Evaluation of combined growth media for in vitro cultivation of oropharyngeal biofilms on prosthetic silicone.

In the upper aerodigestive tract, biofilm deposits by oropharyngeal microbes can cause failure of medical polymer devices like voice prostheses. Previous studies on testing of inhibitive strategies still lack of comparability due to varying study protocols concerning growth media, microbial species and growth conditions. Goal of the study was therefore to test cultivation of a mixed biofilm of isolated oropharyngeal microbes under in vitro growth conditions using mixtures of common growth media. Mixtures of yeast peptone dextrose medium (YPD), fetal bovine serum (FBS), RPMI 1640, Yeast nitrogen base medium (YNB) and brain heart infusion (BHI) were tested to grow mixed biofilm deposits of Candida albicans, Candida tropicalis, Staphylococcus aureus, Streptococcus epidermidis, Rothia dentocariosa and Lactobacillus gasseri on medical grade silicone. Periodic assessment of living biofilm was performed over 22 days by a digital microscope and the cultivated biofilm structures were analyzed by scanning electron microscopy after completion of the study. Mixtures of BHI, YPD and FBS improved microscopic growth of multispecies biofilm deposits over time, while addition of RPMI and YNB resulted in reduction of visible biofilm deposit sizes. A mixtures of FBS 30% + YPD 70% and BHI 30% + YPD 70% showed enhanced support of permanent surface growth on silicone. Growth kinetics of in vitro multispecies biofilms can be manipulated by using mixtures of common growth media. Using mixtures of growth media can improve growth of longterm multispecies oropharyngeal biofilm models used for in vitro testing of antibiofilm materials or coatings for voice prostheses.

In vitro effects of particulate bone substitute materials on the resorption activity of human osteoclasts.

Much research has been done on bone cells, but only a few studies deal with biomaterial-induced effects on human osteoclasts, which may take on an important role in the successful regeneration of bone. In order to highlight such effects, human peripheral blood mononuclear cells (PBMCs) were extracted from venous blood, differentiated to osteoclasts and then cultured in, the presence of five particulate hydroxyapatite (HA)/ß-tricalcium phosphate (TCP) biomaterials, on bovine bone slices and glass cover slips. The biomaterials, AlgOSS 50/50 (50 % HA/50 % TCP), AlgOSS 20/80 (20 % HA/80 % TCP), Algipore (98 % HA), Cerasorb (100 % TCP) and Bio-Oss (100 % HA) were chosen to assess their influence on cell morphology and numbers. Light microscopic evaluation was performed during ongoing cell culture. After 21 d of cultivation, the biomaterial-induced effects on osteoclastic resorption of the bone slices were evaluated by scanning electron microscopy (SEM). Osteoclast-like cells were identified by TRAP staining. All five biomaterials showed larger area fractions of resorbed bone than the control (5.6 ± 6.8 %), as measured on SEM images. The purely hydroxyapatite-based Algipore (9.8 ± 9.7 %) and Bio-Oss (7.9 ± 8.8 %) showed significantly elevated area fraction rates (p ≤ 0.05) of bone resorption. Light microscope evaluation revealed a significant, but inhibiting effect of Cerasorb (p = 0.05). These data indicated that introducing of small biomaterial hydroxyapatite particles may have improved the performance of bone substitute materials.

Development and validation of a 3D-printed interfacial stress sensor for prosthetic applications.

A novel capacitance-based sensor designed for monitoring mechanical stresses at the stump-socket interface of lower-limb amputees is described. It provides practical means of measuring pressure and shear stresses simultaneously. In particular, it comprises of a flexible frame (20 mm × 20 mm), with thickness of 4mm. By employing rapid prototyping technology in its fabrication, it offers a low-cost and versatile solution, with capability of adopting bespoke shapes of lower-limb residua. The sensor was first analysed using finite element analysis (FEA) and then evaluated using lab-based electromechanical tests. The results validate that the sensor is capable of monitoring both pressure and shear at stresses up to 350 kPa and 80 kPa, respectively. A post-signal processing model is developed to induce pressure and shear stresses, respectively. The effective separation of pressure and shear signals can be potentially advantageous for sensor calibration in clinical applications. The sensor also demonstrates high linearity (approx. 5-8%) and high pressure (approx. 1.3 kPa) and shear (approx. 0.6 kPa) stress resolution performance. Accordingly, the sensor offers the potential for exploitation as an assistive tool to both evaluate prosthetic socket fitting in clinical settings and alert amputees in home settings of excessive loading at the stump-socket interface, effectively preventing stump tissue breakdown at an early stage.

Tricalcium phosphate-based biocomposites for mandibular bone regeneration--A histological study in sheep.

The present study investigated the suitability of three different absorbable biocomposites for the repair of critical sized bone defects created at the mandibular angle of adult sheep. Each biocomposite was composed of a three-dimensional individualized polylactide scaffold, containing a tricalcium phosphate biomaterial (chronOS). Either autologous bone marrow (chOS/BoneMarrow) or coagulation factor XIII (chOS/FactorXIII) was added to the biomaterial for osteopromotion. Venous whole blood (chOS/Blood) added to the biomaterial served as a control. A total of 18 adult sheep were used for implantation studies, subdivided into three groups of six animals each. After 12 weeks of observation, the animals were sacrificed and the mandibles were retrieved for qualitative and quantitative histologic assessment within three regions of interest (transitional zone, center, and periphery) throughout the biocomposites. Successful bone regeneration was defined by the absence of scaffold deformation and the presence of new bone formation within the biocomposites. In histomorphometry, only chOS/BoneMarrow showed elevated area fractions of newly formed bone in all regions of interest (transitional zone 50.7 ± 7.5, center 31.9 ± 9.3, periphery 23.1 ± 13.5). This led to preservation of the macroscopic scaffold structure in all specimens. Zero hurdle regression confirmed this by validating the factor biocomposite as significant (p < 0.001) for regeneration success. In our experiment, chOS/BoneMarrow was the only biocomposite passing the hurdle of regeneration in all three regions of interest. In contrast, bone formation was less pronounced and uniform in chOS/FactorXIII and chOS/blood-containing specimens. In these groups, scaffolds showed obvious to significant deformation. Overall, autologous bone marrow showed the most promising results in our experimental setting. As opposed to reports in the literature, we could not confirm the suitability of coagulation factor XIII to promote bone formation, since bone formation rates were comparable only to those of the control venous blood.

Histomorphologic findings on human bone samples six months after bone augmentation of the maxillary sinus with Algipore.

Sinus grafting, a popular and standard treatment for maxillary atrophy, uses a variety of grafting materials. In this study, specimens obtained 6 months after sinus grafting with Algipore were evaluated under light microscopy and showed osseoformation, xenograft degradation, and bone ingrowth into particles. Osteoblastic cells were embedded in the intracorpuscular bone matrix, which indicated that xenograft particles are an osseoconductive scaffold and stimulate matrix deposition. Acute inflammatory responses after insertion of Algipore did not occur. Particles were degraded during physiologic bone remodeling, and newly formed bone gradually replaced resorbed biomaterial.

Guided bone regeneration with titanium membranes: a clinical study.

Guided bone regeneration using barrier membranes is useful in bone augmentation. Because the commonly used polytetrafluoroethylene (PTFE, Gore-Tex (R), WL Gore, Flagstaff, AZ, USA) membranes or resorbable membranes tend to collapse, more stable membranes are desirable. A titanium membrane (FRIOS(R) BoneShield, Friatec, Mannheim, Germany) was evaluated in a clinical study of 52 patients. Most of them had particulate bone grafts or phycogene hydroxyapatite (Algipore(R), Friatec, Mannheim, Germany) or both stabilized with titanium membranes. In 78 procedures, 23 membranes (29%) became exposed, but only seven of these (9%) led to failure of the graft with a considerable loss of augmented material. The time interval between operation and possibly exposure was responsible for the result. Early exposures (within a few weeks) led to poor formation of new bone within the grafts, whereas if exposure was later, results were as good as in procedures in which the membranes did not become exposed.

Interaction effect of D4 dopamine receptor gene and serotonin transporter promoter polymorphism on the cortisol stress response.

Genetic variation of the serotonin transporter (SCL6A4, 5-HTT) has been associated with fear- and anxiety-related behaviors, while a polymorphism in exon III of the D4 dopamine receptor gene (DRD4) has been linked to novelty seeking. The dopaminergic and the serotonergic neurotransmitter system have been found to modulate the amygdala-connected circuitries that are crucial in emotional modulation and response to fearful stimuli. Additionally, reactivity of amygdala-innervated effector systems is also essential for our understanding of anxiety-related behaviors. Here, we used the stress-induced activation of the hypothalamic-pituitary-adrenal axis to investigate the impact of 5-HTTLPR and DRD4 on the cortisol stress response in 84 healthy adults. Saliva cortisol was measured during and after the Trier Social Stress Test. We found a significant main effect of DRD4: Carriers of the 7R allele exhibited lower cortisol responses. Additionally, a DRD4 by 5-HTTLPR interaction emerged: 5-HTTLPR LA/LA homozygotes showed a lower cortisol response than did S or LG allele carriers but only if they possessed at least one copy of the DRD4 7R allele. The results point to independent and joint effects of these polymorphisms on stress responsivity.

Bone regeneration using a naturally grown HA/TCP carrier loaded with rh BMP-2 is independent of barrier-membrane effects.

The present study investigated whether bone regeneration and biomaterial replacement would be improved by loading of biogenous biphasic biomaterial scaffolds (HA/TCP ratio 30/70) with rhBMP-2, and whether the placement of three barrier membranes differing in structure and porosity (prototyped SLA Ti specimens, GORE RESOLUT Adapt specimens, and titanized TiMESH light specimens) would have a synergistic effect. A rabbit calvarial model was used for the implantation studies. Histological specimens were obtained after 12 weeks and evaluated quantitatively for differences between the various material combinations. Loading of the biomaterials with rhBMP-2 significantly enhanced the amount of regenerated bone and caused a pronounced biomaterial replacement. While BMP-induced bone had formed uniformly over the surgical defects, bone regeneration in the absence of BMP depends on bone promotion from the margins of the defects toward the center. No positive effect on bone regeneration was seen for any of the placed barrier membranes. While the present study had shown that rhBMP-2 loading significantly increases bone regeneration using the investigated biomaterial, barrier-membrane placement may be useful in predetermining the final shape of the regenerative site but provides no additional beneficial impact on the amount and quality of the bone regeneration induced by rhBMP-2.

Haploinsufficiency of the SERPINA6 gene is associated with severe muscle fatigue: A de novo mutation in corticosteroid-binding globulin deficiency.

Corticosteroid-binding globulin (SERPINA6) deficiency is an extremely rare hereditary disorder characterized by reduced corticosteroid-binding capacity with normal or low plasma corticosteroid-binding globulin concentration, and normal or low basal cortisol levels associated with hypo-/hypertension and muscle fatigue. Here, we present a patient with severe muscle fatigue, normal blood pressure, and abnormal high saliva cortisol levels following a standardized stress test. This patient was found heterozygous for a de novo 367 asparagine-encoding variant of the corticosteroid-binding globulin gene, previously described as "transcortin Lyon". Both parents were homozygous for the ("wildtype") 367 aspartate-encoding allele. To the best of our knowledge, this case represents the first de novo mutation reported for corticosteroid-binding globulin deficiency, implicating a pathogenic role of variants of SERPINA6 in some cases of muscle fatigue.

Effect of anticoagulation on blood membrane interactions during hemodialysis.

BACKGROUND: Adequate anticoagulation is a precondition to prevent extracorporeal blood clotting and to improve biocompatibility during hemodialysis. In this study, we performed a morphologic analysis by using scanning electron microscopy to compare three modes of anticoagulation-conventional unfractionated heparin (UFH), low molecular weight heparin (LMWH; dalteparin sodium), or sodium citrate during hemodialysis-on membrane-associated coagulation activation. METHODS: Fifteen patients on regular hemodialysis therapy were investigated. Five patients received UFH, five patients LMWH, and five patients sodium citrate as an anticoagulant during a standardized hemodialysis protocol using a single-use polysulfone capillary dialyzer. Membrane-associated clotting was evaluated using a scanning electron microscope. A dialyzer clotting score was used for quantitative description of coagulation activation on membrane segments. RESULTS: Using UFH as an anticoagulant revealed the most pronounced cell adhesion and thrombus formation and the highest dialyzer clotting score (11.5 +/- 1.3 of a maximal 20 points). LMWH had a lower dialyzer clotting score than UFH (10.4 +/- 1.2 of 20 points). During the use of sodium citrate, a negligible thrombus formation and the lowest dialyzer clotting score (1.6 +/- 0.6 of 20 points, P < 0.05) were observed. CONCLUSION: The results of this investigation indicate that using sodium citrate as an anticoagulant during hemodialysis induces a lower activation of coagulation than both conventional and fractionated heparin, which might contribute to an improvement of biocompatibility of hemodialysis extracorporeal circulation.

New range of light-activated surgical adhesives for tissue repair.

A new range of light-activated surgical adhesives has been developed for laser-assisted tissue repair. The surgical adhesive is composed of a polymer scaffold doped with the traditional protein solder mix of serum albumin and a chromophoric dye. Problems associated with inflexibility in conforming to various tissue geometries, instability in a hydrated environment due to non-uniform tissue adhesive denaturation, and solubility in physiological fluids prior to denaturation are overcome using the adhesives. The new adhesives offer a viable alternative to conventional fasteners, including sutures, staples and clips, currently used for tissue repair. In addition, it could be possible to use patches prepared from the adhesive in the field as a simple and effective method to stop bleeding and repair tissue quickly in an emergency situation. First, studies were conducted to improve the flexibility of traditional protein solders to enable them to be tailored to a wide range of clinically relevant geometry's including tubes, crescents and tape. Second, the creation of a chromophore concentration gradient across the thickness of the adhesive was investigated as a means to allow control of the heat source gradient through the adhesive. Increased deposition of the laser energy near the vital solder/tissue interface was thus achieved. Finally, predenaturation of the adhesive was investigated as a means for enhancing its stability in a hydrated environment thus improving the handling characteristics of the adhesive for clinical application. The application of the new surgical adhesives to augment laser tissue repairs is shown to enhance edge co-optation, improve repair strength and to reduce thermal tissue injury. The moldable, absorption controllable and flexible nature of the new adhesives greatly improves the clinical applicability of laser-solder tissue repair.

[Evidence of osteocalcin expression in osteoblast cells of mandibular origin growing on biomaterials with RT-PCR and SDS-PAGE/Western blotting]. / Nachweis der Osteokalzinexpression osteoblastärer Zellen mandibulären Ursprungs, wachsend auf Biomaterialien, mittels RT-PCR und SDS-PAGE/Western Blotting.

A new approach to addressing difficult tissue reconstructive or replacement problems in the oral cavity is to engineer new tissue by using selective cell transplantation on polymer scaffolds. The current study characterized the osteoblastic nature of adherent mandibular cells on biomaterials, which could have a potential use as scaffolds for tissue engineering strategies. Cells of mandibular origin from one patient were cultivated on three different biomaterials (PepGen P-15 trade mark, Frios Algipore, and OsteoGraf/LD-700) for 7 and 14 days and osteocalcin expression was demonstrated by RT-PCR and SDS-PAGE/Western blotting. In order to explicitly characterize only the adherent cells on the biomaterials, we first separated the biomaterials with adherent cells from the culture plate before trypsinization. We could demonstrate that cell growth of adherent mandibular osteoblast-like cells was significantly higher on biomaterials with an organic component (PepGen P-15 trade mark ) in comparison to Frios Algipore and OsteoGraf/LD-700, respectively. In conclusion, only the explicit study of adherent cells at the gene and protein levels gives information about the osteoconductivity of biomaterials.

Thrombus formation on the balloon of heparin-bonded pulmonary artery catheters: an ultrastructural scanning electron microscope study.

OBJECTIVE: To investigate heparin-bonded pulmonary artery catheters with respect to thrombus formation and platelet aggregation at the balloon and the shaft using a scanning electron microscope in critically ill patients. DESIGN: Prospective study. SETTINGS: Critical care unit and research laboratories. PATIENTS: Pulmonary artery catheters were inserted in critically ill patients (n = 10). INTERVENTIONS: Pulmonary artery catheters were removed after 24, 48, 72, or 120 hrs, and the ultrastructure was investigated in specialized research laboratories. MEASUREMENTS AND MAIN RESULTS: Balloon and shaft were investigated using a scanning electron microscopic technique. Area of thrombus formation was quantified using image analysis. Heparin release of the catheters was measured. The frequency of balloon inflations was investigated in in vitro experiments by inflating catheters different times (0, 10, 20, and 30 times). Twenty-four hours after catheter insertion, scanning electron microscopic images showed thrombus formation and platelet aggregation at the site of the balloon. Seventy-two hours after catheter insertion, a thrombus started to detach. The areas of thrombus formation did not differ, but thrombus organization changed dramatically 72 and 120 hrs after catheter insertion. The shaft was colonized by single cells only. Cracks of the balloon could be observed after 72 hrs, whereas no cracks could be found in in vitro controls. In vitro, heparin release of the pulmonary artery catheters decreased significantly after 24 hrs. CONCLUSIONS: Scanning electron microscopic images of heparin-bonded pulmonary artery catheters demonstrate thrombus formation on the balloon 24 hrs after pulmonary artery catheter insertion, increasing dramatically at 72 and 120 hrs. The shaft was colonized by single cells only. The thrombus size is not significantly different during the observation time, but the grade and quality of thrombus formation differ.