Extracellular vesicles, exosomes and shedding vesicles in regenerative medicine - a new paradigm for tissue repair.

Tissue regeneration by stem cells is driven by the paracrine activity of shedding vesicles and exosomes, which deliver specific cargoes to the recipient cells. Proteins, RNA, cytokines and subsequent gene expression, orchestrate the regeneration process by improving the microenvironment to promote cell survival, controlling inflammation, repairing injury and enhancing the healing process. The action of microRNA is widely accepted as an essential driver of the regenerative process through its impact on multiple downstream biological pathways, and its ability to regulate the host immune response. Here, we present an overview of the recent potential uses of exosomes for regenerative medicine and tissue engineering. We also highlight the differences in composition between shedding vesicles and exosomes that depend on the various types of stem cells from which they are derived. The conditions that affect the production of exosomes in different cell types are deliberated. This review also presents the current status of candidate exosomal microRNAs for potential therapeutic use in regenerative medicine, and in applications involving widely studied organs and tissues such as heart, lung, cartilage and bone.

Orientation and conformation of anti-CD34 antibody immobilised on untreated and plasma treated polycarbonate.

The conformation and orientation of proteins immobilised on synthetic materials determine their ability to bind their antigens and thereby the sensitivity of the microarrays and biosensors employing them. Plasma immersion ion implantation (PIII) of polymers significantly increases both their wettability and protein binding capacity. This paper addresses the hypothesis that a PIII treated polymer surface modifies the native protein conformation less significantly than a more hydrophobic untreated surface and that the differences in surface properties also affect the protein orientation. To prove this, the orientation and conformation of rat anti-mouse CD34 antibody immobilized on untreated and PIII treated polycarbonate (PC) were investigated using ToF-SIMS and FTIR-ATR spectroscopy. Analysis of the primary structure of anti-CD34 antibody and principal component analysis of ToF-SIMS data were applied to detect the difference in the orientation of the antibody attached to untreated and PIII treated PC. The difference in the antibody conformation was analysed using deconvolution of the Amide I peak (in FTIR-ATR spectra) and curve-fitting. It was found that compared to the PIII treated sample, the antibody immobilized on the untreated PC sample has a secondary structure with a lower fraction of ß-sheets and a higher fraction of α-helices and disordered fragments. Also, it was found that anti-CD34 antibody has a higher tendency to occur in the inactive 'tail-up' orientation when immobilized on an untreated PC surface than on a PIII treated surface. These findings confirm the above hypothesis.

Surface characteristics and microbial adherence ability of modified polymethylmethacrylate by fluoridated glass fillers.

BACKGROUND: The current study objectives were to evaluate the influence of fluoridated glass fillers loading on the surface roughness, wettability, and adherence of candida and bacteria with and without saliva presence to a polymethylmethacrylate (PMMA) denture base material surface. METHODS: Four concentrations of fluoridated glass fillers were added to PMMA: 1%, 2.5%, 5% and 10% by weight pre-polymerization and 0% was the control. Discs of each concentration were fabricated (n = 5 for each variable). Surface roughness (Ra ) was measured using atomic force microscopy (AFM). Wettability was assessed by measuring the contact angle of a sessile drop of water. Specimens were incubated with Candida albicans, or Streptococcus mutans with and without saliva coating. Adherence was presented as a percentage of the colonized surface area, counted using an optical microscope at x100 magnification. RESULTS: The 10% group showed significantly greater roughness than the control and 1% groups; however, no significant differences in contact angle values were detected. The microbial adhesion was inversely proportional to the fluoridated glass fillers concentration where 10% concentration significantly decreased candidal and bacterial adhesion compared to others. Saliva coating significantly decreased microbial adhesion. CONCLUSIONS: It was concluded that fluoridated glass fillers could decrease microbial adhesion to acrylic denture base without adversely affecting surface properties.

Antibiotic eluting clay mineral (Laponite®) for wound healing application: an in vitro study.

Different materials in form of sponge, hydrogel and film have been developed and formulated for treating and dressing burn wounds. In this study, the potential of Laponite, a gel forming clay, in combination with an antimicrobial agent (mafenide), as a wound dressing material was tested in vitro. Laponite/mafenide (Lap/Maf) hydrogel was formulated in three different ratios of Lap/Maf 1:1, 1:2, 1:3. Laponite/mafenide/alginate (Lap/Maf/Alg) film was also formulated by combining Lap/Maf gel (1:1) with alginate. Intercalation rate of mafenide into the layers of Laponite nanoparticles and physico-chemical properties, including wound dressing characteristics of materials were studied using various analytical methods. Furthermore, the degradation of materials and the release profile of mafenide were investigated in simulated wound exudates fluid and antibacterial effectiveness of the eluted mafenide was tested on a range of bacterial species. The cytotoxicity of materials was also evaluated in skin fibroblast culture. The results showed that mafenide molecules were intercalated between the nano-sized layers of Laponite. The eluted mafenide showed active antibacterial effects against all three tested bacteria. All intercalated mafenide released from Lap/Maf 1:1 and 1:2 gel formulations and nearly 80% release from 1:3 formulation during test period. No significant difference was observed in release profile of mafenide between Lap/Maf/Alg film and Lap/Maf formulations. Wound dressing tests on Lap/Maf/Alg film showed it is a breathable dressing and has capacity to absorb wound exudates. The study showed that prepared Lap/Maf composite has the potential to be used as an antibiotic eluting gel or film for wound healing application. Additionally, Laponite has shown benefits in wound healing processes by releasing Mg(2+) ions and thereby reducing the cytotoxic effect of mafenide on fibroblast cells.

A mini-review on novel intraperiodontal pocket drug delivery materials for the treatment of periodontal diseases.

Periodontal disease is defined as chronic inflammatory condition characterized by the destruction of the periodontal tissues causing loss of connective tissue attachment, loss of alveolar bone, and the formation of pathological pockets around the diseased teeth. The use of systemic antibiotics has been advocated for its treatment, but concerns emerged with respect to adverse drug reactions and its contribution to bacterial resistance. Thus local drug delivery devices have been developed that aim to deliver a high concentration of antimicrobial drugs directly to the affected site, while minimizing drug's systemic exposure. A burst release of antimicrobial agent from carrier, resulting in a short and inadequate exposure of bacteria residing in periodontal pocket to the agent, remains the main challenge of current local delivery systems for the treatment of periodontal disease. This review aims to investigate and compare different local antimicrobial delivery systems with regard to the treatment of periodontal disease.

Biointerface: protein enhanced stem cells binding to implant surface.

The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment.

Biocompatible, smooth, plasma-treated nickel-titanium surface--an adequate platform for cell growth.

High nickel content is believed to reduce the number of biomedical applications of nickel-titanium alloy due to the reported toxicity of nickel. The reduction in nickel release and minimized exposure of the cell to nickel can optimize the biocompatibility of the alloy and increase its use in the application where its shape memory effects and pseudoelasticity are particularly useful, e.g., spinal implants. Many treatments have been tried to improve the biocompatibility of Ni-Ti, and results suggest that a native, smooth surface could provide sufficient tolerance, biologically. We hypothesized that the native surface of nickel-titanium supports cell differentiation and insures good biocompatibility. Three types of surface modifications were investigated: thermal oxidation, alkali treatment, and plasma sputtering, and compared with smooth, ground surface. Thermal oxidation caused a drop in surface nickel content, while negligible chemistry changes were observed for plasma-modified samples when compared with control ground samples. In contrast, alkali treatment caused significant increase in surface nickel concentration and accelerated nickel release. Nickel release was also accelerated in thermally oxidized samples at 600 °C, while in other samples it remained at low level. Both thermal oxidation and alkali treatment increased the roughness of the surface, but mean roughness R(a) was significantly greater for the alkali-treated ones. Ground and plasma-modified samples had 'smooth' surfaces with R(a)=4 nm. Deformability tests showed that the adhesion of the surface layers on samples oxidized at 600 °C and alkali treatment samples was not sufficient; the layer delaminated upon deformation. It was observed that the cell cytoskeletons on the samples with a high nickel content or release were less developed, suggesting some negative effects of nickel on cell growth. These effects were observed primarily during initial cell contact with the surface. The most favorable cell responses were observed for ground and plasma-sputtered surfaces. These studies indicated that smooth, plasma-modified surfaces provide sufficient properties for cells to grow.

Double-blind, placebo-controlled evaluation of extended-release bupropion in elderly patients with major depressive disorder.

Major depressive disorder in the elderly is associated with increased morbidity and reduced quality of life. This 10 week, placebo-controlled study investigated the efficacy and tolerability of extended-release bupropion (150-300 mg once daily) in depressed patients aged 65 years or older. The statistical assumptions necessary for the validity of the protocol-specified analysis of covariance were not met for the analysis of the primary outcome variable (Montgomery-Asberg Depression Rating Scale total score at Week 10, last observation carried forward). Alternative statistical methods used for the analysis of this variable demonstrated statistical significance. Statistically significant improvements were observed on the majority of secondary end points when compared with placebo, including the health outcome measures for motivation and energy, and life satisfaction and contentment. Adverse events were generally mild to moderate and similar between treatment groups. This study demonstrated that the extended-release bupropion is an effective, well-tolerated treatment for major depression in the elderly.

Controlled delivery of antimicrobial gallium ions from phosphate-based glasses.

Gallium-doped phosphate-based glasses (PBGs) have been recently shown to have antibacterial activity. However, the delivery of gallium ions from these glasses can be improved by altering the calcium ion concentration to control the degradation rate of the glasses. In the present study, the effect of increasing calcium content in novel gallium (Ga2O3)-doped PBGs on the susceptibility of Pseudomonas aeruginosa is examined. The lack of new antibiotics in development makes gallium-doped PBG potentially a highly promising new therapeutic agent. The results show that an increase in calcium content (14, 15 and 16 mol.% CaO) cause a decrease in degradation rate (17.6, 13.5 and 7.3 microg mm(-2) h(-1)), gallium ion release and antimicrobial activity against planktonic P. aeruginosa. The most potent glass composition (containing 14 mol.% CaO) was then evaluated for its ability to prevent the growth of biofilms of P. aeruginosa. Gallium release was found to reduce biofilm growth of P. aeruginosa with a maximum effect (0.86 log(10) CFU reduction compared to Ga2O3-free glasses) after 48 h. Analysis of the biofilms by confocal microscopy confirmed the anti-biofilm effect of these glasses as it showed both viable and non-viable bacteria on the glass surface. Results of the solubility and ion release studies show that this glass system is suitable for controlled delivery of Ga3+. 71Ga NMR and Ga K-edge XANES measurements indicate that the gallium is octahedrally coordinated by oxygen atoms in all samples. The results presented here suggest that PBGs may be useful in controlled drug delivery applications, to deliver gallium ions in order to prevent infections due to P. aeruginosa biofilms.

Eight-week, placebo-controlled, double-blind comparison of the antidepressant efficacy and tolerability of bupropion XR and venlafaxine XR.

The efficacy, safety and tolerability of bupropion XR and venlafaxine XR was assessed and compared with placebo in adult outpatients with major depressive disorder (MDD). Adults meeting DSM-IV criteria for MDD with a minimum Hamilton Depression Rating Scale (HAMD) 17-Item total score of > or =18 were randomized to eight weeks of double-blind treatment with either bupropion XR (150 mg/day), venlafaxine XR (75 mg/day) or placebo. At the end of the fourth week of treatment, a dosage increase to bupropion XR 300 mg/day or venlafaxine XR 150 mg/day was allowed if, in the opinion of the investigator, response was inadequate. The primary efficacy endpoint was mean change from baseline at week 8 in the Montgomery-Asberg Depression Rating Scale (MADRS) total score last observation carried forward (LOCF). Mean changes from baseline at week 8 (LOCF) in MADRS total score were statistically significant for bupropion XR and venlafaxine XR patients compared to the placebo group: -16.0 for bupropion XR (P = 0.006 vs placebo), -17.1 for venlafaxine XR (P < 0.001 vs placebo) and -13.5 for placebo. Secondary outcomes (including CGI-S, HAM-A, MEI, Q-LES-Q-SF, responder and remitter analyses) also improved significantly for both active treatment groups compared with placebo. The most frequently reported adverse events were dry mouth and insomnia for bupropion XR, and nausea, hyperhidrosis, fatigue, and insomnia for venlafaxine XR. In this double-blind, placebo-controlled trial, bupropion XR at doses up to 300 mg/day and venlafaxine XR at doses up to 150 mg/day demonstrated comparable antidepressant efficacy.

Surface preparation of bioactive Ni-Ti alloy using alkali, thermal treatments and spark oxidation.

The primary aim of this study was to compare different surface treatments used for bioactivation of pure titanium surfaces--thermal, alkali treatment and spark oxidation, and to assess their suitability as treatments for Ni-Ti alloys. This was considered by examining the surface properties, calcium phosphate precipitation from a physiological solution, and nickel ion release. Additionally, changes in the transformation temperature were measured for thermally treated samples. These studies indicate that the native surface of Ni-Ti alloy is highly bioactive when assessing the precipitation of calcium phosphates from Hank's solution. Low temperature heat treatments also produced promising surfaces while high temperature treatment resulted in a very low rate of Ca and P precipitation. Alkali treatment and spark oxidation resulted in some bioactivity. Nickel ion release was greatest for alkali treated and sparks oxidized samples, and the rate of its release from these two samples was on the verge of daily safe dose for adolescent human. The other analyzed samples revealed very low rates of nickel ion release. Heat treatment at 400 degrees C resulted in significant increase in the transformation temperatures, and a further increase of the treatment temperature up to 600 degrees C caused a drop of the transformation temperature.

Vitreoretinal surgery in diabetic patients on hemodialysis.

The present paper reports our first results after pars plana vitrectomy in patients with diabetic retinopathy and hemodialysis with a follow-up of 6 to 24 months. Between January 1992 and October 1994 we performed vitreoretinal surgery with silicone oil tamponade in nine eyes of seven patients with diabetic nephropathy on hemodialysis. All patients had had type I diabetes for 19-32 years. Over the observation period the retina was completely attached in eight eyes. Final visual acuity of 0.1-0.7 was attained in four eyes, 0.06 two, hand movements in one eye. Two eyes had no useful final visual acuity because of redetachment of the retina or secondary glaucoma with rubeosis iridis. The small number of complications shows that pars plana vitrectomy can be done in diabetic patients with nephropathy on hemodialysis. This significantly improves their quality of life.

[Results of two year use of dipyridamole and metindol in patients with primary focal and (or) segmental glomerulosclerosis]. / Wyniki dwuletniego stosowania dipirydamolu i metindolu w leczeniu chorych na pierwotne, ogniskowe i (albo) segmentalne szkliwiejace klebkowe zapalenie nerek.

Primary focal or segmental glomerulosclerosis is neuropathy diagnosed in 10% of children and 20% of adult patients with nephrotic syndrome. Immunosuppressive therapy was applied during two years period. The very good clinical results were obtained in 20% and good results in 40% of patients after indomethacin and dipyridamole therapy.

[Immune complexes and C3 and C4 of the complement in urine of patients with glomerulonephritis]. / Kompleksy immunologiczne oraz C3 I C4 dopelniacza w moczu chorych na klebuszkowe zapalenia nerek.

The circulating immune complexes (CIC), urinary immune complexes (UIC), C3 and C4 in urine (UC3 and UC4) in 99 patients with immunologic glomerular diseases: 13 with extracapillaris GN (ExGN), 38 with membranoproliferative GN (MPGN), 33 with mesangial proliferative GN (MesPGN), 5 with focal segmental glomerulosclerosis (FSGS), 5 with membranous nephropathy (MN), and 3 with minimal change nephropathy (MC) were investigated in the study. Depending on the (IS) immunosuppressive treatment all patients were classified into 3 groups. Group I: 61 recently biopsied patients with glomerular disease consisted of patients attending our renal department, group II: 24 patients with biopsy proven glomerular disease IS treated in the past but with GN with restrained disease activity, group III: 14 patients with active glomerulopathy who have been treated for some months. Nephelometry C1q binding test was used for CIC detection and 3.5% polyethylene glycol precipitate for its detection in urine by C1q binding test was applied. Radial immunodiffusion (NANORID) method was used for urine C3 and C4 detection. UC3 were detected in urine from 37% of all patients: in 39% patients of group I (GN at time of diagnosis), 38% of group II (GN with restrained disease activity) and 36% of group III (active GN received immunosuppressive therapy for several months). It suggest that nonimmunological-mechanism induce C3 detected in the rine of such patients. According to histological findings UC3 was detected in 3 patients with ExGN of group I, in patient with ExGN of group II and in about half patients with MPGN from group I and II, in about 25% patients with MesPGN from group I and II. About half patients with MesPGN of group III, one patient with MPGN of group III and a few patients with other histological findings of group I were UC3 positive. Simultaneous excretion of C4 in urine was detected in some UC3 positive patients (in about 5% patients). At the same time in about 50% UC3 positive patients was observed urinary excretion of IC. CIC and UIC were detected in 25% of all patients: in 29% of group I, in 17% of group II, and in 29% patients of group III. According to histological findings CIC was detected in 3 patients with ExGN, in 7 patients with MPGN from 26 of group I, and 3 from 7 with MPGN of group II, and 3 patients with MesPGN of group I and 1 patients with MesPGN of group II, and 33% patients with MesPGN of group III with high proteinuria. These findings suggested that urinary IC reflected the immunological activity of glomerulopathy and their presence in patients urine after IS treatment suggests incomplete response to this therapy while urinary C3 and C4 were connected with urinary protein excretion and may be of importance in tubulointerstitial injury and progression of renal insufficiency.

[Tumor necrosis factor (TNF) and interleukin-6 (IL-6) in patients with glomerulonephritis]. / Czynnik martwicy nowotworu (TNF) i interleukina-6 (IL-6) w klebuszkowych zapaleniach nerek.

Serum and urinary tumor necrosis factor-alpha (SeTNF and UTNF) and interleukin-6 (SeIL-6 and UIL-6) by ELISA method were determined in 99 patients with glomerulonephritis (GN): 13 with extracapillaris GN (ExGN), 38 with membranoproliferative GN (MPGN), 33 mesangial proliferative GN (MesPGN), 5 with focal segmental glomerulosclerosis (FSGS), 5 with membranous nephropathy (MN), 3 with minimal change nephropathy (MC) and in 32 healthy adults. The higher levels of Se TNF than those in the healthy were in 25 patients: in nearly all with ExGN, in 8 with MPGN and in single patients with other GN. In all patients with high SeTNF were many extra renal organs involvement. Measurable levels of UTNF were in 30 patients (30%) (in 12 with ExGN, 9 with MPGN, 7 with MesPGN, 1 with MN, and 1 with FSGS). Most patients with high SeTNF belonged to group I. The higher levels of SeIL-6 than those in healthy were in 17 patients belonging to group I, in which high SeIL-6 were in 3 patients with ExGN, 6 with MPGN, 3 with MesPGN, 2 with MN, and 3 with FSGS. Measurable urinary IL-6 levels were in 27 (27%) patients, mainly in group I, and in single patients in other groups. The majority of patients with ExGN and MPGN from group I and UIL-6 positive suffered from renal insufficiency and histologically had proliferative GN. We conclude that the elevation of TNF alpha and/or IL-6 in plasma may reflect a secondary consequence of immune cells activation while urinary TNF alpha and/or IL-6 may be secreted by activated glomerular cells. Thus, high levels of TNF alpha and/or IL-6 in serum of patients with GN and extra renal organs involvement, peculiary with infections, suggested antibiotics therapy, because infection may stimulated cytokines production and they are important in pathogenesis and progress of GN. High urinary levels of IL-6 and (or) TNF alpha in patients with proliferative GN suggest great disease activity and is useful in the evaluating of IS treatment.

[Anti-neutrophil cytoplasmic antibodies (ANCA) in the glomerulonephritis]. / Przeciwciala przeciw cytoplazmie granulocytów obojetnochlonnych (ANCA) w klebuszkowych zapaleniach nerek.

Ninety nine patients with biopsy-proven diagnosis of immunologic glomerular disease were included in this study: 13 with extracapillaris glomerulonephritis (ExGN), 38 with membranoproliferative GN (MPGN), 33 with mesangial proliferative GN (MesPGN), 5 with focal segmental glomerulosclerosis (FSGS), 5 with membranous nephropathy (MN), and 3 with minimal change nephropathy (MC). Sera of these patients were tested for the presence of anti-neutrophil cytoplasmic antibodies (ANCA) by standard indirect immunofluorescence (IIF). Specific antiproteinase 3 (anti-Pr 3) and anti-myeloperoxidase (anti-MPO) by ELISA were assayed in ANCA-positive patients. ANCA were detected by IIF and ELISA in sera from 28 patients (28%). Of the 28 patients, 3 had cytoplasmic ANCA (c-ANCA), while 25 had perinuclear pattern (p-ANCA). Two c-ANCA positive patients with anti-Pr3 belonged to group I (GN at time of diagnosis). Another c-ANCA positive patients suffering from GN belonged to group II (GN with restrained disease activity). Of the 25 p-ANCA positive patients, 23 belonged to group I, 1 to group II, and I to group III (active GN received immunosuppressive therapy for several months). Majority of ANCA positive patients with GN were complicated by extrarenal organs involvement. One c-ANCA positive patient had ExGN and two patients had MesPGN. Of the 25 p-ANCA positive patients, 7 had ExGN, 10 MPGN, 4 MesPGN, 2 FSGS, and 1 had MC. Five p-ANCA positive patients with ExGN were temporary dialysis-dependent. One of them died due to cardiac infarction, one became dialysis-dependent, there of them (treated also with plasma exchange) responded favorably to this treatment and after 3 to 12 months had life-sustaining renal function (s-creatinine < 265 umol/L; < 3 mg%). Renal-limited disease had 3 p-ANCA positive patients (anti-MPO) with ExGN another ANCA positive patient had extrarenal organs involvement. The majority of patients with low titer of ANCA by IIF and low concentration p-ANCA by ELISA had different morphological GN, in most cases MPGN. All c-ANCA positive patients had extrarenal organs involvement, mainly respiratory tract. One of them was dialysis-dependent, and two responded favorably to immunosuppressive therapy. It is concluded that the IIF and ELISAs for anti-Pr3, and anti-MPO have an acceptable performance and are useful in the diagnostic of patients with vasculitis. Vasculitis can be primary in patients with pauci-immune crescentic GN, or secondary to other disease processes including immune complex-mediated GN. IS treatment (methylpredisolone + cyclophospamide) of ANCA positive patients with renal insufficiency nad normal or enlarged renal size is favourable. In the patients with extrarenal organs involvement and without improvements in renal function, however, requirement of plasma exchange is considered.

[A rare case of renal osteodystrophy as a consequence of end stage renal failure and secondary hyperparathyroidism]. / Rzadki przypadek osteodystrofii nerkowej w przebiegu krancowej niewydolnosci nerek i wtórnej nadczynnosci przytarczyc.

A case of rare facial dysplasia due to renal osteodystrophy of end stage renal failure is described in a 19-year old boy treated with chronic haemodialyses. The favourable effects of surgical correction are shown.