Current tissue engineering strategies to heal critical-size bone defects through direct bone formation are limited by incomplete integration of grafts with host bone and incomplete graft ...vascularization. An alternative strategy for bone regeneration is the use of cartilage grafts that form bone through endochondral ossification. Endochondral cartilages stimulate angiogenesis and are remodeled into bone, but are found in very small quantities in growth plates and healing fractures. We sought to develop engineered endochondral cartilage grafts using osteoarthritic (OA) articular chondrocytes as a cell source. Such chondrocytes often undergo hypertrophy, which is a characteristic of endochondral cartilages.
We compared the ability of unmodified human OA (hOA) cartilage and cartilage grafts formed in vitro from hOA chondrocytes to undergo endochondral ossification in mice. Scaffold-free engineered chondrocyte grafts were generated by pelleting chondrocytes, followed by culture with transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4. Samples derived from either primary or passaged chondrocytes were implanted subcutaneously into immunocompromised mice. Grafts derived from passaged chondrocytes from three patients were implanted into critical-size tibial defects in mice. Bone formation was assessed with histology after 4 weeks of implantation. The composition of tibial repair tissue was quantified with histomorphometry.
Engineered cartilage grafts generated from passaged OA chondrocytes underwent endochondral ossification after implantation either subcutaneously or in bone. Cartilage grafts integrated with host bone at 15 out of 16 junctions. Grafts variably remodeled into woven bone, with the proportion of bony repair tissue in tibial defects ranging from 22% to 85% (average 48%). Bony repair tissue bridged the tibial defects in half of the animals. In contrast, unmodified OA cartilage and engineered grafts formed from primary chondrocytes did not undergo endochondral ossification in vivo.
hOA chondrocytes can adopt an endochondral phenotype after passaging and TGF-β superfamily treatment. Engineered endochondral cartilage grafts can integrate with host bone, undergo ossification, and heal critical-size long-bone defects in a mouse model. However, additional methods to further enhance ossification of these grafts are required before the clinical translation of this approach.
ABSTRACT
Coculture of mesenchymal stem cells (MSCs) with articular chondrocytes (ACs) increases glycosaminoglycan (GAG) accumulation compared to monoculture. MSCs might (1) differentiate into ACs ...(progenitor role) and/or (2) stimulate AC matrix metabolism (trophic role). MSCs lose the ability to undergo chondrogenesis after extended passaging. We hypothesized that MSCs act predominantly as progenitors, and that late‐passage MSCs without chondrogenic potential would be unable to increase GAG in coculture. Early‐ and late‐passage human MSCs (hMSCs) were grown in pellet monoculture under chondrogenic conditions and in pellet coculture with bovine ACs. Chondrogenesis was assessed with GAG quantification, safranin‐O staining, and quantitative PCR (qPCR). Contributions of human and bovine cells were assessed with species‐specific qPCR and human‐specific immunostaining. Late‐passage hMSCs did not undergo chondrogenesis in monoculture with chondrogenic stimuli or in coculture with ACs. Early‐passage hMSCs underwent chondrogenesis only in response to chondrogenic stimuli. Coculture pellets in both cases accumulated as much GAG/DNA as monoculture AC pellets. Aggrecan transcription was not increased in coculture. Late‐passage hMSCs that do not undergo chondrogenesis are capable of stimulating GAG accumulation in coculture with ACs. This supports a trophic effect of hMSCs on ACs. hMSCs may have therapeutic utility even after prolonged passaging. Published 2013 by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 31:1936–1942, 2013
Poly(lactic-
-glycolic acid) (PLGA) particles are very widely used, particularly for drug delivery, including commercial clinical formulations. Adding perfluorocarbon (PFC) enables
imaging and ...quantification of the PLGA particles through
F NMR, MRS or MRI. PFCs are both hydrophobic and lipophobic at the same time. This property makes their encapsulation in particles challenging, as it requires the addition of a third immiscible phase during the emulsification process. Here we explore how different parameters affect the miniemulsion formation of particles loaded with perfluoro-15-crown-5-ether (PFCE). By changing the concentration of surfactant and type of solvent, we were able to control the radius of synthesized particles, between 85-200 nm. We assessed stability and release from the particles at different pH values, showing that hydrophobic agents are released from the particles by diffusion rather than degradation. With cell experiments, we show that primary human dendritic cells take up the particles without any apparent effect, including on cell migration. In summary, the control of synthesis conditions leads to particles with sufficient PFCE encapsulation, which are suitable for drug loading and cell labeling, and do not affect cell viability or functionality. Finally, these nanoparticles can be produced at GMP-grade for clinical use.
Abstract only
Galectin‐9 and galectin‐1 both kill thymocytes, peripheral T cells and T cell lines. However, differences in target cell specificity, glycoprotein receptor requirements, and ...intracellular death pathways for these two galectins are not well understood. We found that galectin‐9 and galectin‐1 required different glycan ligands and glycoprotein receptors to trigger T cell death. They also triggered different intracellular death pathways, as only galectin‐9 T cell death was blocked by intracellular bcl‐2, while only galectin‐1 T cell death was blocked by intracellular galectin‐3. Galectin‐9 and galectin‐1 were both expressed in murine thymus, but killed different thymocyte subsets, with mature thymocytes more susceptible to galectin‐9. To define the structural features responsible for distinct activities of galectin‐1 and galectin‐9, we created a series of bivalent neo‐galectins with galectin‐1 and galectin‐9 carbohydrate recognition domains on different peptide linkers. The N‐terminal carbohydrate recognition domain and linker peptide contributed to potency of the galectins. Strikingly, the C‐terminal carbohydrate recognition domain determined receptor recognition, death pathway signaling and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency all contribute to specific effects of different galectins in regulating T cell death.
Poly(lactic-
co
-glycolic acid) (PLGA) particles are very widely used, particularly for drug delivery, including commercial clinical formulations. Adding perfluorocarbon (PFC) enables
in vivo
imaging ...and quantification of the PLGA particles through
19
F NMR, MRS or MRI. PFCs are both hydrophobic and lipophobic at the same time. This property makes their encapsulation in particles challenging, as it requires the addition of a third immiscible phase during the emulsification process. Here we explore how different parameters affect the miniemulsion formation of particles loaded with perfluoro-15-crown-5-ether (PFCE). By changing the concentration of surfactant and type of solvent, we were able to control the radius of synthesized particles, between 85-200 nm. We assessed stability and release from the particles at different pH values, showing that hydrophobic agents are released from the particles by diffusion rather than degradation. With cell experiments, we show that primary human dendritic cells take up the particles without any apparent effect, including on cell migration. In summary, the control of synthesis conditions leads to particles with sufficient PFCE encapsulation, which are suitable for drug loading and cell labeling, and do not affect cell viability or functionality. Finally, these nanoparticles can be produced at GMP-grade for clinical use.
The influence of different synthesis parameters on the characteristics of polymeric particles with a third perfluorocarbon phase.
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