Comparative analysis of biofilm models to determine the efficacy of antimicrobials.

Biofilms are one of the greatest challenges in today's treatment of chronic wounds. While antimicrobials kill platonic bacteria within seconds, they are rarely able to harm biofilms. In order to identify effective substances for antibacterial therapy, cost-efficient, standardized and reproducible models that aim to mimic the clinical situation are required. In this study, two 3D biofilm models based on human plasma with immune cells (lhBIOM) or based on sheep blood (sbBIOM) containing S. aureus or P. aeruginosa, are compared with the human biofilm model hpBIOM regarding their microscopic structure (scanning electron microscopy; SEM) and their bacterial resistance to octenidine hydrochloride (OCT) and a sodium hypochlorite (NaOCl) wound-irrigation solution. The three analyzed biofilm models show little to no reaction to treatment with the hypochlorous solution while planktonic S. aureus and P. aeruginosa cells are reduced within minutes. After 48 h, octenidine hydrochloride manages to erode the biofilm matrix and significantly reduce the bacterial load. The determined effects are qualitatively reflected by SEM. Our results show that both ethically acceptable human and sheep blood based biofilm models can be used as a standard for in vitro testing of new antimicrobial substances. Due to their composition, both fulfill the criteria of a reality-reflecting model and therefore should be used in the approval for new antimicrobial agents.

Efficacy of antiseptics in a novel 3-dimensional human plasma biofilm model (hpBIOM).

The increasing incidence of non-healing wounds constitutes a pivotal socio-economic burden. 60-80% of chronic wounds are colonized by pathogenic microorganisms within a protective extracellular polymeric substance, bearing a great challenge in wound management. Human plasma was used to prepare the biofilm model (hpBIOM), adding pathogens to the plasma and forming Coagula-like discs with integrated pathogens were produced. The antiseptics Octenisept and Lavasorb were tested regarding their antibacterial properties on clinically relevant biofilm-growing bacteria (MRSA, P. aeruginosa) in the hpBIOM. Biofilm-typical glycocalyx-formation was confirmed using immunohistochemical staining. Treatment of a 12 h-maturated biofilm with Octenisept resulted in complete eradication of P. aeruginosa and MRSA after 48 h. Lavasorb proved less effective than Octenisept in this setting. In more mature biofilms (24 h), both antiseptics showed a delayed, partially decreased efficacy. Summarized, the hpBIOM provides essential factors for a translational research approach to be used for detailed human biofilm analyses and evaluation of antimicrobial/-biofilm properties of established and novel therapeutic strategies and products. Octenisept and Lavasorb showed an attenuated efficacy in the hpBIOM compared to planktonic conditions and previously published biofilm-studies, prompting the question for the necessity of introducing new international standards and pre-admission requirements on a translational base.

Impact of probiotics on pathogen survival in an innovative human plasma biofilm model (hpBIOM).

BACKGROUND: Despite of medical advances, the number of patients suffering on non-healing chronic wounds is still increasing. This fact is attended by physical and emotional distress and an economic load. The majority of chronic wounds are infected of harmful microbials in a protecting extracellular matrix. These biofilms inhibit wound healing. Biofilm-growing bacteria developed unique survival properties, which still challenge the appropriate wound therapy. The present in-vitro biofilm models are not suitable for translational research. By means of a novel in-vivo like human plasma biofilm model (hpBIOM), this study systematically analysed the influence of 3 probiotics on the survival of five clinically relevant pathogenic microorganisms. METHODS: Human plasma was used to produce the innovate biofilm. Pathogenic microorganisms were administered to the plasma. By stimulating the production of a fibrin scaffold, stable coagula-like discs with integrated pathogens were produced. The five clinically relevant pathogens P. aeruginosa, S. aureus, S. epidermidis, E. faecium and C. albicans were challenged to the probiotics L. plantarum, B. lactis and S. cerevisiae. The probiotics were administered on top of the biofilm and the survival was quantified after 4 h and 24 h of incubation. For statistics, two-way ANOVA with post-hoc Tukey's HSD test was applied. P-value > 0.05 was considered to be significant. RESULTS: SEM micrographs depicted the pathogens on the surface of the fibrin scaffold, arranged in close proximity and produced the glycocalyx. The application of probiotics induced different growth-reducing capacities towards the pathogens. B. lactis and S. cerevisiae showed slight bacteria-reducing properties. The survival of C. albicans was not affected at all. The most antimicrobial activity was detected after the treatment with L. plantarum. CONCLUSIONS: This study successfully reproduced a novel human biofilm model, which provides a human wound milieu and individual immune competence. The success of bacteriotherapy is dependent on the strain combination, the number of probiotics and the activity of the immune cells. The eradicating effect of L. plantarum on P. aeruginosa should be emphasized.

Long-term effects of enriched environment on neurofunctional outcome and CNS lesion volume after traumatic brain injury in rats.

To determine whether the exposure to long term enriched environment (EE) would result in a continuous improvement of neurological recovery and ameliorate the loss of brain tissue after traumatic brain injury (TBI) vs. standard housing (SH). Male Sprague-Dawley rats (300-350 g, n=28) underwent lateral fluid percussion brain injury or SHAM operation. One TBI group was held under complex EE for 90 days, the other under SH. Neuromotor and sensorimotor dysfunction and recovery were assessed after injury and at days 7, 15, and 90 via Composite Neuroscore (NS), RotaRod test, and Barnes Circular Maze (BCM). Cortical tissue loss was assessed using serial brain sections. After day 7 EE animals showed similar latencies and errors as SHAM in the BCM. SH animals performed notably worse with differences still significant on day 90 (p<0.001). RotaRod test and NS revealed superior results for EE animals after day 7. The mean cortical volume was significantly higher in EE vs. SH animals (p=0.003). In summary, EE animals after lateral fluid percussion (LFP) brain injury performed significantly better than SH animals after 90 days of recovery. The window of opportunity may be wide and also lends further credibility to the importance of long term interventions in patients suffering from TBI.

Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.

Small animal fracture models have gained increasing interest in fracture healing studies. To achieve standardized and defined study conditions, various variables must be carefully controlled when designing fracture healing experiments in mice or rats. The strain, age and sex of the animals may influence the process of fracture healing. Furthermore, the choice of the fracture fixation technique depends on the questions addressed, whereby intra- and extramedullary implants as well as open and closed surgical approaches may be considered. During the last few years, a variety of different, highly sophisticated implants for fracture fixation in small animals have been developed. Rigid fixation with locking plates or external fixators results in predominantly intramembranous healing in both mice and rats. Locking plates, external fixators, intramedullary screws, the locking nail and the pin-clip device allow different degrees of stability resulting in various amounts of endochondral and intramembranous healing. The use of common pins that do not provide rotational and axial stability during fracture stabilization should be discouraged in the future. Analyses should include at least biomechanical and histological evaluations, even if the focus of the study is directed towards the elucidation of molecular mechanisms of fracture healing using the largely available spectrum of antibodies and gene-targeted animals to study molecular mechanisms of fracture healing. This review discusses distinct requirements for the experimental setups as well as the advantages and pitfalls of the different fixation techniques in rats and mice.

The effects of parathyroid hormone applied at different regimes on the trochanteric region of the femur in ovariectomized rat model of osteoporosis.

This study aims to investigate the effects of two application frequencies of parathyroid hormone on the trochanteric region of rat femur. Forty-three-month-old female Sprague-Dawley rats were divided into 4 groups (n = 10/group). Three groups were ovariectomized, and 8 weeks later they were administered the following treatments (5 weeks): soy-free diet (OVX), subcutaneously injected PTH (0.040 mg/kg) 5 days a week (PTH 5x/w), subcutaneously injected PTH (0.040 mg/kg) every 2 days (PTH e2d), and a sham group. The values of the biomechanical and histomorphometric parameters showed higher results in 5x/w animals in comparison to the OVX and PTH 2ed groups. The ratio between bone diameter/marrow diameter (B.Dm/Ma.Dm) in subtrochanteric cross sections did not show any significant differences between PTH 5x/w and PTH e2d. The increased bone formation rate was observed under PTH treatment in both groups mainly at the endosteal side. The endosteum seems here to be one of the targets of PTH with an accelerate bone formation and a pronounced filling-in of intracortical cavities with higher intensity for the PTH 5x/w in comparison to PTH e2d rats.

Effect of parathyroid hormone on hypogonadism induced bone loss of proximal femur of orchiectomized rat.

PURPOSE: Management of hypogonadism-induced osteoporosis in elderly men is still a challenge. We investigated the short-term effects of parathyroid hormone (PTH) treatments on strength, micro-architecture, and mineral density of trochanteric region of orchiectomized rat femur. METHODS: Eight-month-old male Sprague-Dawley rats (n = 44) were divided into two groups: (1) orchiectomized (ORX) and (2) sham group. Twelve weeks after orchiectomy, half of the orchiectomized animals were treated with daily subcutaneously injected PTH (0.040 mg/kg/BW) (ORX-PTH) for 5 weeks. The other half remained untreated (ORX). The sham-operated group was divided and treated in the same way (sham, sham-PTH). After 5 weeks, both femurs were excised for biomechanical and histomorphometric analysis, trabecular measurements, mineral content assessment, and immunofluorescence analysis. RESULTS: The femoral trochanteric strength after PTH treatment was enhanced in the breaking test (ORX-F(max) = 158.7 N vs. ORX + PTH-F(max) = 202 N). Stiffness of treated ORX animals reached nearly the levels observed in untreated sham rats. PTH therapy improved the trabecular connectivity, width, and area (ORX-Tb.Ar = 47.79% vs. ORX + PTH-Tb.Ar = 68.47%, P < 0.05) in the proximal femur. The treated rats showed significantly improved mineral content in ashed femurs (ORX-mineral content = 43.73% vs. ORX + PTH-mineral content = 49.49%) when compared to the untreated animals. A comparison of widths of fluorescence bands in cortical bone of the subtrochanteric cross-sections showed a significant increase in oppositions after the PTH therapy. CONCLUSIONS: Our finding supports the hypothesis that PTH therapy seems to be a rational therapy in patients with hypogonadism induced bone loss and improves the bone strength of trochanteric region of rat femur.

Improvement of femoral bone quality after low-magnitude, high-frequency mechanical stimulation in the ovariectomized rat as an osteopenia model.

The treatment and prevention of osteoporosis involve great challenges. Nonpharmacological and supportive therapy procedures, sport, and physical exercises seem to prevent bone loss and improve bone mass. In the present study, we examined the effect of whole-body vertical vibration (WBVV) on femoral intertrochanteric bone quality in the rat osteoporosis model. Sixty female Sprague-Dawley rats, 3-month old, were ovariectomized (OVX) or sham-operated. After 3 months, each group was divided into two subgroups. In one of the subgroups, rats were treated with WBVV at 90 Hz (3.9 g) for 35 days; the second subgroup remained untreated. After killing the animals, biomechanical strength and trabecular bone architecture of the proximal region of femurs were analyzed. New cortical bone appositions and mineral density of femurs were additionally measured. Treatment with WBVV resulted in improved biomechanical properties. Maximal load and stiffness of the intertrochanteric region of femurs after WBVV were significantly enhanced. Maximal load and stiffness in treated OVX animals reached the levels observed in untreated sham rats. WBVV significantly improved all measured histomorphometric parameters in the trabecular area. Treated rats showed significantly improved mineral content in ashed femurs compared to untreated animals. A comparison of widths of fluorescence bands in cortical bone of subtrochanteric cross sections did not show any significant differences between the groups after WBVV. Low-magnitude, high-frequency mechanical stimulation improves bone strength in the proximal femur and may be a possible nonpharmacologic treatment option for postmenopausal osteoporosis.

Evaluation of bone quality and quantity in osteoporotic mice--the effects of genistein and equol.

The technology of gene manipulation is often used in mice. A crucial point for osteoporosis research is the evaluation of biomechanical and morphologic parameters. These parameters, however, are difficult to measure in mice. Nevertheless, this study demonstrates the capability of using techniques for the evaluation of bone quality and quantity after various treatments in osteopenic mice. After ovariectomy, 60 C57BL/6J mice were divided into 4 groups and were fed a soy-free diet (C) supplemented with estradiol, genistein or equol for 3 months. To analyze the osteoprotective effects of the tested supplements, we evaluated the bone biomechanical properties, histomorphometric changes and bone mineral density of the proximal tibiae metaphysis. The biomechanical parameters of genistein (GEN) were shown to be similar to those levels observed with estradiol (E). The biomechanical parameters of both GEN and E were significantly superior to those observed with C. Supplementation with equol (EQO) demonstrated higher mean biomechanical values than those observed with C. The histomorphometric evaluation demonstrated an increased number of nodes in mice treated with GEN and E as compared to the mice treated with EQO and C. Treatment with E and EQO led to improved cortical bone, which was only partly seen with the mice treated with GEN. The analysis of the bone mineral density (BMD) demonstrated that treatment with GEN and E resulted in a significant improvement as compared to the mice treated with C, while the cancellous density was significantly increased in all of the supplementation groups. This study conclusively demonstrated that bone quality and quantity parameters can be measured in mice. Furthermore, biomechanical and morphologic evaluations were shown to be reliable for use in mice. Further studies may combine these techniques with gene manipulation technology to better understand osteoporosis. Treatment with GEN resulted in improved biomechanical results and enhancement of morphologic parameters.

Effects of isoflavones equol and genistein on bone quality in a rat osteopenia model.

Phytoestrogens might be an alternative medication in prophylaxis and treatment of osteoporosis. In this study, the osteoprotective effects of genistein (GEN) and equol (EQO) were evaluated. After ovariectomy, 44 rats received soy-free food (Control, C) and developed substantial osteoporosis over the course of two months. After that period, the rats were divided into different groups and fed estradiol (E), GEN or EQO for 35 days. To analyze the osteoprotective effects of the tested substances, bone biomechanical properties and histomorphometric changes of the lumbar vertebrae were evaluated. In analyzing the vertebral body compression strength, we found that the EQO (103.8%) and GEN (96.8%) groups reached similar levels relative to the E group, while the C group reached 77.7% of the biomechanical properties of the E group. EQO was significantly superior to C. The histomorphometric evaluation demonstrated an increased number of nodes in EQO- and E-treated rats compared to GEN- and C-treated rats. E led to an improvement of cortical as well as trabecular bone, an advantage that was only partly seen in the other groups. Treatment with phytoestrogens induced improved bone quality. EQO and GEN might be alternatives for hormone replacement therapy, although further studies are needed to elucidate possible side effects.

Improvement of trochanteric bone quality in an osteoporosis model after short-term treatment with parathyroid hormone: a new mechanical test for trochanteric region of rat femur.

UNLABELLED: We have examined the changes induced in the trochanteric region of femur of ovariectomized rat after administration of estradiol and parathyroid hormone. We have developed a reproducible biomechanical test and produced trochanteric fractures to evaluate stiffness and strength of this region in addition to histomorphometry. INTRODUCTION: We investigated the short-term effects of parathyroid hormone (PTH) and estrogen (E) on the strength of the rat trochanteric region in a new mechanical test. METHODS: Forty-four 3-month-old female Sprague-Dawley rats were ovariectomized and 8 weeks later treated with soy-free diet (C), daily applications of orally supplied E (0.5 mg/kg food) or subcutaneously injected PTH (0.014 mg/kg), for 5 weeks, and an additional untreated group was added as sham-operated. The femurs were examined for biomechanical and histomorphometric changes. RESULTS: Our new mechanical test was validated in a right-left comparison. The PTH treatment induced significantly superior biomechanical results (F (max) = 225.3 N, stiffness = 314.9 N/mm) compared to E (F (max) = 182.9 N, stiffness = 237.2 N/mm), C (F (max) = 166.03 N, stiffness = 235.56 N/mm), and sham (F (max) = 192.1 N, stiffness = 267.2 N/mm). Animals of the PTH group demonstrated a significantly improved trabecular bone structure and area (75.67%) in comparison to the E (61.04%) and C (57.18%) groups. CONCLUSION: Our new biomechanical test is valid and produces trochanteric fracture. Our results show that the short-term antiosteoporotic effects of PTH are in the trochanteric region of ovariectomized rat superior to E.

Estrogen and raloxifene improve metaphyseal fracture healing in the early phase of osteoporosis. A new fracture-healing model at the tibia in rat.

BACKGROUND: Fracture healing in osteoporosis is delayed. Quality and speed of fracture healing in osteoporotic fractures are crucial with regard to the outcome of patients. The question arises whether established antiosteoporotic drugs can further improve fracture healing. MATERIALS AND METHODS: Osteoporosis manifests predominantly in the metaphyseal bone. Nevertheless, an established metaphyseal fracture model is lacking. A standardized metaphyseal fracture-healing model with stable plate fixation was developed for rat tibiae. The healing process was analyzed by biomechanical, gene expression, and histomorphometric methods in ovariectomized (OVX) and sham-operated rats (SHAM), compared to standardized estrogen (E)- and raloxifene (R)-supplemented diets. RESULTS: Estrogen and raloxifene improved the biomechanical properties of bone healing compared to OVX (Yield load: SHAM = 63.1 +/- 20.8N, E = 60.8 +/- 17.9N, R = 44.7+/-17.5N, OVX = 32:5 +/- 22.0N). Estrogen vs OVX was significant based on a denser trabecular network. Raloxifene greatly induced total callus formation ((R = 5.3 +/- 0.9 mm2, E = 4.7 +/- 0.5 mm2, SHAM = 4.51 +/- 0.61 mm2, OVX =4.1 +/- 0.6 mm2), whereas estrogen mainly enhanced new endosteal bone formation. There was no correlation between the gene expression (osteocalcin, collagen1alpha1, IGF-1, tartrate-resistant phosphatase) in the callus and the morphology and quality of callus formation. CONCLUSION: Raloxifene and estrogen improve fracture healing in osteoporotic bone significantly with regard to callus formation, resistance, and elasticity. The biomechanically stable metaphyseal osteotomy model with T-plate fixation presented here has proven to be appropriate to investigate fracture healing in osteoporosis.

Effects of low-magnitude, high-frequency mechanical stimulation in the rat osteopenia model.

SUMMARY: In this study, short-term, whole-body vertical vibration at 90 Hz improved trabecular bone quality. There was an improvement of bone quality and density in both osteoporotic and control rats. This treatment may therefore be an attractive option for the treatment of osteoporosis. INTRODUCTION: Aside from pharmacological treatment options, physical exercise is known to augment bone mass. In this study, the effects of whole-body vertical vibration (WBVV) on bone quality and density were evaluated using an osteoporotic rat model. METHODS: Sixty female Sprague Dawley rats were ovariectomized (C) or sham (SHAM) operated at the age of 3 months. After 3 months, both groups were divided into two subgroups that received either WBVV at 90 Hz for 35 days or no treatment. After sacrificing the rats, we evaluated vertebral bone strength, histomorphometric parameters, and bone mineral density (BMD). RESULTS: Treatment with WBVV resulted in improved biomechanical properties. The yield load after WBVV was significantly enhanced. According to yield load and Young's modulus, the treated OVX rats reached the level of the untreated SHAM animals. In all measured histomorphometric parameters, WBVV significantly improved bone density. Treatment with WBVV demonstrated greater effects on the trabecular bone compared to the cortical bone. The ash-BMD index showed significant differences between treated and untreated rats. CONCLUSION: Using WBVV as a non-pharmacological supportive treatment option for osteoporosis demonstrated an enhancement of bone strength and bone mass. This procedure may be an attractive option for the treatment of osteoporosis.

Effect of testosterone, raloxifene and estrogen replacement on the microstructure and biomechanics of metaphyseal osteoporotic bones in orchiectomized male rats.

INTRODUCTION: Currently, osteoporosis research is rarely undertaken in males but an increase in male life expectancy in the company of hypogonadism suggests the necessity for potential therapeutic options. MATERIALS AND METHODS: In this study, the changes in bone structure under standardized testosterone- (T), raloxifene- (R) and estrogen (E)-supplemented diets were analyzed in osteoporotic castrated male rats. RESULTS: Unexpected biomechanical results could be only explained by the histomorphometry, but not by BMD measurements obtained from the qCT. All tested substances showed a significant improvement in the trabecular network (trabecular bone area for C: 2.55 mm(2), T: 4.25 mm(2), R: 4.22 mm(2) and E: 4.28 mm(2)), and suggests that the bone structure was preserved. For the metaphyseal cortical bone, a significant loss was detected in T (CBP: 18.7%) compared to R (CBP: 30.0%), E (CBP: 26.8%) and even to the osteoporotic control (CBP: 28.6%). This explains the observed early mechanical final failure after T supplementation. However, due to the preserved trabecular bone in T, the occurrence of the first microfractures (yL: 49 +/- 21.4 N) was significantly later than in the osteoporotic control (yL: 39.5 +/- 15.5 N). Raloxifene performed well in hindering the bone loss associated with osteoporosis. However, its effect (yL: 83.3 +/- 16.5 N) did not approach the protective effect of E (yL: 99.2 +/- 21.1 N). CONCLUSION: Testosterone only preserved the deterioration of the trabecular bone but not of the cortical bone. Raloxifene prevented the bone loss associated with osteoporosis at all bony structures. This effect did not approach the protective effect of estrogen on trabecular bone, but it is more suitable for male individuals because it has no feminizing effects on the subject.

Vitex agnus castus as prophylaxis for osteopenia after orchidectomy in rats compared with estradiol and testosterone supplementation.

Osteoporosis research undertaken in males is rare and there are only a few therapeutic options. Phytoestrogens might be a safe alternative for prophylaxis. Sixty 3-month-old male rats were orchidectomized and divided into five groups. The groups either received soy-free food (C), estradiol (E), testosterone (T) or Vitex agnus castus in different concentrations (AC high/AC low) for 12 weeks. The tibia metaphysis was tested biomechanically and histomorphometrically. The AC high group reached 87% of the biomechanical values of the estradiol group and was significantly superior to the control group. Testosterone supplementation resulted in poor biomechanical properties. The cortical bone parameters of the AC group were similar to the control group, while supplementation with estradiol and testosterone demonstrated a reduction of cortical bone. The AC high group reached 88.4% of trabecular bone area, 80.7% of trabecular number and 66.9% of the number of trabecular nodes compared with estradiol supplementation. Vitex agnus castus demonstrated osteoprotective effects in males. It preserves the cortical as well as the trabecular bone and might be a safe alternative for HRT. Testosterone supplementation has positive effects on trabecular bone, which are concurrently counteracted by the loss of cortical bone.

[Feasibility of flat-panel volumetric computed tomography (fpVCT) in experimental small animal imaging of osteoporosis - initial experience]. / Erste Erfahrungen mit einem Flächendetektor-Volumen-CT (fpVCT) in der experimentellen Osteoporosediagnostik am Kleintiermodell.

BACKGROUND: Flat-panel volumetric computed tomography (fpVCT) is a new, noninvasive CT imaging modality with increased isotropic resolution. Technical details, potential applications, and our initial experience with a fpVCT prototype scanner in the imaging of osteoporosis in a rat model are presented. METHODS: To date, 21 rats have been investigated in vivo with fpVCT. Pharmacologic effects on bone mineral density (BMD) and structure were of special interest. Image evaluation focussed on the second lumbar vertebra and the left femoral bone. To validate measurement results, BMD values calculated with fpVCT were correlated with results of BMD measurements from ashing of the second lumbar vertebra and femoral bones. RESULTS: Our initial results show that fpVCT is capable of detecting differences in BMD between ovariectomized rats treated with estradiol and a control group with high statistical significance (p<0.05), corresponding to ashing as the gold standard. CONCLUSIONS: In a rat model, fpVCT imaging is especially useful in longitudinal in vivo investigations of BMD measures. Spatial resolution of up to 150 microm allows imaging of the trabecular structure only in human cadaveric bones.