Salispheres from Different Major Salivary Glands for Glandular Regeneration.

Dysfunctional salivary glands (SGs) are a clinical challenge due to the lack of effective treatments. Cell therapy with stem/progenitor cells may improve this situation by providing promising therapeutic solutions. Therefore, exploring abundant cellular sources is important. Three major pairs of SGs are located in different anatomic regions: the parotid glands, the submandibular glands, and the sublingual glands. Although SG stem/progenitor cells can be isolated and cultivated from all major SGs as salispheres, the differences among SG origins remain unclear. In this study, salispheres were successfully isolated from all major SGs. The salispheres demonstrated unique cellular features that originated from their native tissues. The characteristic expression profiles and cellular features of SG stem cells were demonstrated in all salispheres. When they were transplanted into irradiated animals, the salispheres were all capable of improving the saliva secretion that was disrupted by irradiation. Typical histologic structures could be observed in most parts of the treated glands, and the fibrotic environments of irradiated submandibular glands were remodeled by all salispheres regardless of origins. This study characterized the cellular features and in vivo effects of salispheres that were derived from different anatomic origins. The results suggest the possibility of functional redundancy among distinct pairs of major SGs, which is useful for the design of cell therapy to treat dysfunctional glandular organs.

Chitosan films promote formation of olfactory neurospheres and differentiation of olfactory receptor neurons.

BACKGROUND: Olfactory dysfunction significantly impairs the life quality of patients. Therefore, a model needs to be developed for anosmia. Chitosan is a biodegradable natural polysaccharide that has been widely studied for regenerative purposes in the nervous system. However, whether chitosan promotes differentiation of olfactory receptor neurons or regulates formation of neurospheres in the olfactory system remains unexplored. METHODOLOGY: Olfactory neuroepithelial cells were isolated from embryonic wistar rats on day 17, and cultured on controls and chitosan films for 12 days. The effects of treatment were assessed using immunocytochemistry, quantitative polymerase chain reaction and western blots following culturing. The substrate of poly-L-lysine-co-laminin was adopted as a control. RESULTS: In contrast to the flat layer on controls, olfactory neuroepithelial cells form olfactory neurospheres on chitosan films with steadily increasing diameter. The olfactory neurospheres contain basal cells, as well as immature and mature olfactory receptor neurons. The expression level of olfactory marker protein is higher on chitosan films than those on controls in gene and protein levels, and the olfactory transduction elements also express a similar trend. Mature olfactory receptor neurons are found predominantly at the periphery of the olfactory neurospheres. CONCLUSIONS: Chitosan films not only facilitate formation of olfactory neurospheres, but also promote differentiation of olfactory receptor neurons. Chitosan is a potential biomaterial to establish an in vitro culture model to treat olfactory dysfunction in future.

Effects of the surface characteristics of nano-crystalline and micro-particle calcium phosphate/chitosan composite films on the behavior of human mesenchymal stem cells in vitro.

Calcium phosphate (CaP) compounds, the main inorganic constituent of mammalian bone tissues, are believed to support bone precursor cell growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the present study, different scales of CaP crystals on chitosan membranes were prepared for culture of human mesenchymal stem cells (hMSCs) in vitro. A series of aqueous CaP suspensions with different concentrations were mixed with chitosan solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. With different weight ratios of CaP in chitosan solution, the various surface characteristics of nano-amorphous (C/CaP 0.1), nano-crystalline (C/CaP 0.5) and micro-particle (C/CaP 2) CaP compounds were examined by scanning electron microscopy and electron dispersion spectroscopy. X-ray diffraction on micro-particles of CaP indicated the formation of crystalline hydroxyapatite. The behavior of hMSCs, including proliferation, cell spreading and osteogenic differentiation, was studied on the C/CaP films. In basal culture medium, the incorporation of CaP into chitosan films could promote the proliferation of hMSCs. The C/CaP 0.5 film with connected CaP nano-crystals had better cellular viability. The fluorescence microscope images at 14 days of culture revealed extensive networks of F-actin filaments of hMSCs on chitosan, C/CaP 0.1 and C/CaP 0.5 films. The cellular morphology on C/CaP 2 film with discrete CaP micro-particles was partly restrained. In osteogenic medium, the alkaline phosphatase (ALP) activity of hMSCs increased and showed the process of osteogenic differentiation. The ALP levels on C/CaP 2 film were higher than those on C/CaP 0.1 and C/CaP 0.5 films. These results demonstrated that the crystallinity and topography of CaP on chitosan membranes could modulate the behaviors of cultured hMSCs in vitro.

Quantitative rat liver function test by galactose single point method.

The purpose of this study was to investigate the galactose single point (GSP) method, a residual liver function test recently recommended by the US Food and Drug Administration, which can be a useful tool for rat liver function measurement. Rats were treated either with carbon tetrachloride (CCl(4)) alone (1 mL/kg, intraperitoneally [i.p.]) for one day or with isoniazid (INH) alone (150 mg/kg, i.p.) or (in order to ameliorate the effects of INH) with a combination of INH and bis-p-nitrophenyl phosphate (BNPP) (25 mg/kg, i.p.) for 21 days. Hepatotoxicity was assayed by plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and scores of histological activity index-necroinflammation (HAI-NI) of the respective liver specimens. The GSP method in rats was defined by the galactose blood level after 60 min. Significant differences in GSP values were observed between controls and the CCl(4)-treated rats. After 21 days of treatment, no significant changes in AST and ALT values were observed among the control, INH and INH-BNPP groups. There were significant differences in average GSP values for controls (P < 0.001) and INH-BNPP (P < 0.001) compared with INH alone. Highly significant correlations (P < 0.001) were obtained between GSP and scores of HAI-NI for all the groups. GSP was concluded to be a more sensitive biomarker of INH-induced hepatotoxicity than AST or ALT in the rats. The GSP method has been proved to be a simple and useful tool for the quantitative determination of liver function in rats, which can possibly be extended to other animals.

Preparation of EVAL membranes with smooth and particulate morphologies for neuronal culture.

In this work, the in vitro interaction of cerebellar granule neurons prepared from 7-day-old Wistar rats and poly ethylene-co-vinyl alcohol (EVAL) membranes was investigated. Cells were cultured in smooth and particulate EVAL membranes for up to 7 days. Particulate membranes were prepared by using 1-octanol to precipitate EVAL solutions in DMSO. Such a membrane was microporous characterized by a packed bed of particles. Voids left between the aggregated particles formed a continuous and interconnected porous network. Crystallization of the EVAL polymer induced by 1-octanol is responsible for the formation of particulate morphology. The membrane structure and its relationship with cells were examined by scanning electron microscopy and the MTT assay. It was observed that the particulate membrane was more favorable for the neuron culture than the smooth membrane. Neurons seeded on the particulate membrane were able to regenerate with formation of an extensive neuritic network. Therefore, the particulate structure may spatially mediate cellular response that can promote neuronal cell attachment, differentiation and neuritic growth, indicating that the particulate structure should be useful as a new polymer scaffold for nerve repair.

Endoscopic comparison of the gastroduodenal safety and the effects on arachidonic acid products between meloxicam and piroxicam in the treatment of osteoarthritis.

Our objective was to evaluate the efficacy, the gastroduodenal safety, and the effects on arachidonic acid products of meloxicam, a new acidic enolic non-steroidal anti-inflammatory drug which preferentially inhibits cyclo-oxygenase-2 over cyclo-oxygenase-1, versus piroxicam in patients with osteoarthritis of the knee. Meloxicam 7.5 mg or piroxicam 20 mg daily was administered for 4 weeks in this double-blind parallel-groups randomised study. The efficacy for pain relief of the two tested medications was assessed by means of visual analogue scale and other clinical parameters. Pre- and post-treatment endoscopies were performed, and the findings were scored and recorded. The gastric fluid was aspirated at each time and prostaglandin E2, thromboxane B2 and leukotriene B4 were determined by ELISA. There was no significant difference between the groups regarding the primary efficacy. Changes in endoscopic findings by means of Lanza score showed statistically significant differences between the two treatment groups in favour of meloxicam at all sites--gastric, duodenal and total. Within-group comparisons showed a statistically significant difference (worsening) in gastric and total score with piroxicam, but no significant difference with meloxicam. The frequency of clinically relevant cases (total score >2) also showed a statistically significant worsening in the piroxicam group. The better GI tolerability of meloxicam was also suggested by fewer adverse GI events and no withdrawals due to adverse events compared with piroxicam. The pre-/post-study gastric juice concentration of PGE2, TXB2, and LTB4 in the meloxicam group was 135.2 +/- 85.8/71.2 +/- 32.2, 116.3 +/- 81.7/99.4 +/- 107.5 and 388 +/- 321/223 +/- 98 pg/ml respectively. The pre-/post-study gastric juice concentration of PGE2, TXB2 and LTB4 in the piroxicam group was 105.7 +/- 43.1/68.2 +/- 34.9, 94.0 +/- 50.9/105.9 +/- 121.1 and 625 +/- 1574/828 +/- 1464 pg/ml, respectively. Both meloxicam and piroxicam significantly inhibited gastric PGE2 levels after 4 weeks' treatment; however, there was no difference between these two groups. Neither of these medications had an effect on TXB2. Only meloxicam inhibited LTB4 concentration significantly, and the between-groups difference was significant. Meloxicam 7.5 mg once daily had better gastrointestinal tolerability and an efficacy comparable to that of piroxicam 20 mg over 4 weeks in patients with osteoarthritis of the knee.

Studies on the effect of surface properties on the biocompatibility of polyurethane membranes.

To study the effect of surface properties on the biocompatibility of biomaterials based on the same material, polyurethane membranes with different surface properties were prepared. Myoblast culture and interleukin-1 (IL-1) generation in an air pouch model and in vitro monocyte culture were used to examine biocompatibility of different polyurethane membranes. Polyurethane membranes were found to exhibit significant differences depending on their surface properties prepared by different fabrication processes. When myoblasts were cultured on polyurethane surfaces, the smooth and hydrophobic membrane (F1), prepared by the solvent evaporation process, showed the greatest inhibition of myoblast adhesion compared with other porous and hydrophilic membranes (F2, F3 and F4), prepared by immersing the polymer solution into a precipitation bath. In contrast, IL-1 generation by monocytes/macrophages on the membrane F1 was more severe than those on the porous and hydrophilic membranes. Based on our results, the interaction of biomaterials with various cells is discussed.

The role of cell density in the survival of cultured cerebellar granule neurons.

The dependence for survival of cerebellar granule neurons on the cell density was examined both experimentally and theoretically. The results of batch experiments revealed that the cell survival index (CSI) was inappreciable, if cell density was below a critical level. If cell density exceeded this critical value, CSI increased with the increase in cell density. In addition, CSI was significantly increased by using a conditioned medium from the dense cultures. This suggests that not only cell density promotes survival of neurons, but also an increased concentration of growth factors produced by neurons has a direct effect on the survival of the neurons. A quantitative model describing the distribution of the growth factor at different cell densities was proposed to investigate the role of cell density in the survival of the neurons. We showed the existence of a critical level for cell density, and good agreement in the improvement of CSI was found between the theoretical prediction and the experimental result. Finally, the average concentration of growth factor necessary for cell survival based on our model was in a reasonable range compared to the practice of the addition of neurotrophic factors to the medium of cultured cerebellar granule neurons.

Development of biodegradable polyesterurethane membranes with different surface morphologies for the culture of osteoblasts.

To evaluate the biocompatibility of biodegradable polyesterurethane membranes with different surface morphologies for their possible use as orthopedic biomaterials, rat osteoblasts were cultured on smooth, sunken, and particulate polyesterurethane membranes. A close interaction between cells and exposed particles on the particulate membranes was found. Cells on the particulate surfaces were well spread and flattened and had the greatest adhesion while cells on the smooth surfaces were more rounded, less spread, and less adhered. In addition, in order to investigate their in vivo degradation rates, the morphologic changes in retrieved membranes from 2, 4, and 8 weeks after subcutaneous implantation were observed by scanning electron microscopy and their average molecular weight changes were determined by gel permeation chromatography. These analyses showed that smooth membranes, compared with the two other surface membrane types, had the greatest rate and degree of molecular weight change. In contrast, the molecular weight of particulate membranes, which favor the osteoblast culture, had not changed significantly at 8 weeks postimplantation. Thus particulate polyesterurethane membrane surfaces may be of use as an orthopedic biomaterial, and polyesterurethane membranes certainly provide an ideal system for further study of the relative contributions to biocompatibility and degradation derived from surface morphology.

Human monocyte adhesion and activation on crystalline polymers with different morphology and wettability in vitro.

This study evaluated the effects of crystalline polyamide (Nylon-66), poly(ethylene-co-vinyl alcohol) (PEVA), and poly(vinylidene fluoride) (PVDF) polymers with nonporous and porous morphologies on the ability of monocytes to adhere and subsequently activate to produce IL-1beta, IL-6, and tumor necrosis factor alpha. The results indicated monocyte adhesion and activation on a material might differ to a great extent, depending on the surface morphology and wettability. As the polymer wettability increases, the ability of monocytes to adhere increases but the ability to produce cytokines decreases. Similarly, these polymers, when prepared with porous surfaces, enhance monocyte adhesion but suppress monocyte release of cytokines. Therefore, the hydrophobic PVDF with a nonporous surface stimulates the most activity in adherent monocytes but shows the greatest inhibition of monocyte adhesion when compared with all of the other membranes. In contrast, the hydrophilic Nylon-66, which has a porous surface, is a relatively better substrate for this work. Therefore, monocyte behavior on a biomaterial may be influenced by a specific surface property. Based on this result, we propose that monocyte adhesion is regulated by a different mechanism than monocyte activation. Consequently, the generation of cytokines by monocytes is not proportional to the number of cells adherent to the surface.

Chronic idiopathic intestinal pseudo-obstruction.

Chronic idiopathic intestinal pseudo-obstruction (CIIP) is a rare heterogeneous clinical syndrome characterized by recurrent episodes of symptoms and signs of intestinal obstruction in the absence of a mechanically obstructing lesion. Dilatation of other viscera, such as the renal pelvis, ureter or urinary bladder, is identified in a minority of patients. We report the cases of two patients with CIIP presenting with abdominal fullness and constipation. Radiologic examination of the first patient revealed dilatation of the esophagus, stomach, duodenum and bowel loops up to the ascending colon. The nerve conduction velocity study of the right extremities revealed polyneuropathy and urinary bladder manometry revealed poor sensation. The patient had been admitted to our hospital three times for symptomatic relief within the prior six months. During the last admission, his symptoms persisted without response to medical treatment. Soon after discharge, the patient underwent surgery at another hospital and died of nutritional problems. The second patient was transferred to our hospital after an exploratory laparotomy was performed one month earlier. A radiographic examination revealed distention of the stomach, duodenum, small intestine and ascending colon, as well as bilateral hydronephrosis. Rheumatologic examination revealed no evidence of autoimmune disorder. The patient also had heavy proteinuria due to minimal change disease that was proven by renal biopsy. After receiving prokinetic, cathartic and corticosteroid medication for kidney disease, symptoms improved, but hydronephrosis persisted.

Properties of the poly(vinyl alcohol)/chitosan blend and its effect on the culture of fibroblast in vitro.

In this work, the properties of poly(vinyl alcohol) (PVA) and PVA/chitosan blended membranes were investigated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and electron spectroscopy for chemical analysis (ESCA). The SEM photographs show the PVA/chitosan blended membrane undergoes dramatic changes on the surface and bulk structure during the membrane formation. The DSC analysis shows that PVA and chitosan are not very compatible in the PVA/chitosan blended membrane, whereas the combination of two polymer chains of constitutionally different features is revealed. In addition, the surface of the PVA/chitosan blended membrane is enriched with nitrogen atoms at the ESCA analysis. These reflect the PVA membrane can be modified by blending with chitosan that in turn may affect the biocompatibility of the blended membrane. Therefore, adhesion and growth of fibroblasts on the PVA as well as PVA/chitosan blended membranes were investigated. Cell morphologies on the membranes were examined by SEM and cell viability was studied using MTT assay. It was observed that the PVA/chitosan blended membrane was more favorable for the cell culture than the pure PVA membrane. Cells cultured on the PVA/chitosan blended membrane had good spreading, cytoplasm webbing and flattening and were more compacting than on the pure PVA membrane. Consequently, the PVA/chitosan blended membrane may spatially mediate cellular response that can promote cell attachment and growth, indicating the PVA/chitosan blended membrane should be useful as a biomaterial for cell culture.

Analysis of ultrahigh molecular weight polyethylene failure in artificial knee joints: thermal effect on long-term performance.

The mechanism resulting in damage to and failure of ultrahigh molecular weight polyethylene (UHMWPE) tibial inserts was investigated on clinically retrieved components. The severity of the subsurface damage increased with the length of time that the component had been implanted. A theoretical analysis was developed to account for the generation of subsurface damage based on a heat transfer model. Friction generates surface heat during articulation of total knee systems. Due to the cooling effect of body fluid on the surface, the rise in temperature on the UHMWPE surface is lower than that below the surface. The peak temperature was estimated to occur on a plane positioned about 1 to 2 mm below the surface. This result was similar to the bulk temperature variation observed during in vivo and in vitro studies by other investigators. Although the difference in temperature on and below the surface is only a few degrees, the thermal effect becomes apparent after a long time and may be explained by the viscoelastic behavior of polymers: the temperature-time equivalence. It is therefore suggested that this thermal effect is another contributory factor to material damage, in addition to high stress and oxidative degradation (in appropriate cases). Therefore, any technological efforts aimed at improving the performance of artificial joint prostheses should minimize the thermal effects at the subsurface of the articular components.