Preclinical In Vivo Safety of Poly-Phosphorylated Superlubrication Vectors for the Treatment of Osteoarthritis.

Osteoarthritis (OA) can lead to a significant functional disability. Poly[2-(methacryloyloxy)ethyl phosphorylcholine] (pMPC) liposomes are a novel treatment modality for OA, intended to restore the natural lubrication properties of articular cartilage. Here, we report on two studies aimed to assess the local and systemic safety and toxicity of pMPCylated liposomes in comparison with physiological saline, in Sprague-Dawley (SD) rats and in sheep after a single intra-articular (IA) injection. The animals were sacrificed after 1 and 6 weeks (rats) and 3 and 6 weeks (sheep). No signs of toxicity or abnormal clinical findings were observed. Histopathological evaluation revealed no signs of reactivity or abnormal findings in the injected joints or in any other organs. In conclusion, a single IA injection of the pMPCylated liposomes demonstrated an excellent safety profile and did not result in local reactivity or systemic toxicity, thus supporting its further development for use in humans.

Percutaneous Left Ventricular Assist Device Leads to Heart Rhythm Stabilisation in Cardiogenic Shock: Results from the Dresden Impella Registry.

BACKGROUND: Severe heart rhythm disturbances (SHRDs) occur regularly in cardiogenic shock (CS). Percutaneous left ventricular assist devices (pLVADs) can actively unload the left ventricle (LV), decreasing left ventricular end-diastolic pressure and wall tension, which are suspected parameters for the induction and maintenance of arrhythmias. The aim of this study was to describe effects of LV unloading on SHRD. METHOD: In the Dresden Impella Registry, 97 patients received an Impella CP in refractory CS. Of them, 19 had SHRDs, which were not stopped by common therapeutic strategies such as electrical defibrillation or antiarrhythmic drugs. They were only stopped after implantation of a micro-axial heart pump. This phenomenon was referred to as heart rhythm stabilisation (HRS). Clinical outcome and laboratory parameters were assessed and risk factors for the occurrence of HRS were identified. RESULTS: All 19 patients with refractory SHRD terminated immediately into a stable heart rhythm after insertion of the micro-axial heart pump. In 37% no additional defibrillation was needed. Of the patients with HRS, CS was mostly caused by myocardial infarction (68%). Resuscitation before pLVAD was performed in 89% for more than 30 minutes. Patients with HRS were resuscitated more frequently and for a longer duration than patients without HRS. After HRS, the serum lactate and norepinephrine dosage decreased in the first 12 hours, whereas left ventricular ejection fraction increased by 95%. CONCLUSIONS: Left ventricular unloading in patients with CS seems to be an option for treating patients with sustained life-threatening tachycardia, who are refractory to common treatment.

Evidence for laser-induced homogeneous oriented ice nucleation revealed via pulsed x-ray diffraction.

The induction of homogeneous and oriented ice nucleation has to date not been achieved. Here, we report induced nucleation of ice from millimeter sized supercooled water drops illuminated by ns-optical laser pulses well below the ionization threshold making use of particular laser beam configurations and polarizations. Employing a 100 ps synchrotron x-ray pulse 100 ns after each laser pulse, an unambiguous correlation was observed between the directions and the symmetry of the laser fields and that of the H-bonding arrays of the induced ice crystals. Moreover, an analysis of the x-ray diffraction data indicates that, in the main, the induced nucleation of ice is homogeneous at temperatures well above the observed and predicted values for supercooled water.

Lubrication of Articular Cartilage.

The major synovial joints such as hips and knees are uniquely efficient tribological systems, able to articulate over a wide range of shear rates with a friction coefficient between the sliding cartilage surfaces as low as 0.001 up to pressures of more than 100 atm. No human-made material can match this. The means by which such surfaces maintain their very low friction has been intensively studied for decades and has been attributed to fluid-film and boundary lubrication. Here, we focus especially on the latter: the reduction of friction by molecular layers at the sliding cartilage surfaces. In particular, we discuss such lubrication in the light of very recent advances in our understanding of boundary effects in aqueous media based on the paradigms of hydration lubrication and of the synergism between different molecular components of the synovial joints (namely hyaluronan, lubricin, and phospholipids) in enabling this lubrication.

Effect of glucosamine sulfate on surface interactions and lubrication by hydrogenated soy phosphatidylcholine (HSPC) liposomes.

Glucosamine sulfate (GAS) is a charged monosaccharide molecule that is widely used as a treatment for osteoarthritis, a joint disease related to friction and lubrication of articular cartilage. Using a surface force balance, we examine the effect of GAS on normal and, particularly, on shear (frictional) interactions between surfaces in an aqueous environment coated with small unilamellar vesicles (SUVs), or liposomes, of hydrogenated soy phosphatidylcholine (HSPC). We examine the effect of GAS solution, pure water, and salt solution (0.15 M NaNO3) both inside and outside the vesicles. Cryoscanning electron microscopy shows a closely packed layer of liposomes whose morphology is affected only slightly by GAS. HSPC-SUVs with encapsulated GAS are stable upon shear at high compressions (>100 atm) and provide very good lubrication when immersed both in pure water and physiological-level salt solutions (in the latter case, the liposomes are exceptionally stable and lubricious up to >400 atm). The low friction is attributed to several parameters based on the hydration lubrication mechanism.

Observed changes in the morphology and phenotype of breast cancer cells in direct co-culture with adipose-derived stem cells.

BACKGROUND: Regarding aesthetics and long-term stability, cell-assisted lipotransfer is a promising method for breast reconstruction. Here, autologous fat grafts enriched with autologous adipose-derived stem cells are transferred. However, as adipose-derived stem cells secrete high amounts of growth factors, potential risks of tumor reactivation remain. In this study, influences of adipose-derived stem cells on inflammatory breast cancer cells were evaluated in a direct co-culture system. METHODS: Human adipose-derived stem cells were isolated and cultivated either alone or in a direct co-culture with the inflammatory breast carcinoma cell line T47D. At different time points, cell morphology was observed by scanning electron microscopy, cell membranes were stained by immunofluorescence, and gene expression was analyzed by real-time polymerase chain reaction. RESULTS: In co-cultures, T47D breast carcinoma cells showed tumorsphere-typical growth surrounded by a monolayer of adipose-derived stem cells. Direct cell-to-cell contacts could be observed between the two different cell types. Immunofluorescence revealed vesicular exchange and fusion between carcinoma cells and adipose-derived stem cells. Expression levels of transcriptional genes for typical malignancy markers were substantially higher in co-cultures compared with single cultures. CONCLUSIONS: Direct intercellular contact between carcinoma cells and adipose-derived stem cells by means of exosomal vesicular exchange was revealed. Breast cancer cells displayed a change towards a more malignant phenotype associated with higher rates of metastasis and worsened prognosis. As cell-assisted lipotransfer is often performed after breast cancer surgery, transfer of adipose-derived stem cells might lead to deterioration of prognosis in case of recurrence as it has been described for inflammatory breast cancer.

Murine embryonic fibroblast cell lines differentiate into three mesenchymal lineages to different extents: new models to investigate differentiation processes.

Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation.

Cultivation of keratinocytes and fibroblasts in a three-dimensional bovine collagen-elastin matrix (Matriderm®) and application for full thickness wound coverage in vivo.

New skin substitutes for burn medicine or reconstructive surgery pose an important issue in plastic surgery. Matriderm® is a clinically approved three-dimensional bovine collagen-elastin matrix which is already used as a dermal substitute of full thickness burn wounds. The drawback of an avital matrix is the limited integration in full thickness skin defects, depending on the defect size. To further optimize this process, Matriderm® has also been studied as a matrix for tissue engineering of skin albeit long-term cultivation of the matrix with cells has been difficult. Cells have generally been seeded onto the matrix with high cell loss and minimal time-consuming migration. Here we developed a cell seeded skin equivalent after microtransfer of cells directly into the matrix. First, cells were cultured, and microinjected into Matriderm®. Then, cell viability in the matrix was determined by histology in vitro. As a next step, the skin substitute was applied in vivo into a full thickness rodent wound model. The wound coverage and healing was observed over a period of two weeks followed by histological examination assessing cell viability, proliferation and integration into the host. Viable and proliferating cells could be found throughout the entire matrix. The presented skin substitute resembles healthy skin in morphology and integrity. Based on this study, future investigations are planned to examine behaviour of epidermal stem cells injected into a collagen-elastin matrix under the aspects of establishment of stem cell niches and differentiation.

TRPV channel expression in human skin and possible role in thermally induced cell death.

Cell death via necrosis and apoptosis is a hallmark of deep dermal to full-thickness cutaneous burn injuries. Keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature via heat-gated transient receptor potential vanilloid (TRPV) ion channels. The aim of this study was to investigate the distribution of TRPV1, 2, 3, and 4 in uninjured and thermally burned skin. The authors investigated warmth-evoked currents in keratinocytes and cell kinetics of thermally injured keratinocytes in culture with agonists and antagonists of TRPV channels. Specimens of uninjured normal skin and discarded tissue of thermally injured skin were stained for TRPV1, 2, 3, and 4. Cultured primary human keratinocytes were heated for 5 minutes at the following temperatures: 37°C (control), 42°C, and 60°C and thereafter cultured for 24 or 48 hours at 37°C. Thermally stressed cells were treated with TRPV antagonists capsazepine or ruthenium red, and cell viability capacity was determined. TRPV1, TRPV2, TRPV3, and TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin. Patch clamp analysis showed a functional response of human keratinocytes at temperatures >40°C. Cell death of keratinocytes after heating at 42°C was reduced by 15 and 5% with ruthenium red and by 20 and 30% by capsazepine at 24 and 48 hours, respectively. Cell death after treatment at 60°C was significantly reduced at 24 hours with capsazepine (22%) or ruthenium red (18%) but only minimally affected after 48 hours postinjury. Interaction with TRPV channels on keratinocytes may offer a new strategy to counteract cell death after thermal injury.

DNA-mediated two-dimensional colloidal crystallization above different attractive surfaces.

We explore the formation of "floating" two-dimensional colloidal crystals above weakly attractive surfaces that are either positively or negatively charged. In particular, we studied crystal formation above positively charged poly-L-lysine-poly(ethylene glycol) surfaces with and without short single-stranded DNA and above negatively charged bovine albumin serum-streptavidin multilayers. Confocal microscopy revealed the evolution of crystals several micrometers above all three surfaces. Interestingly, the "flying height" of crystals was found to depend on the surface coating. All crystalline structures remained remarkably stable over weeks, even under high salt conditions. Neither lifting the crystals nor lowering them by means of buoyancy forces destroyed them.

Direct observation of size fractionation during colloidal crystallization.

We present a confocal microscopy study of the quasi-two-dimensional crystallization of a binary mixture of spherical colloids coated with long DNA strands. Our experiments show that in the crystalline phase the two colloidal species are completely demixed. Analysis of the lattice spacings in the two types of colloidal crystal shows that the diameters of the two species of colloids differ by 10%. We argue that the demixing in the crystalline phase is due to size segregation during crystallization. This phenomenon had been predicted in several theoretical studies. To our knowledge, the present study provides the first 'real-space' experimental confirmation of this effect.

Percutaneous collagen induction: minimally invasive skin rejuvenation without risk of hyperpigmentation-fact or fiction?

BACKGROUND: Photoaging is generally treated by ablative procedures that injure the epidermis and basal membrane and lead to fibrosis of the papillary dermis. Damaging the epidermis significantly can cause potential adverse effects such as dyspigmentation. It was recently shown in clinical trials that percutaneous collagen induction therapy is an alternative for safely treating wrinkles and scars and for smoothening the skin without the risk of dyspigmentation. METHODS: The purpose of this study was to increase current knowledge regarding whether percutaneous collagen induction therapy presents an effective means for skin rejuvenation without risk of dyspigmentation, as the authors' clinical data suggested. Fifty-six rats were assigned to three groups: group A (n = 24), percutaneous collagen induction therapy plus skin care; group B (n = 24), skin care; and group C (n = 8) controls. The authors evaluated the effect of percutaneous collagen induction therapy on the epidermis, melanocytes, and the pigmentation markers interleukin-10 and melanocyte-stimulating hormone. RESULTS: Percutaneous collagen induction therapy left the epidermis intact without any damage to the stratum corneum, any other layers of the epidermis, or the basal membrane. No signs of dermabrasive reduction of epidermal thickness were evident 24 hours after the procedure. The number of melanocytes neither increased nor decreased in any of the groups. DNA microarray experiments demonstrated that interleukin-10 was increased in percutaneous collagen induction therapy-treated skin after 2 weeks. Concerning the MC1R (melanocyte-stimulating hormone) gene, gene expression microarray analysis indicated a faint down-regulation both 24 hours and 2 weeks after percutaneous collagen induction therapy. CONCLUSION: Percutaneous collagen induction therapy offers a modality with which to rejuvenate and improve skin appearance and quality without risk of dyspigmentation.