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Human monocytes have been grouped into classical (CD14++CD16−), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

Integrated image-based design and solid free-form fabrication can create scaffolds that attain desired elasticity and permeability while fitting any 3D craniofacial defect. The scaffolds could be manufactured from degradable polymers, calcium phosphate ceramics and titanium. The designed scaffolds supported significant bone regeneration for all pore sizes ranging from 300 to 1200 microns. These results suggest that designed scaffolds are clinically applicable for complex craniofacial reconstruction.

IntroductionMesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct.MethodsHuman MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-β3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-β1 and TIMP1(tissue inhibitor of metalloproteinases-1)).ResultsSignificantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFβ groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-β1 and decreased expression of sox-9 and collagen II relative to the TGFβ group was observed.ConclusionsTogether, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.

Mesenchymal stem cells (MSCs) are thought to occupy a perivascular niche where they are exposed to signals originating from vascular cells. This study focused on the effects of endothelial cell (EC)-derived signals on MSC differentiation toward vascular cell lineages. Upon co-culture with two types of ECs, macrovascular (macro) ECs and microvascular (micro) ECs, the former caused MSCs to increase expression of both EC and smooth muscle cell (SMC) markers, while the latter induced expression of EC markers only. These marker changes in MSCs were linked to the extracellular matrixes secreted by the ECs (EC-matrix) rather than soluble EC-secreted factors. Beyond enhanced marker expression, EC-matrix also induced functional changes in MSCs indicative of development of a genuine vascular cell phenotype. These included enhanced incorporation into vessels and cytoskeletal localization of vascular SMC-specific contractile elements. The bioactivity of EC-matrix was sensitive to EDTA washes and required sulfated glycosaminoglycans. However, neither soluble VEGF nor substrate surfaces coated with fibronectin, collagen type IV, or laminin recreated the effects of EC-matrix on MSC vascular differentiation. In conclusion, these results identified EC-matrix as a critical regulator of vascular cell differentiation of MSCs. Elucidating these MSC-EC-matrix interactions and identifying the specific EC-matrix components involved will shed light on the perivascular signals seen by MSCs in vivo.

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumor’s inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells.

lage often necessitate surgical intervention after injury or degenerative disease. [1][2][3][4][5] However, current therapies such as osteochondral grafting, chondroplasty, and prosthetic joint replacement have only partial or temporary success due to inadequate donor tissue availability, donor site morbidity, the risk of infection, abrasion of the cartilage surface, loosening of implants, and limited dura-bility of prosthetics. [6][7][8] Tissue-engineering approaches have the potential to overcome the lack of donor tissue and to create a graft that contains biologically and mechanically functional tissue. A variety of tissue-engineering techniques have been developed to regenerate bone and cartilage. [9][10][11][12][13][14][15][16] These studies have illustrated that engineered cartilage must integrate with the host tissue, provide a smooth and natural articulating surface, and bear functional mechanical loads. In attempts to satisfy these requirements, investigators have used a variety of