Biochemical Characterization of Human Thy 1

The present studies were undertaken in order to a) verify the presence of a human Thy 1 antigen, b) demonstrate its similarity to murine Thy 1, and c) biochemically characterize the molecule. Previously, a 25,000 molecular weight molecule had been isolated from a human T lymphoblastoid cell line, Molt 3, using isolation procedures most frequently used for the isolation of rodent Thy 1. The human Thy 1 (p25) was demonstrated to be biochemically similar to murine Thy 1.2 by molecular weight and ability to bind Lens Culinaris lentil lectin. Recently, we confirmed similarity between the murine and the human molecule by peptide maps and amino acid compositions. In addition, a strong cross-reactivity using ananti-p25 antiserum was established with human IgG subclasses. The antiserum did not react with human IgM or IgA, nor with primate immunoglobulins or a battery of other antigens. Through a variety of immunoabsorption experiments, through the use of various digestions, and known amino acid sequences, the shared areas of homology were tentatively determined to be in the intact disulfide bonds of the first and third domains of human IgG and the 9-112 disulfide bond of Thy 1. A form of the p25 antigen noted and partially characterized was the p40 antigen, the dimerized form of the p25. The p25 and p40 are essentially identical in several aspects, such as amino acid compositions, peptide maps, carbohydrate compositions, reactivity to anti-p25 serum, and in aggregation studies. It was determined that the detergent sodium dodecyl sulfate tends to convert the p25 molecule into the p40 form through some other chemical means than disulfide bond interchange. The method of isolation and reagents used unequivocally determines observed molecular weight. Another form detected is the p16 which also reacted with anti-p25 serum, was similar by amino acid composition, slightly different by peptide mapping and definitely contained less carbohydrate than the p25 antigen. The p16 is possibly a cryptic antigen, a breakdown form of the p25 molecule. A 40,000 MW molecule was isolated from human thymus which binds lentil lectin, reacts with antiserum made to the p25 antigen and possesses an amino acid composition very similar to that of the p25 antigen. Data suggests that the Thy 1 antigen from human thymocytes tends to dimerize more readily than that found on Molt 3 cells and that this antigen is present in considerably smaller amounts on human thymocytes as compared with Molt 3 cells

The Role of the Osteocyte in Bone and Non-bone Disease

When normal physiological functions go awry, disorders and disease occurs. This is universal, even for the osteocyte, a cell embedded within the mineralized matrix of bone. It was once thought that this cell was simply a place-holder in bone. However, within the last decade, the number of studies of osteocytes has dramatically increased leading to the discovery of novel functions of these cells. But with the discovery of novel physiological functions came the discoveries of how these cells can also be responsible for not only bone diseases and disorders, but also those of kidney, heart, and potentially muscle

Late osteoblast/early osteocyte-like cell line for visualizing collagen assembly in living cells [abstract]

Immortal cell lines representing the late osteoblast/early osteocyte phenotype have been generated that stably express a collagen-GFP or collagen-mCherry fusion protein to fluorescently label type I collagen fibrils either red or green. These novel cell lines allow visualization of collagen fibril assembly in living cells over time, which is not possible with existing technologies. The only other approaches that have been used for monitoring collagen assembly in living cells include using fluorescently labeled antibodies to collagen or a fluorescently labeled recombinant bacterial protein that binds to collagen. These have the disadvantage over our new invention that they may potentially interfere with the protein function and that they only label a population of fibrils at one point in time, which can then be followed (i.e. they do not necessarily label new collagen as it is synthesized). The specificity of the bacterial binding protein for type I collagen as opposed to other collagens is unclear and neither of these probes can be used to follow intracellular steps in the collagen assembly pathway, as they do not cross the cell membrane. Therefore our collagen-GFP and collagen-mCherry probes represent a significant improvement over existing technologies. Potential Areas of Applications: Potential commercial applications of this invention include using these cell lines to screen for drugs that enhance collagen assembly and could therefore have potential as bone anabolic treatments for diseases such as osteoporosis. The cells can also be used to screen for drugs that inhibit collagen assembly and therefore have potential as treatments to prevent fibrosis, etc. The cells also have many potential uses in developing approaches for tissue engineering of bone tissues. For example, the cells can be seeded onto scaffolds and the assembly of collagen can be monitored in real time in the living cultures. Mineral deposition on collagen can be monitored simultaneously using vital dyes for calcium deposition. These cell lines may also have commercial application for looking at mechanisms of tissue destruction, such as degradation of matrix proteins by proteases, such as occurs during inflammation

Multi-Staged Regulation of Lipid Signaling Mediators during Myogenesis by COX-1/2 Pathways

Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis. Knocking down COXs, particularly COX-1, significantly reduced the release of PGs from muscle cells, especially PGE2 and PGF2α, as well as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA). Moreover, COXs could interplay with LOXs to regulate the signaling of hydroxyeicosatetraenoic acids (HETEs). The changes in LMs are associated with the expression of genes, such as Itrp1 (calcium signaling) and Myh7 (myogenic differentiation), in skeletal muscle. In conclusion, both COX-1 and -2 contribute to LMs production during myogenesis in vitro, and COXs could interact with LOXs during this process. These interactions and the fine-tuning of the levels of these LMs are most likely important for skeletal muscle myogenesis, and potentially, muscle repair and regeneration

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient‐rich oxygenated blood through the vasculature to support cell metabolism within most cell‐dense tissues. Since scaffold‐free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue‐like structures, we generated a generalizable biofabrication method resulting in self‐supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO‐A5 osteoblast‐based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico

Biomaterial Compositions

Biomaterial compositions comprising organosilicon monomers (such as silorane monomers) and chemical curing systems or dual chemical/light curing systems, in conjunction with optional tetraoxaspiro[5.5]undecanes (“TOSUs”) and/or fillers. The present invention is directed to biomaterial compositions, as well as methods for manufacturing the same, and methods of using the compositions. The biomaterial composition comprises one or more organosilicon monomers (such as a silorane) and a chemical curing system or dual chemicaVlight curing system for polymerizing the 10 monomer(s). The compositions may include one or more tetraoxaspiro[5.5]undecanes “TOSUs”) and/or fillers. Accelerators (such as photoacids), photosensitizers, and/or electron donors may also be included in the composition as appropriate

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation

UMKC Center of Excellence in the Study of Dental and Musculoskeletal Tissues (CEMT) [abstract]

Biomedical Tissue Engineering, Biomaterials, & Medical Devices Poster SessionDiseases of mineralized tissues such as bone and teeth or of the muscles that control bone movement result in significant health costs in terms of suffering, loss of work and productivity, and even death. There is a tremendous need for new approaches to treating musculoskeletal diseases. The UMKC Center of Excellence in the Study of Dental and Musculoskeletal Tissues has formed a multidisciplinary and interdisciplinary center that includes investigators from the UMKC Schools of Dentistry, Medicine, Nursing, Biological Sciences, and Computing and Engineering that focus on dental and musculoskeletal health. The Center of Excellence integrates basic and clinical investigators into a powerful translational team to prevent and treat diseases of mineralized tissue, which includes teeth, cartilage, bone, and muscle. Findings are being applied to biomaterials and composite research, medical devices, diagnostics, and clinical imaging, as related to diagnostics and treatment of human and animal dental and bone disease. The major objective is to expand the ability to make new discoveries in the basic sciences, translate these findings into treatments, diagnoses, and therapies for mineralized tissue disease into the clinics, the community, and into the commercial sector. This Center provides the infrastructure and personnel to move basic science discoveries into patient care. The development of the Center of Excellence provides an infrastructure for taking the outstanding basic research now occurring in this area and converting those findings into useful clinical treatments and applications leading to improved health outcomes. Several patents are being applied for based on finding from investigators in the center including diagnostics for bone loss leading to osteoporosis, devices for healing both bone and muscle, and basic research tools. Visit website http://cemt.umkc.edu

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen

Periostin Is Essential for the Integrity and Function of the Periodontal Ligament During Occlusal Loading in Mice

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141255/1/jper1480.pd