Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.     

Efficacy and safety of selenium nanoparticles administered intraperitoneally for the prevention of growth of cancer cells in the peritoneal cavity

Peritoneal implantation of cancer cells, particularly postoperative seeding metastasis, frequently occurs in patients with primary tumors in the stomach, colon, liver, and ovary. Peritoneal carcinomatosis is associated with poor prognosis. In this work, we evaluated the prophylactic effect of intraperitoneal administration of selenium (Se), an essential trace element and a putative chemopreventive agent, on peritoneal implantation of cancer cells. Elemental Se nanoparticles were injected into the abdominal cavity of mice, into which highly malignant H22 hepatocarcinoma cells had previously been inoculated. Se concentrations in the cancer cells and tissues, as well as the efficacy of proliferation inhibition and safety, were evaluated. Se was mainly concentrated in cancer cells compared to Se retention in normal tissues, showing at least an order of magnitude difference between the drug target cells (the H22 cells) and the well-recognized toxicity target of Se (the liver). Such a favorable selective distribution resulted in strong proliferation suppression without perceived host toxicity. The mechanism of action of the Se nanoparticle-triggered cytotoxicity was associated with Se-mediated production of reactive oxygen species, which impaired the glutathione and thioredoxin systems. Our results suggest that intraperitoneal administration of Se is a safe and effective means of preventing growth of cancer cells in the peritoneal cavity for the above-mentioned high-risk populations.     

Characterization of implants from Dupuytren's contracture tissue into the nude (athymic) mouse

Dupuytren's contracture tissues were obtained from six patients as excess surgical material. Pieces of these tissues (a total of 38 implants) were placed into subcutaneous pockets in the suprascapular area of nude (athymic) mice. The objective was to determine whether the implant tissues would be maintained in the mouse with the characteristics of Dupuytren's tissue. The implants were removed for study at 14-179 days after implantation. Microvascular anastomosis between implant and host skin was established within the first 14 days. Histologic character and electron microscopic structure of the implants did not change during the course of the study. The implants became reduced in size with time. However, neither the spatial pattern of collagen nor the appearance of fibroblast cells changed. The original high levels of chondroitin-4-sulfate were significantly decreased in the 66- to 179-day postimplantation group, but were not significantly different from the values for normal fascial bands. The hyaluronic acid of the implants increased significantly with time of implantation, but never reached the level found in the normal fascial bands. The use of implants into nude mice may be useful for further experimental studies of Dupuytren's contracture.     

Growth of mouse fibroblasts in the presence of irradiated and lyophilized monolayers of the same type of cells

Certain cells, such as 3T3 mouse embryo fibroblasts, are inhibited from dividing when they grow to a characteristic cell density on a surface in tissue culture. We asked whether the inhibition of cell division could be attributed to the inert chemical composition of neighboring cells, that is, whether the residues of lyophilized cells retained the ability to inhibit the division of normal cells. In addition, we wanted to know whether cells in which DNA synthesis was imparied by irradiation would retain the capacity to effectively inhibit normal cells. To answer these questions, confluent and non-confluent layers of 3T3 cells were prepared in tissue culture dishes and the cells were either lyophilized or irrariated in situ. Fresh 3T3 cells were then added to these prepared layers and their growth was followed using radioactive label. There was no growth of added cells on the confluent monolayers of either untreated or irradiated cells. Growth was unimpeded on the monolayers of lyophilized cells. When cells were added to non-confluent cultures of either normal or irradiated cells the added cells grew until they had covered the remaining surface of the culture dish and had come into contact with the pre-existing cells. In the discussion, consideration is given to the role of available surface over which the cells can spread as well as to the possible interactions between neighboring cells.     

In vivo liberation of gold ions from gold implants. Autometallographic tracing of gold in cells adjacent to metallic gold

For some years, the implantation of small pieces of gold has been used as an unauthorised remedy for osteoarthritis and pain. The aim of the present study was to evaluate whether gold ions are released from gold implants. Pieces of pure gold were placed in the connective tissue of skin, bone and brains of anaesthetised animals. Ten days to several months later the animals were anaesthetised and killed by transcardial perfusion. Tissue blocks containing the gold pieces were cut, and the sections were silver-enhanced by autometallography. It was found that gold ions are released from the implanted gold and diffuse out into the surrounding tissue. The gold-containing cells in connective tissues were macrophages, mast cells and fibroblasts. In the brain, gold accumulated in astrocytes and neurons. Proton-induced X-ray emission spectroscopy analysis of the tissue surrounding gold implants confirmed that gold ions are liberated. The findings suggest that the gold implant technique, on a local scale, mimics systemic treatment with a gold-containing drug.     

Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo

Prospective skin ectoderm is underlaid by a relatively thick (100 +/- 20 micrometer) avascular zone of mesoderm in most regions of the early embryo. To determine whether or not the ectoderm exercises a role in the establishment and maintenance of the avascular zone, trypsin-isolated pieces of backskin ectoderm from chick or quail embryos were implanted as a sheet into a slit cut deep into the capillary bed of the wing bud of host chick embryos of stages 19-23. In sham operations, slits were cut at various anteroposterior levels, and the wing was allowed to heal. At intervals of 3-48 hr after these operations, embryos were injected with India ink, fixed, and cleared. Implants formed flattened vesicles, usually in continuity with host ectoderm, but sometimes completely internalized. Periderm cells from each side of the vesicle faced each other, and the cells of the cuboidal layer faced an avascular mesodermal layer at least 100 micrometer thick at all points. The implantation of prospective skin ectoderm resulted in the formation of an avascular zone in normally vascularized mesoderm of the wing bud. In contrast, the vascular bed of the limb bud abutted directly on implants of Millipore filters or of Silastic silicone (Dow Corning). Likewise, the capillary bed came in direct contact with implants of retinal pigment epithelium, an ectodermal derivative normally in close contact with the vascular choroid coat of the eye. These results, taken in conjunction with earlier experiments that show the necessity of the apical ectodermal ridge for the formation of the marginal veins of the limb bud, suggest that epithelial-mesenchymal interactions are involved in important aspects of vasculogenesis in early embryos.     

冲击波诱导人骨髓基质细胞体内成骨研究

为了研究冲击波(SW)诱导人骨髓基质细胞(hMSCs)在动物体内成骨作用,根据前期工作结果,应用适宜能量冲击波(10kV,500次)处理体外培养的hMSCs,将SW组和对照组hMSCs与羟基磷灰石(HA)载体复合后体外培养2周,应用扫描电镜(SEM)检测细胞在载体表面的生长情况.将hMSCs-HA载体复合体植入裸鼠皮下,分别于术后4周、8周取材进行组织学、四环素荧光标记、SEM观察、碱性磷酸酶测定、RT-PCR检测骨钙素mRNA表达.结果表明,SW组及对照组细胞与HA载体体外复合后生长良好,且SW组细胞分泌较多的细胞基质;细胞载体复合体植入动物体内后,SW组载体表面有类骨组织形成,而对照组HA载体表面无骨组织形成;SW组与对照组的hMSCs-HA载体复合体碱性磷酸酶表达有显著性差异(P<0.01);SW组hMSCs-HA载体复合体术后4周与8周表达骨钙素mRNA,而对照组则无表达.提示hMSCs经适宜能量冲击波作用后与HA载体复合植入裸鼠体内具有成骨作用,适宜能量的冲击波作为一种新的促进hMSCs成骨分化的方法,可应用于组织工程领域.     

A method of quantitating Paneth cell metaplasia of the stomach by image analysis

Paneth cells are one of the histologic components of intestinal metaplasia of the stomach, as are mucin-producing goblet cells. With the aid of an image quantifier, the distribution of Paneth cells histochemically labeled with acid fuchsin was analyzed for a gastrectomy specimen containing an adenocarcinoma of the intestinal type; the topographic distribution of goblet cells histochemically labeled with Alcian blue (pH 2.5) was also analyzed. The specimen was cut into 63 blocks (0.5 X 4.0 cm) in four zones; antrum (zone I), intermediate region (zone II) and fundus (zones III and IV). Paneth cells were found only in sections containing mucin-producing goblet cells. Paneth cells were found in 12.5% of the 16 sections from the antral zone I containing Alcian blue-positive goblet cells. The rates were 44.4% for the intermediate zone II and 55.5% for the distal fundic zone III. The total area occupied by Paneth cells was significantly lower in the gastric mucosa as compared to the duodenal mucosa. The "Paneth cell index" (total Paneth cell area/total goblet cell area) was highest in the duodenum, followed by the distal fundic zone III. This method of quantitating Paneth cell metaplasia of the stomach will be used to investigate the topographic distribution of those cells in populations with low and high incidences of intestinal metaplasia.     

Implantation of embryonic pancreas into the anterior chamber of healthy and diabetic rats

The possibility of pancreatic-islet embryonic implantation into the frontal chamber of the eye in Wistar rats has been demonstrated. The implantation was followed by the induction of alloxan diabetes. Clinical diabetic compensation has been shown in a number of experimental animals (maximum time of experiments--45 days). The formation of islet-like structures in the frontal chamber of the eye was confirmed microscopically. The structures contained B cells in their cytoplasm. Cellular differentiation leading to the appearance of functionally active endocrine cells has been noted in the implants.     

The identification of myogenic cells in regenerating skeletal muscle. I. Early anuran regenerates.

Muscle regeneration is usually considered to involve two easily recognizable mononuclear cell populations: (a) irregularly shaped phagocytic macrophages; and (b) fusiform myogenic cells which reside beneath the basal lamina of the injured myofibers. The present study finds that anuran macrophages can mimic early myogenic cells by adopting a fusiform shape and a sublaminar position during the initial stages of phagocytic infiltration. Implants of minced gastrocnemius muscle from adult Rana pipiens and R. clamitans were used. Macrophage cytology was examined in normally regenerating implants and implants mixed with carbon tracer particles. Implants of nonliving muscle, lyophilized or frozen repeatedly prior to implantation, were also prepared to study the process of macrophage invasion free from contamination by any endogenous myogenic cells. Ultrastructural examination of normal implants finds no clear morphological boundaries by which early myogenic cells and macrophages can reliably be separated. Studies of carbon-marked implants demonstrate the occurrence of numerous, carbon-labeled fusiform sublaminar macrophages. Fusiform macrophages are also found in the myofibers of the implanted muscle tissues killed by previous freezing or lyophilization. These observations indicate that the criteria of cell shape and location traditionally used for myogenic cell identification are of doubtful validity and must be reevaluated.     

First results of the investigation of the stability and tissue integration of a degradable, elastomeric copolymer in an animal model]

The stability and tight integration into adjacent tissue of a novel, degradable, elastic copolymer were examined in an animal model. The biomaterial was used for the reconstruction of a gastric wall defect in Sprague-Dawley rats (n=42) to test the polymeric material under the extreme chemical, enzymatical and mechanical conditions of the stomach. In the control group (n=21) the same defect of the gastric wall was primarily closed without biomaterial implantation. In the baseline group (n=21) the animals were kept under standard conditions without any surgical procedure. The implantation periods were 1 week, 4 weeks and 6 months. The animals' weight was determined preoperatively and before explantation. After explantation, air was pumped into the stomach and the pressure was measured by using a pressure-gauge in order to test whether the surgically produced union of the stomach wall and the polymer patch was gas-tight. After 1 week of implantation time a statistically significant increase of the body weight of the animals was found only in the baseline group. Four weeks and 6 months after the abdominal surgical procedure, a statistically significant increase of the animals' weight was found in the implantation group, the control and the baseline group. Gastrointestinal complications like fistula, perforation or peritonitis did not occur in any of the animals. The measurement of the stomach pressure after maximal gas insufflation did not show significant differences between the implantation group, the control and the baseline group in any of the time periods investigated. Despite very high strains of the gastric wall, no gas leakage was detected. There was a tight connection between the polymer and the adjacent stomach wall in all animals investigated. An adequate mechanical stability of the biomaterial was detectable under the extreme pathophysiological conditions of the stomach milieu. A fast and unfavourable degradation of the degradable polymer was not found in any of the animals. Further investigations are needed to analyse the mechanisms of the tissue integration of the biomaterial as well as the degradation kinetic of the polymer and the process of the tissue remodeling. The knowledge of these processes is necessary to adapt the novel biomaterial and thus prepare it for the use and implantation in different body locations and to develop novel therapeutical options in medicine.     

Experimental effects on surrounding fibrous capsule formation from placing steroid in a silicone bag-gel prosthesis before implantation.

Soft-gel miniprostheses of silicone were implanted subcutaneously into 75 male rats. Groups of prostheses were preinjected with saline, a commercial form of triamcinolone acetonide, or a suspension of crystalline triamcinolone acetonide. The softness of the prosthesis mound later was measured objectively, and the capsules surrounding the implants were analyzed by histology, SEM, and chemistry, at various intervals up to 120 days after implantation. The control implants developed a normal laminar capsule. With an incidence increasing up to 100 percent at 120 days, the steroid-treated implants were surrounded by capsules lacking an inner membrane. The inner membrane of the laminar capsule had a high protein content (relative to normal tissue) and a relatively reduced collagen content, while the diffuse capsule resulting from the TA treatment had a high protein content and a high collagen content (about the same as normal tissue). No differences were found in the softness of the mounds of the implanted prostheses. The effect of the TA treatment was explained on the basis of its collagenolytic effect, which could gradually erode the normal capsule membrane. Capsule firmness could not be related to the architecture or the protein or collagen content in our findings. We hypothesize that normal capsule firmness may be related to the amount and kind of interconnection between the loose outer zone of connective tissue and the surrounding tissue.     

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days. With the unembedded microspheres releasing faster than those embedded in CMC In vivo. the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2 PLGA microspheres in CMC was an effective implantable protein-delivery system for the use in bone repair. Published: October 7. 2001.     

Correlation between the cellular content of mobile water and the viability of lyophilized yeast cells

Spin-echo NMR studies showed that lyophilized yeast cells contain isolated mobile water (IMW), whose content varied from 0.25% (of the dry weight of cells) in the lyophilized exponential-phase yeast cells to 3.8% in the lyophilized lag-phase and stationary-phase yeast cells. The viability rate of yeast cells varied from 20% in the lyophilized preparation of exponential-phase cells to 86% in the lyophilized preparation of early-stationary-phase cells. In the lyophilized preparation of yeast cells grown in a chemostat mode at a constant specific rate, the content of IMW depended on the growth-limiting factor, being minimal in the case of growth limitation by carbon source. In the latter case, the viability rate of cells was also minimal. The data obtained show that there is a correlation between the IMW content and the viability rate of yeast cells in lyophilized preparations.     

Autoradiographic study of human scirrhous gastric carcinoma during cultivation in diffusion chambers]

The growth of the epithelial and connective tissue cells was noted in cultivation of human schirrous carcinoma of the stomach in diffuse chambers. In difference to the initial tissue of the tumour in vitro in which no incorporation of thymidine-H3 into the connective tissue cells was noted, these cells proved to be labeled under conditions of growth in the chambers. One hour after the administration of thymidine-H3 the percentage of cells with labeled nuclei averaged 25.1% this considerably exceeding the value of the label index determined under conditions of incubation in vitro of the initial tumour tissue (6.6%). Under conditions of cultivation there was revealed a rapidly proliferating subpopulation of cells with the mitotic cycle duration of 14.8 hours.     

Regeneration of splenic tissue after autologous subcutaneous implantation: Development of non-lymphoid cells in the white pulp of the rat spleen

Summary Regeneration of splenic tissue after autologous subcutaneous implantation provides a useful model for studying the development of splenic tissue. The development of the various non-lymphoid cells of the white pulp in the rat is described. It appears that regeneration of the implants is initiated by ingrowing vessels and a newly formed reticulum, which forms the microenvironment for the homing lymphocytes. Marginal metallophils are found at their characteristic location at the inner border of the marginal sinus five weeks after implantation. Trapping of antigen-antibody complexes reappears when the first primary follicles can be recognized.     

Helicobacter suis causes severe gastric pathology in mouse and mongolian gerbil models of human gastric disease

Background

“Helicobacter (H.) heilmannii” type 1 is the most prevalent gastric non-H. pylori Helicobacter species in humans suffering from gastric disease. It has been shown to be identical to H. suis, a bacterium which is mainly associated with pigs. To obtain better insights into the long-term pathogenesis of infections with this micro-organism, experimental infections were carried out in different rodent models.

Methodology/Principal Findings

Mongolian gerbils and mice of two strains (BALB/c and C57BL/6) were infected with H. suis and sacrificed at 3 weeks, 9 weeks and 8 months after infection. Gastric tissue samples were collected for PCR analysis, histological and ultrastructural examination. In gerbils, bacteria mainly colonized the antrum and a narrow zone in the fundus near the forestomach/stomach transition zone. In both mice strains, bacteria colonized the entire glandular stomach. Colonization with H. suis was associated with necrosis of parietal cells in all three animal strains. From 9 weeks after infection onwards, an increased proliferation rate of mucosal epithelial cells was detected in the stomach regions colonized with H. suis. Most gerbils showed a marked lymphocytic infiltration in the antrum and in the forestomach/stomach transition zone, becoming more pronounced in the course of time. At 8 months post infection, severe destruction of the normal antral architecture at the inflamed sites and development of mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions were observed in some gerbils. In mice, the inflammatory response was less pronounced than in gerbils, consisting mainly of mononuclear cell infiltration and being most severe in the fundus.

Conclusions/Significance

H. suis causes death of parietal cells, epithelial cell hyperproliferation and severe inflammation in mice and Mongolian gerbil models of human gastric disease. Moreover, MALT lymphoma-like lesions were induced in H. suis-infected Mongolian gerbils. Therefore, the possible involvement of this micro-organism in human gastric disease should not be neglected.     

Modification of macroporous titanium tracheal implants with biodegradable structures: tracking in vivo integration for determination of optimal in situ epithelialization conditions

Previously, we showed that macroporous titanium implants, colonized in vivo together with an epithelial graft, are viable options for tracheal replacement in sheep. To decrease the number of operating steps, biomaterial-based replacements for epithelial graft and intramuscular implantation were developed in the present study. Hybrid microporous PLLA/titanium tracheal implants were designed to decrease initial stenosis and provide a surface for epithelialization. They have been implanted in New Zealand white rabbits as tracheal substitutes and compared to intramuscular implantation samples. Moreover, a basement membrane like coating of the implant surface was also designed by Layer-by-Layer (LbL) method with collagen and alginate. The results showed that the commencement of stenosis can be prevented by the microporous PLLA. For determination of the optimum time point of epithelialization after implantation, HPLC analysis of blood samples, C-reactive protein (CRP), and Chromogranin A (CGA) analyses and histology were carried out. Following 3 weeks the implant would be ready for epithelialization with respect to the amount of tissue integration. Calcein-AM labeled epithelial cell seeding showed that after 3 weeks implant surfaces were suitable for their attachment. CRP readings were steady after an initial rise in the first week. Cross-linked collagen/alginate structures show nanofibrillarity and they form uniform films over the implant surfaces without damaging the microporosity of the PLLA body. Human respiratory epithelial cells proliferated and migrated on these surfaces which provided a better alternative to PLLA film surface. In conclusion, collagen/alginate LbL coated hybrid PLLA/titanium implants are viable options for tracheal replacement, together with in situ epithelialization.     

Implant size and fixation mode strongly influence tissue reactions in the CNS

The function of chronic brain machine interfaces depends on stable electrical contact between neurons and electrodes. A key step in the development of interfaces is therefore to identify implant configurations that minimize adverse long-term tissue reactions. To this end, we here characterized the separate and combined effects of implant size and fixation mode at 6 and 12 weeks post implantation in rat (n = 24) cerebral cortex. Neurons and activated microglia and astrocytes were visualized using NeuN, ED1 and GFAP immunofluorescence microscopy, respectively. The contributions of individual experimental variables to the tissue response were quantified. Implants tethered to the skull caused larger tissue reactions than un-tethered implants. Small diameter (50 µm) implants elicited smaller tissue reactions and resulted in the survival of larger numbers of neurons than did large diameter (200 µm) implants. In addition, tethering resulted in an oval-shaped cavity, with a cross-section area larger than that of the implant itself, and in marked changes in morphology and organization of neurons in the region closest to the tissue interface. Most importantly, for implants that were both large diameter and tethered, glia activation was still ongoing 12 weeks after implantation, as indicated by an increase in GFAP staining between week 6 and 12, while this pattern was not observed for un-tethered, small diameter implants. Our findings therefore clearly indicate that the combined small diameter, un-tethered implants cause the smallest tissue reactions.     

Correlation between the Cellular Content of Mobile Water and the Viability of Lyophilized Yeast Cells

Spin-echo NMR studies showed that lyophilized yeast cells contain isolated mobile water (IMW), whose content varied from 0.25% (of the dry weight of cells) in lyophilized exponential-phase yeast cells to 3.8% in lyophilized lag-phase and stationary-phase yeast cells. The viability rate of yeast cells varied from 20% in a lyophilized preparation of exponential-phase cells to 86% in a lyophilized preparation of early-stationary-phase cells. In a lyophilized preparation of yeast cells grown in a chemostat mode at a constant specific rate, the content of IMW depended on the growth-limiting factor, being minimal in the case of growth limitation by the carbon source. In the latter case, the viability of cells was also minimal. The data obtained show that there is a correlation between the IMW content and the viability of yeast cells in lyophilized preparations.