Mineralized Tissue Engineering, Stem Cell Therapies and Proteomics Approaches

Cellular therapy holds tremendous potential in regeneration of mineralized tissues such as bones and teeth. We have characterized and identified pericytes as a unique population of dental pulp stem cells (DPSCs) that can be sorted by CD146+CD34-CD45-CD56-, expanded in culture, and differentiated into osteogenic, chondrogenic, and adipogenic lineages. A well-characterized stem cell source and an appropriate microenvironment containing growth factors and/or extracellular matrix (ECM) proteins to stimulate differentiation and mineralization are required for successful cellular therapies. To understand cell-ECM protein interaction, we studied the signaling role of phosphophoryn (PP), an ECM protein found in dentin and bone. PP signals through integrins, mitogen activated protein kinase (MAPK), and Smad pathways. There is also signaling crosstalk between the MAPK and Smad pathways. To better understand the complex signaling pathways involved in stem cell differentiation during dentin or bone formation, we have utilized quantitative proteomic strategies to study stem cell differentiation triggered by PP and BMP-2. Proteins upregulated and downregulated during differentiation were identified by mass spectrometry. With the ultimate goal of better enabling the regeneration of diseased or damaged mineralized tissue, our findings in this study have enhanced our understanding in stem cell differentiation to the osteoblastic/odontoblastic lineages and lay foundations for the development of future craniofacial regeneration

In-situ Tissue Engineering of the Intervertebral Disc

A possible approach to stimulate proteoglycan and collagen synthesis for treating intervertebral disc degeneration (IDD) is introduction of growth factors. The objective of Part I of this study was to screen the effect of human recombinant bone morphogenetic protein (BMP)-2 and BMP-12 on nucleus pulposus (NP) cells and to investigate the effect of Ad/BMP-12 on NP and anulus fibrosus (AF) cells. Cells were isolated from degenerated human discs and cultured in monolayer. RhBMP-2 (25, 50, 100, 200, 300, ng/ml) and rhBMP-12 (25, 50, 100 ng/ml) stimulated NP cells in serumless media (1% ITS) for 2 days. Ad/BMP-12 (50, 100, 150 MOI) transduced NP and AF cells, then pellets (150,000 cells/pellet) were formed and incubated in serumless media (1% ITS) for 6 days. Proteoglycan, collagen, and non-collagenous protein synthesis were measured. RhBMP-2 had a more substantial effect on upregulating matrix synthesis than rhBMP-12. Ad/BMP-12 significantly increased matrix synthesis. Total DNA content was increased pellets stimulated by Ad/BMP-12 when compared to control. The increase in matrix synthesis was attributed to both an increase in cell number and in matrix synthesis per cell. Intervertebral disc (IVD) is the largest avascular organ in the body, it has been suggested that lack of nutrition may be one of the cause of IDD. The goals of the second part of this study were to develop a rabbit disc organ culture method to study the effect of FBS concentration in disc metabolism and to attempt gene therapy in the organ culture. Twenty-seven rabbit lumber intervertebral discs were harvested and cultured for 2 weeks in serumless media (1% ITS) or F-12/DMEM (5%, 10%, 15% FBS). NP and AF wet weight, dry weight, % hydration, glycosaminoglycans (GAG), DNA, and lactate content were measured. In 25 discs, 6x106 PFU of Ad/lacZ or Ad/Luciferase was injected into the NP 2 days after the culture to determine the effect of gene transfer. Histology and viability staining of 10 discs were used to show cell morphology and viability. Due to low cell viability, we did not observe successful gene transfer in the organ culture.NP and AF lactate content and AF DNA content were significantly higher in the 15% FBS group than serumless group

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Degradation of Cell Wall Polysaccharides during Traditional and Tank Fermentation of Chinese Liupao Tea

The increase of polysaccharides in the dark tea pile process is thought to be connected to the cell wall polysaccharides’ breakdown. However, the relationship between tea polysaccharides (TPSs) and tea cell wall polysaccharides has not been further explored. In this study, the structural changes in the cell wall polysaccharides [e.g., cellulose, hemicellulose (HC), and pectin] in Liupao tea were characterized before and after traditional fermentation and tank fermentation. Additionally, the degradation mechanism of tea cell wall polysaccharides during fermentation was assessed. The results showed that cellulose crystallinity decreased by 11.9–49.6% after fermentation. The molar ratio of monosaccharides, such as arabinose, rhamnose, and glucose in HC, was significantly reduced, and the molecular weight decreased. The esterification degree and linearity of water-soluble pectin (WSP) were reduced. TPS content increases during pile fermentation, which may be due to HC degradation and the increase in WSP caused by cell wall structure damage. Microorganisms were shown to be closely associated with the degradation of cell wall polysaccharides during fermentation according to correlation analyses. Traditional fermentation had a greater effect on the cellulose structure, while tank fermentation had a more noticeable impact on HC and WSP

Degradation of Cell Wall Polysaccharides during Traditional and Tank Fermentation of Chinese Liupao Tea

The increase of polysaccharides in the dark tea pile process is thought to be connected to the cell wall polysaccharides’ breakdown. However, the relationship between tea polysaccharides (TPSs) and tea cell wall polysaccharides has not been further explored. In this study, the structural changes in the cell wall polysaccharides [e.g., cellulose, hemicellulose (HC), and pectin] in Liupao tea were characterized before and after traditional fermentation and tank fermentation. Additionally, the degradation mechanism of tea cell wall polysaccharides during fermentation was assessed. The results showed that cellulose crystallinity decreased by 11.9–49.6% after fermentation. The molar ratio of monosaccharides, such as arabinose, rhamnose, and glucose in HC, was significantly reduced, and the molecular weight decreased. The esterification degree and linearity of water-soluble pectin (WSP) were reduced. TPS content increases during pile fermentation, which may be due to HC degradation and the increase in WSP caused by cell wall structure damage. Microorganisms were shown to be closely associated with the degradation of cell wall polysaccharides during fermentation according to correlation analyses. Traditional fermentation had a greater effect on the cellulose structure, while tank fermentation had a more noticeable impact on HC and WSP

Placental Perivascular Cells for Human Muscle Regeneration

Perivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin. In vitro-cultured placental villi released rapidly adhering and migratory CD146+CD34−CD45−CD56− cells of putative perivascular origin that expressed mesenchymal stem cell markers. CD146+CD34−CD45−CD56− perivascular cells isolated and purified from the placental villi by flow cytometry were indeed highly myogenic in culture, and generated dystrophin-positive myofibers, and they promoted angiogenesis after transplantation into SCID/mdx mouse muscles. These observations confirm the existence of mesenchymal progenitor cells within the walls of human blood vessels, and suggest that the richly vascularized human placenta is an abundant source of perivascular myogenic cells able to migrate within dystrophic muscle and regenerate myofibers