It May Seem Inflammatory,but Some T Cells Are Innately Healing to the Bone

Among the most significant developments to have taken place in osteology over the last few decades is an evolution from treating and viewing bone disorders primarily through an endocrine lens to instead seeing them as metabolic disorders that interface at the molecular and cellular level with the immune system. Osteoimmunology was officially born in response to accumulating evidence that the immune system is integrally involved in bone remodeling, but much of the early work focused on the role of conventional αβ T cells in driving bone loss. There is, however, emerging data indicating that innate lymphocytes, in particular γδ T cells, may in fact be important for bone regeneration. We first observed that bisphosphonate‐associated osteonecrosis of the jaw (ONJ), a rare but serious adverse drug effect characterized by nonhealing necrotic bone tissue of the mandible or maxilla, was linked to a deficiency in a subset of γδ T cells found in human peripheral blood. Patients who developed ONJ while on bisphosphonate therapy not only lacked the main subset of circulating γδ T cells, but they also all had underlying conditions that compromised their immune integrity. A number of recent studies have unraveled the role of γδ T cells (and lymphocytes sharing their characteristics) in bone regeneration—particularly for fracture healing. These findings seem to contradict the prevailing view of such “inflammatory” T cells as being bone degenerative rather than restorative. This viewpoint melds together the emerging evidence of these so‐called inflammatory T cells in bone remodeling and healing—showing that they are not in fact “all bad to the bone.” © 2016 American Society for Bone and Mineral Research.     

The diverse origin of bone-forming osteoblasts

Osteoblasts are the only cells that can give rise to bones in vertebrates. Thus, one of the most important functions of these metabolically active cells is mineralized matrix production. Because osteoblasts have a limited lifespan, they must be constantly replenished by preosteoblasts, their immediate precursors. Because disruption of the regulation of bone-forming osteoblasts results in a variety of bone diseases, a better understanding of the origin of these cells by defining the mechanisms of bone development, remodeling, and regeneration is central to the development of novel therapeutic approaches. In recent years, substantial new insights into the origin of osteoblasts—largely owing to rapid technological advances in murine lineage-tracing approaches and other single-cell technologies—have been obtained. Collectively, these findings indicate that osteoblasts involved in bone formation under various physiological, pathological, and therapeutic conditions can be obtained from numerous sources. The origins of osteoblasts include, but are not limited to, chondrocytes in the growth plate, stromal cells in the bone marrow, quiescent bone-lining cells on the bone surface, and specialized fibroblasts in the craniofacial structures, such as sutures and periodontal ligaments. Because osteoblasts can be generated from local cellular sources, bones can flexibly respond to regenerative and anabolic cues. However, whether osteoblasts derived from different cellular sources have distinct functions remains to be investigated. Currently, we are at the initial stage to aptly unravel the incredible diversity of the origins of bone-forming osteoblasts. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Dynamics of cellular entry and drug delivery by dendritic polymers into human lung epithelial carcinoma cells

Dendrimers and hyperbranched polymers are emerging as potentially ideal drug delivery vehicles because they provide a significant amount of tailorability and a large density of functional groups. This study explores the dynamics of cellular entry of dendrimers and hyperbranched polymers alone, and in the complexed form with ibuprofen, into A549 human lung epithelial carcinoma cells using UV/Vis spectroscopy, flow cytometry and fluorescence microscopy. Both dendrimers and hyperbranched polymers appear to enter these cells rapidly. The polyamidoamine (PAMAM) dendrimers, with NH₂ and OH end functionalities appear to enter cells (in approx. 1 h) faster than the hyperbranched polyol (OH functionality) (in approx. 2 h). Cellular entry of PAMAM-NH₂ was detected as early as 5 min. All branched polymers and their ibuprofen complexes entered A549 lung epithelial cells rapidly when compared to the pure drug. The drug payload was about 50% by weight in the complexes formed by PAMAM-NH₂ dendrimers and was about 30% in the encapsulated form for Polyol-OH and PAMAM-OH. The complexation and encapsulation of ibuprofen with the polymers appear to facilitate rapid cellular entry of ibuprofen. The anti-inflammatory effect of the polymer-complexed drug was demonstrated by more rapid suppression of COX-2 mRNA levels than that achieved by the pure drug. This suggests that these dendritic polymers can act as efficient drug carriers, delivering high 'payloads' of drug even with complexation and encapsulation.     

Porcine chromaffin cells,culture, and transplant for antinociceptive effects in rodents and primates

Abstract

It has been shown that xenografts and allografts of spinally transplanted adrenal chromaffin cells produce antinociception in animals and pain relief in patients with cancer pain. As there is a very limited availability of human adrenal tissue to serve as allografts, the clinical need for xenogeneic chromaffin cells as transplants is obvious. Bovine adrenal glands as a steady source of chromaffin cells have been extensively studied. There is however concern about the possible infection in humans with retrovirus following transplantation. The purpose of this study is to use the pig as a preferred donor animal species for xenotransplantation into rat and monkey. As pigs have been cloned, this opens the door to gene-targeted technologies and allows for genetic modifications, which possibly could improve the efficacy and safety of chromaffin cell transplantation. Porcine chromaffin cells were isolated from adrenal glands of 6–8-month-old pigs. After culturing cells for 1 week in a medium containing serum, the release of met-enkephalin and norepinephrine from the cells was detected by high-performance liquid chromatography and radioimmunoassay with nicotine stimulation, lasting ~3 weeks. Transplantation of these cells into the subarachnoid space of rats produced antinociceptive effects on A δ and C fiber-mediated responses lasting 2–3 weeks. Similar findings were observed in studies with macaque monkeys. Compared with the same number of bovine chromaffin cells, porcine chromaffin cells showed a more robust and longer antinociceptive effect, and could be a better source of cells for human transplantation.     

Possible Mechanism of Increase in Gastric Mucosal PGE2 and PGI2 Generation Induced by Ecabet Sodium, a Novel Gastroprotective Agent

The gastroprotective agent ecabet sodium(ecabet, 12-sulfodehydroabietic acid monosodium salt)increases the formation of prostaglandin (PG)E₂ and I₂ by gastric mucosa. Inthe present study, we examined the effect of ecabet on metabolism ofarachidonic acid (AA) in rat gastric mucosal cells.Ecabet (0.1-10 mM) concentration- and time-dependentlypotentiated the release of [¹⁴C]AA fromgastric mucosal cells prelabeled with [¹⁴C]AA andsimultaneously increased the production ofPGE₂ and PGI₂. The ecabet-mediatedincreases in [¹⁴C]AA release andPGE₂ production were both partly depressed bymepacrine (30 and 100 M) and Ca²⁺ chelation.Ecabet, however, showed no effect on gastricphospholipase A₂ (PLA₂) activityand [Ca²⁺]_i in the gastric mucosalcells. Ecabet and other dehydroabietic acid derivatives, 12-carboxydehydroabietic acid monosodium saltand mono[16-(12-sulfodehydroabietyl)]succinic acidmonosodium salt, which potentiated the liberation of[¹⁴C]AA, increased the membrane fluidity ofgastric mucosal cells assessed by usingdiphenylhexatrienepropionic acid (DPH-PA) as the probe,while 12-sulfamoyldehydroabietic acid showed no effecton either the AA liberation or the membrane fluidity.Ecabet (0.1-10 mM) increased the membrane fluidityconcentration- and time-dependently in accordance withits facilitating effect on AA release. In conclusion,ecabet increases the synthesis of PGE₂ andPGI₂ by gastric mucosal cells through promoting the release ofAA, which is partly dependent on PLA₂ andCa²⁺. The ecabet-induced increase in membranefluidity may be involved in part 2 in the liberation ofAA from the gastric mucosal cells.     

Local and Systemic Liberation of Proinflammatory Cytokines in Ulcerative Colitis

Determination of plasma and tissue cytokinelevels in inflammatory bowel disease have frequentlyresulted in conflicting data. In the present study wedetermined in patients with ulcerative colitis (UC), the levels of the proinflammatory cytokinesinterleukin (IL)-1, IL-6, interferon(IFN)-, and tumor-necrosis factor (TNF)-liberated by peripheral blood mononuclear cells (PBMC)and lamina propria mononuclear cells (LPMC) after 48-hrculture with pokeweed mitogen (PWM). IL-1, IL-6,IFN- and TNF- in the supernatant weredetected by ELISA. Results show low basal levels ofIL-1 secretion by PBMC and LPMC, and a considerableincrease after mitogen stimulation. Basal IL-6production by PBMC was higher in UC patients than incontrols [2029 pg/ml, CI₉ (–165 to4223) vs 572 pg/ml (–383 to 1527) respectively, P = 0.05] and also afterPWM activation [14,995 pg/ml (7759 -22230) vs 6598 pg/ml(3240-9956), respectively, P = 0.05]. In LPMC, nodifferences in IL-6 secretion were observed. TNF- in activated PBMC of patients with UC was notsignificantly increased in relation to control (P =0.09). No constitutive secretion of IFN- wasobserved in mononuclear cells. IFN- levelssecreted by activated LPMC were lower in patients withUC than in controls [1571 pg/ml (–108 to 3251) vs7953 pg/ml (3851-12,055), respectively, P = 0.03]. Theseresults suggest that IL-6, IL-1, and TNF- participate as mediators in the inflammatoryphenomena observed in UC. Further studies are necessaryto evaluate the role of IFN- in thiscondition.     

Expansion Conditions for Early Hepatic Progenitor Cells from Embryonal and Neonatal Rat Livers

Long-term primary cultures were established fromfetal or neonatal livers by using cell suspensionsdepleted of red blood cells and by culturing the cellsin hormonally defined medium containing dimethyl sulfoxide. Two distinct populations of hepaticprogenitor cells were evident in the cultures, based onmorphology, proliferative ability, and liver-specificgene expression. Most colonies consisted of immature hepatic progenitors: small, blastlike cells,weakly expressing alpha-fetoprotein, albumin, and-glutamyltranspeptidase, and showing evidence ofproliferation as measured by bromodeoxyuridineincorporation. At the perimeter of these colonies of immaturecells and forming some colonies by themselves were moremature hepatic progenitor cells: larger cells, withincreased cytoplasmic to nuclear ratios, little proliferation, and strongly expressing albumin,alpha-fetoprotein, and -glutamyltranspeptidase.The latter two proteins were localized to the bilecanalicular membranes of these cells. Glycogen deposits were present in the mature cells from day 14embryos after eight days of culture. Thus, DMSOtreatment of hepatic parenchymal progenitors provides anovel system for studies of liver development.     

Collagen–PCL Sheath–Core Bicomponent Electrospun Scaffolds Increase Osteogenic Differentiation and Calcium Accretion of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hASCs) are an abundant cell source capable of osteogenic differentiation, and have been investigated as an autologous stem cell source for bone tissue engineering applications. The objective of this study was to determine if the addition of a type-I collagen sheath to the surface of poly(ε-caprolactone) (PCL) nanofibers would enhance viability, proliferation and osteogenesis of hASCs. This is the first study to examine the differentiation behavior of hASCs on collagen–PCL sheath–core bicomponent nanofiber scaffolds developed using a co-axial electrospinning technique. The use of a sheath–core configuration ensured a uniform coating of collagen on the PCL nanofibers. PCL nanofiber scaffolds prepared using a conventional electrospinning technique served as controls. hASCs were seeded at a density of 20 000 cells/cm² on 1 cm² electrospun nanofiber (pure PCL or collagen–PCL sheath–core) sheets. Confocal microscopy and hASC proliferation data confirmed the presence of viable cells after 2 weeks in culture on all scaffolds. Greater cell spreading occurred on bicomponent collagen–PCL scaffolds at earlier time points. hASCs were osteogenically differentiated by addition of soluble osteogenic inductive factors. Calcium quantification indicated cell-mediated calcium accretion was approx. 5-times higher on bicomponent collagen–PCL sheath–core scaffolds compared to PCL controls, indicating collagen–PCL bicomponent scaffolds promoted greater hASC osteogenesis after two weeks of culture in osteogenic medium. This is the first study to examine the effects of collagen–PCL sheath–core composite nanofibers on hASC viability, proliferation and osteogenesis. The sheath–core composite fibers significantly increased calcium accretion of hASCs, indicating that collagen–PCL sheath–core bicomponent structures have potential for bone tissue engineering applications using hASCs.     

In Vitro Biocompatibility of New PVA-Based Hydrogels as Vitreous Body Substitutes

In order to synthesize injectable hydrogels suitable as vitreous body substitutes, a new method based on the use of trisodium trimetaphosphate (STMP) to cross-link PVA was recently proposed. Hydrogels with different molar ratios between STMP and PVA were realised. The aim of the present study was the evaluation of the biocompatibility of the different STMP/PVA hydrogels synthesised by analysing the effects of their in vitro interaction with cultures of mouse fibroblasts NIH3T3, primary human microvascular endothelial cells adult (HMVECad) and human lens cells. Cytotoxicity of hydrogels was first evaluated by analysing cell density and proliferation. Morphological and morphometric analysis of cell in contact with hydrogels was then performed using light microscopy and scanning electron microscopy, respectively. Moreover, cell adhesion and growth onto the hydrogels surface was evaluated and correlated to the amount of adsorbed proteins. At last, the biocompatibility of the sheared STMP/PVA 1:8 hydrogel was tested. The in vitro data of all the STMP/PVA hydrogels demonstrated their good biocompatibility, and indicated that the 1:8 sample was the most promising as vitreous body substitute.     

Improvement of Stem Cell Viability in Hyaluronic Acid Hydrogels Using Dextran Microspheres

Although hyaluronic acid (HA) has been widely used in clinics as an injectable biomaterial, it may not be appropriate as an injectable stem cell carrier because highly hydrophilic HA hydrogels provide an unfavorable environment in which the encapsulated stem cells are likely to be constrained to a round shape, thereby losing their native morphology. Herein, we hypothesized that dextran microspheres (DMs) can improve stem cell viability in HA hydrogels because they can act as substrates for stem cell adhesion, spreading and proliferation. DMs with a mean diameter of 80 μm were mixed with HA hydrogels. Human adipose-derived stem cells (hASCs) were isolated from human adipose tissue and seeded into the DM-incorporated HA hydrogels. When compared with the hydrogels alone, the number of viable cells was significantly increased in the presence of the DMs. Initially, hASCs appeared to be round in the HA hydrogels. At 12 h after seeding, the hASCs apparently attached onto the DMs and became slightly flattened. One day after seeding, the hASCs seemed to spread onto the surface of the DMs. Fluorescence micrography of live and dead cells confirmed that the cell viability was significantly improved by use of the DMs in HA hydrogels. Overall results demonstrated that the microsphere/hydrogel composite supported stem cell survival and spreading. These characteristics show the potential for use of the composite in cell-delivery and tissue-engineering applications.