS parameters and the HPLC measurement of PENT, validating the use of TRFS as an alternative method of PENT detection. Osteoblastic gene expression was significantly reduced in MSCs seeded on ribose DMs at days 7 and 14. However, no significant differences in calcium deposition were detected between control and ribose DMs. These data demonstrate the efficacy of nondestructive fluorescence spectroscopy to examine the formation of nonenzymatic collagen cross-links within biomimetic culture platforms and showcase one example where an improved biomimetic substrate can be used to probe cell-ECM interactions in the presence of collagen cross-links
Hydrogel biophysical properties instruct coculture-mediated osteogenic potential
Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.U.S. National Institutes of HealthNational Institute of Dental and Craniofacial Research [R03-DE021704]AO Foundation (Davos, Switzerland) [C10-39L]American Heart Association Western States Affiliate Pre-doctoral FellowshipUniv Calif Davis, Dept Biomed Engn, Davis, CA 95616 USAUniv Calif Davis, Sch Med, Dept Orthopaed Surg, Davis, CA 95616 USAUniv Fed Sao Paulo, Dept Biophys, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biophys, Sao Paulo, BrazilU.S. National Institutes of Health, National Institute of Dental and Craniofacial Research Grant R03-DE021704, and the AO Foundation (Davos, Switzerland) (C10-39L to J.K.L.). K.M. was supported by the American Heart Association Western States Affiliate Pre-doctoral FellowshipWeb of Scienc
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Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts.
Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation
Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging.
We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable
Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in <i>Mycobacterium fortuitum</i>-Infected Fish Macrophages
<div><p><i>Mycobacterium fortuitum</i> causes ‘mycobacteriosis’ in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca<sup>+2</sup>)-signalling cascade on <i>M</i>. <i>fortuitum-</i>induced apoptosis in headkidney macrophages (HKM) of <i>Clarias</i> sp. <i>M</i>. <i>fortuitum</i> could trigger intracellular-Ca<sup>+2</sup> influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKII<i>g</i>). Gene silencing and inhibitor studies established the role of CaM in <i>M</i>. <i>fortuitum</i> pathogenesis. We noted that CaMKII<i>g</i> activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKII<i>g</i> in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKII<i>g</i> to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of <i>M</i>. <i>fortuitum</i> pathogenesis. We propose that <i>M</i>. <i>fortuitum</i> triggers intracellular Ca<sup>+2</sup> elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKII<i>g</i> resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of <i>M</i>. <i>fortuitum</i>-infected HKM.</p></div
<i>M</i>. <i>fortuitum</i> alters intracellular Ca<sup>+2</sup> homeostasis leading to activation of CaM and PKC-α.
<p>(A) HKM were pre-treated with or without BAPTA/AM followed by <i>M</i>. <i>fortuitum</i> infection and cytosolic Ca<sup>+2</sup> elevation was measured using Fluo-4 at 1 h of infection. (B) HKM transfected with indicated siRNA or pre-treated separately with indicated inhibitors and at 4 h p.i. CaM-protein expression detected by EIA. (C) Fold changes in CaM-mRNA expression was determined in HKM transfected with specific siRNA or non-targeted siRNA followed by <i>M</i>. <i>fortuitum</i> infection 4 h p.i by qPCR. (D) HKM were pre-treated separately with or without indicated inhibitors and PKC activity determined at 2 h p.i. Vertical bars represent mean ± SE (n = 3).*<i>P</i><0.05, compared to HKM; <sup>γ</sup><i>P</i><0.05, compared to HKM+Sc; <sup>#</sup><i>P</i><0.05, compared to HKM+MF; <sup>•</sup><i>P</i><0.05, compared to HKM+MF+Sc. HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+MF, HKM infected with <i>M</i>. <i>fortuitum</i>; HKM+MF+Sc, HKM transfected with scrambled siRNA infected with <i>M</i>. <i>fortuitum</i>; HKM+MF+siRNA-CaM, HKM transfected with siRNA-CaM infected with <i>M</i>. <i>fortuitum</i>; HKM+MF+BAPTA/AM, HKM+MF+CMZ, HKM+MF+CC, HKM+MF+Gö6976, HKM were pre treated with BAPTA/AM, CMZ, CC, Gö6979 respectively followed by infection with <i>M</i>. <i>fortuitum</i>.</p