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The healing of predominantly metaphyseal fractures in postmenopausal osteoporosis is delayed and comparatively poor. Due to the potential side effects of HRT, natural alternatives are appealing. The aim of this study was to determine whether Cimicifuga racemosa extract BNO 1055 improves metaphyseal fracture healing in severe osteopenic bone in rats. Thirty-three 12-week-old female rats developed severe osteopenia during 10 weeks after ovariectomy. After metaphyseal tibial-osteotomy and standardized T-plate-osteosynthesis, the healing periods in ovariectomized rats (C), 17-[alpha]-estradiol (E) and Cimicifuga racemosa (CR) supplemented diets were assessed for 35 days. Changes in callus morphology were evaluated qualitatively by biomechanical testing and quantitatively in microradiographies and fluorochrome-labeled histological sections. The CR-supplementation slightly improved callus quality and trabecular bone formation. It significantly enhanced the endosteal callus density compared to C group (Cl.Dn.e C: 59.08 ± 21.89, E: 45.95 ± 18.39, CR: 60.85 ± 18.66*), though most of the other morphological parameters examined showed no improvement. The time course of fracture healing did not change due to CR. Estrogen-supplementation enhanced the biomechanical properties of the fracture site. Trabecular bone was improved indicating the physiological endosteal healing process. The CR-supplementation did not exhibit positive effects in severe (senile) osteopenic fracture healing as seen in early (postmenopausal) osteoporosis in rats. Callus formation was slightly improved under CR. Estrogen improved fracture healing in severe osteopenic bone, while the extent of callus formation played a minor role. Copyright © 2010 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Objectives: Patient-specific diagnostic and therapeutic approaches are important in the care of people with chronic wounds. The heterogeneity of underlying disease profiles and the diversity of the wound micro-environment make generalized approaches difficult. While high-throughput molecular diagnostic methods are increasingly widespread and available, the analysis of objective biomolecular disease patterns has not found its way into everyday wound management. The aim of this study is to evaluate the use of wound swab samples for the analysis of biomarkers and disease patterns in people with chronic wounds. Methods: A sample cohort from the multicenter "Wound-BIOME" project was analyzed. The project aims to comprehensively investigate the local micro-environment of chronic wounds of various entities, healing tendencies and regeneration stages at the biomolecular level. A sample collection and handling protocol suitable for everyday use was tested and evaluated regarding feasibility for multiplex immunoassay, proteomics, small RNA sequencing (miRNA) and metagenome analyses (microbiomics). Results: It could be shown that standard wound swabs are well-suited for the analysis of the complex wound micro-environment using various high-throughput methods. Despite the sample heterogeneity, the quality was adequate to analyze biomolecular patterns. Conclusions: Initial analyses of protein signatures, microbial wound communities and miRNA patterns show promising results for future individualized diagnostics and targeted interventions.

In this study, the potential of silk fibroin biomaterials for enhancing wound healing is explored, focusing on their integration into a human 3D ex vivo wound model derived from abdominoplasties. For this purpose, cast silk fibroin membranes and electrospun nonwoven matrices from Bombyx mori silk cocoons were compared to untreated controls over 20 days. Keratinocyte behavior and wound healing were analyzed qualitatively and quantitatively by histomorphometric and immune histochemical methods (HE, Ki67, TUNEL). Findings reveal rapid keratinocyte proliferation on both silk fibroin membrane and nonwoven matrices, along with enhanced infiltration in the matrix, suggesting improved early wound closure. Silk fibroin membranes exhibited a significantly improved early regeneration, followed by nonwoven matrices ( p < 0.05) compared to untreated wounds, resulting in the formation of multi-layered epidermal structures with complete regeneration. Overall, the materials demonstrated excellent biocompatibility, supporting cell activity with no signs of increased apoptosis or early degradation. These results underscore silk fibroin's potential in clinical wound care, particularly in tissue integration and re-epithelialization, offering valuable insights for advanced and-as a result of the electrospinning technique-individual wound care development. Furthermore, the use of an ex vivo wound model appears to be a viable option for pre-clinical testing.

Objective: Hard-to-heal (chronic) wounds are common in patients with diabetes and are associated with a decrease in quality of life (QoL). Pathogenic bacteria often colonise hard-to-heal wounds and hinder the healing process which poses a high risk for (systemic) infections. In this study, we aim to prove that probiotics are capable of displacing human pathogenic bacteria, ameliorating inflammation and positively influencing the microenvironment/microbiome of skin and mucosa., Method: In this pilot study, patients with diabetes and hard-to-heal wounds with a duration of 2-120 months received an oral multispecies probiotic daily for six months. Changes in oral, stool and wound microbiome were investigated, and the effects of the probiotic intervention on wound healing, periodontitis and wound-specific quality of life (Wound-QOL-17) were analysed throughout the course of this clinical study., Results: In total, seven of the 20 patients included were unable to complete the study. After six months of oral probiotic intake supplementation in five out of the remaining 13 patients, the wounds had healed completely. Most patients reported an improvement in wound-specific QoL, with particular positive effects on pain and mobility. Microbiome analysis revealed a reduction in Staphylococcus aureus and Pseudomonas aeruginosa , and Staphylococcus epidermis in healed wounds., Conclusion: This findings of this study provide evidence for the beneficial effects of the oral application of a multispecies probiotic over six months in patients with diabetes and hard-to-heal wounds on wound closure, wound microbial pattern, QoL, and on dental health. A randomised, placebo-controlled, double-blinded clinical trial is required to verify the results.

The treatment of chronic wounds still represents a major challenge in wound management. Recent estimates suggest that 60-80% of chronic wounds are colonized by pathogenic microorganisms, which are strongly considered to have a major inhibiting influence on the healing process. By means of an innovative biofilm model based on human plasma, the time-dependent behavior of various bacterial strains under wound-milieu-like conditions were investigated, and the growth habits of different cocci species were compared. Undescribed fusion events between colonies of MRSA as well as of Staphylococcus epidermidis were detected, which were associated with the remodeling and reorganization of the glycocalyx of the wound tissue. After reaching a maximum colony size, the spreading of individual bacteria was observed. Interestingly, the combination of different cocci species with Pseudomonas aeruginosa in the human plasma biofilm revealed partial synergistic effects in these multispecies organizations. RT-qPCR analyses gave a first impression of the relevant proteins involved in the formation and maturation of biofilms, especially the role of fibrinogen-binding proteins. Knowledge of the maturation and growth behavior of persistent biofilms investigated in a translational human biofilm model reflects a starting point for the development of novel tools for the treatment of chronic wounds.

Biofilms are a major problem in hard-to-heal wounds. Moreover, they are composed of different species and are often tolerant to antimicrobial agents. At the same time, interspecific synergy and/or competition occurs when some bacterial species clash. For this reason, the tolerance of two dual-species wound biofilm models of Pseudomonas aeruginosa and Staphylococcus aureus or Enterococcus faecium against antimicrobials and antimicrobial dressings were analyzed quantitatively and by confocal laser scanning microscopy (CLSM). The results were compared to findings with planktonic bacteria. Octenidine-dihydrochloride/phenoxyethanol and polyhexamethylene biguanide (PHMB) irrigation solutions showed a significant, albeit delayed reduction in biofilm bacteria, while the PHMB dressing was not able to induce this effect. However, the cadexomer-iodine dressing caused a sustained reduction in and killed almost all bacteria down to 10 2 cfu/mL within 6 days compared to the control (10 10 cfu/mL). By means of CLSM in untreated human biofilm models, it became evident that P. aeruginosa dominates over E. faecium and S. aureus . Additionally, P. aeruginosa appeared as a vast layer at the bottom of the samples, while S. aureus formed grape-like clusters. In the second model, the distribution was even clearer. Only a few E. faecium were visible, in contrast to the vast layer of P. aeruginosa . It seems that the different species avoid each other and seek their respective niches. These mixed-species biofilm models showed that efficacy and tolerance to antimicrobial substances are nearly species-independent. Their frequent application appears to be important. The bacterial wound biofilm remains a challenge in treatment and requires new, combined therapy options.

Objective: Wound biofilms are one of the greatest challenges in the therapy of hard-to-heal (chronic) wounds, as potent antimicrobial substances fail to eradicate bacteria within short incubation periods. Preclinical investigations using novel model systems that closely mimic the human wound environment and wound biofilm are required to identify new and effective therapeutic options. This study aims to identify bacterial colonisation patterns that are relevant for diagnosis and therapy., Method: In this study, a recently established human plasma biofilm model (hpBIOM) was incorporated into a wound within human dermal resectates after abdominoplasty. The interaction of the biofilm-forming bacteria meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa with the skin cells was investigated. Possible effects on wound healing processes in correlation with the persistence of the biofilm in the wound environment were analysed in patients with leg ulcers of different aetiologies and biofilm burden., Results: Using haematoxylin and eosin staining, species-dependent infiltration modes of the bacteria into the wound tissue were determined for the pathogens MRSA and Pseudomonas aeruginosa . The spreading behaviour correlated with clinical observations of the spatial distributions of the bacteria. In particular, the clinically prominent Pseudomonas aeruginosa -specific distension of the wound margin was identified as epidermolysis due to persistent infiltration., Conclusion: The hpBIOM applied in this study represents a potential tool for preclinical analyses dealing with approval processes for new antimicrobial applications. In terms of clinical practice, a microbiological swabbing technique including the wound margin should be routinely applied to prevent wound exacerbation.

Current standards insufficiently acknowledge the influence of the wound micro-environment on the efficacy of antimicrobial agents. To address this, octenidine/phenoxyethanol, polyhexanide, povidone-iodine, and sodium-hypochloride/hypochlorous acid solutions were submitted to standard-based (DIN-EN-13727) or modified peptide-based challenges and compared to a simulated clinical reference using human acute or chronic wound exudate (AWF/CWF). Antimicrobial efficacy against S. aureus and P. aeruginosa was compared using a quantitative suspension method. Agreement between methods were investigated using Bland-Altman (B&A) analysis. Different substances and challenges demonstrated diverging results, depending on class and concentration of agent and challenge. Highly concentrated antiseptics maintained a high efficacy under complex challenges, while especially chlorine-based irrigation solutions showed a remarkably reduced antimicrobial effect. Composition of challenge substance proved more relevant than pure concentration. Therefore, the current standard challenge conditions did not adequately reflect the wound micro-environment with over- or under-estimating antimicrobial efficacy, whilst the modified peptide-challenge showed a higher level of agreement with simulated realistic conditions (AWF/CWF). The results emphasize that a "one-fits-all" approach is not feasible to generalize antimicrobial efficacy, as certain aspects of the complex micro-environment pose a differing influence on varying agents. Based on these results, revision and target focused adaptation of the current standards should be considered.

Concern about microbial tolerance and resistance to established antimicrobials drives research into alternatives for local antiseptic wound treatment. Precise efficacy profiles are thereby important in the evaluation of potential alternative antimicrobials, and protein interference ("protein error") is a key factor. Here, the antimicrobial efficacy of cetylpyridinium chloride (CPC) and miramistin (MST) was compared to the established antimicrobials octenidine (OCT), povidon-iodine (PVP-I), polyhexamethylene-biguanide (PHMB) and chlorhexidine (CHX). Efficacy was evaluated after 0.5, 1, 3, 5 and 10 min against Staphylococcus aureus , Pseudomonas aeruginosa, Escherichia coli , Enterococcus faecium and Candida albicans using an in vitro quantitative suspension method (based on DIN EN 13727). To investigate protein interference, 0.3% or 3% bovine albumin was used as the challenge. OCT and PVP-I demonstrated a significant efficacy within 0.5 min, regardless of the microbial organism and protein challenge ( p < 0.01). CPC and MST showed no inferiority in efficacy, with only MST needing up to 3 min to achieve the same microbial reduction. PHMB and CHX also achieved significant reduction rates over the tested time-course, yet demonstrated a necessity for prolonged exposure (up to 10 min) for comparable reduction. A protein interference was predominantly observed for PHMB against S. aureus , but without statistically significant differences in antimicrobial efficacy between the 0.3% and 3% protein challenges. All other tested agents showed no relevant interference with the presence of protein. CPC and MST proved to be non-inferior to established wound antiseptics agents in vitro. In fact, CPC showed a more efficient reduction than PHMB and CHX despite there being an introduced protein challenge. Both agents demonstrated no significant "protein error" under challenging conditions (3% albumin), posing them as valid potential candidates for alternative antimicrobials in wound management., Competing Interests: Conflict of interest: All authors declare no conflict of interest regarding this paper., (© 2022 the Author(s), licensee AIMS Press.)

Objectives: Treating infected or chronic wounds burdened with biofilms still is a major challenge in medical care. Healing-stimulating factors lose their efficacy due to bacterial degradation, and antimicrobial substances negatively affect dermal cells. Therefore, alternative treatment approaches like the pulsed low intensity laser therapy (LILT) require consideration., Methods: The effect of pulsed LILT (904 nm, in three frequencies) on relevant human cells of the wound healing process (fibroblasts (BJ), keratinocytes (HaCaT), endothelial cells (HMEC), monocytes (THP-1)) were investigated in in-vitro and ex-vivo wound models with respect to viability, proliferation and migration. Antimicrobial efficacy of the most efficient frequency in cell biological analyses of LILT (3200 Hz) was determined in a human biofilm model (lhBIOM). Quantification of bacterial load was evaluated by suspension method and qualitative visualization was performed by scanning electron microscopy (SEM)., Results: Pulsed LILT at 904 nm at 3200 Hz ± 50% showed the most positive effects on metabolic activity and proliferation of human wound cells in vitro (after 72 h - BJ: BPT 0.97 ± 0.05 vs. 0.75 ± 0.04 (p = 0.0283); HaCaT: BPT 0.79 ± 0.04 vs. 0.59 ± 0.02 (p = 0.0106); HMEC: 0.74 ± 0.02 vs. 0.52 ± 0.04 (p = 0.009); THP-1: 0.58 ± 0.01 vs. 0.64 ± 0.01 (p > 0.05) and ex vivo. Interestingly, re-epithelialization was stimulated in a frequency-independent manner. The inhibition of metabolic activity after TNF-α application was abolished after laser treatment. No impact of LILT on monocytes was detected. Likewise, the tested LILT regimens showed no growth rate reducing effects on three bacterial strains (after 72 h - PA: -1.03%; SA: -0.02%; EF: -1,89%) and one fungal (-2.06%) biofilm producing species compared to the respective untreated control. Accordingly, no significant morphological changes of the biofilms were observed after LILT treatment in the SEM., Conclusions: Frequent application of LILT (904 nm, 3200 Hz) seems to be beneficial for the metabolism of human dermal cells during wound healing. Considering this, the lack of disturbance of the behavior of the immune cells and no growth-inducing effect on bacteria and fungi in the biofilm can be assigned as rather positive. Based on this combined mode of action, LILT may be an option for hard to heal wounds infected with persistent biofilms., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The treatment of acute and chronic infected wounds with residing biofilm still poses a major challenge in medical care. Interactions of antimicrobial dressings with bacterial load, biofilm matrix and the overall protein-rich wound microenvironment remain insufficiently studied. This analysis aimed to extend the investigation on the efficacy of a variety of antimicrobial dressings using an in vitro biofilm model (lhBIOM) mimicking the specific biofilm-environment in human wounds. Four wound dressings containing polyhexanide (PHMB), octendine di-hydrochloride (OCT), cadexomer-iodine (C-IOD) or ionic silver (AG) were compared regarding their antimicrobial efficacy. Quantitative analysis was performed using a quantitative suspension method, separately assessing remaining microbial counts within the solid biofilm as well as the dressing eluate (representing the absorbed wound exudate). Dressing performance was tested against P. aeruginosa biofilms over the course of 6 days. Scanning electron microscopy (SEM) was used to obtain qualitative visualization on changes in biofilm structure. C-IOD demonstrated superior bacterial reduction. In comparison it was the only dressing achieving a significant reduction of more than 7 log 10 steps within 3 days. Neither the OCT- nor the AG-containing dressing exerted a distinct and sustained antimicrobial effect. PHMB achieved a non-significant microbicidal effect (1.71 ± 0.31 log 10 steps) at day 1. Over the remaining course (6 days) it demonstrated a significant microbistatic effect compared to OCT, AG and the control. Quantitative results in the dressing eluate correlate with those of the solid biofilm model. Overall, AG- and OCT-containing dressings did not achieve the expected anti-biofilm efficacy, while C-IOD performed best. Chemical interaction with the biofilms extrapolymeric substance (EPS), visualized in the SEM, and dressing configuration (agent concentration and release pattern) are suspected to be responsible. The unexpected low and diverse results of the tested antimicrobial dressings indicate a necessity to rethink non-debridement anti-biofilm therapy. Focussing on the combination of biofilm-disruptive (for EPS structure) and antimicrobial (for residing microorganisms) features, as with C-IOD, using dehydration and iodine, appears reasonably complementary and an optimal solution, as suggested by the here presented in vitro data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stuermer, Plattfaut, Dietrich, Brill, Kampe, Wiencke, Ulatowski, Geffken, Rembe, Naumova, Debus and Smeets.)

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Biofilms pose a relevant factor for wound healing impairment in chronic wounds. With 78% of all chronic wounds being affected by biofilms, research in this area is of high priority, especially since data for evidence-based selection of appropriate antimicrobials and antiseptics is scarce. Therefore, the objective of this study was to evaluate the anti-biofilm efficacy of commercially available hypochlorous wound irrigation solutions compared to established antimicrobials. Using an innovative complex in-vitro human plasma biofilm model (hpBIOM), quantitative reduction of Pseudomonas aeruginosa , Staphylococcus aureus , and Methicillin-resistant S. aureus (MRSA) biofilms by three hypochlorous irrigation solutions [two <0.08% and one 0.2% sodium hypochlorite (NaClO)] was compared to a 0.04% polyhexanide (PHMB) irrigation solution and 0.1% octenidine-dihydrochloride/phenoxyethanol (OCT/PE). Efficacy was compared to a non-challenged planktonic approach, as well as with increased substance volume over a prolonged exposure (up to 72 h). Qualitative visualization of biofilms was performed by scanning electron microscopy (SEM). Both reference agents (OCT/PE and PHMB) induced significant biofilm reductions within 72 h, whereby high volume OCT/PE even managed complete eradication of P. aeruginosa and MRSA biofilms after 72 h. The tested hypochlorous wound irrigation solutions achieved no relevant penetration and eradication of biofilms despite increased volume and exposure. Only 0.2% NaClO managed a low reduction under prolonged exposure. The results demonstrate that low-dosed hypochlorous wound irrigation solutions are significantly less effective than PHMB-based irrigation solution and OCT/PE, thus unsuitable for biofilm eradication on their own. The used complex hpBIOM thereby mimics the highly challenging clinical wound micro-environment, providing a more profound base for future clinical translation., (Copyright © 2020 Rembe, Huelsboemer, Plattfaut, Besser and Stuermer.)

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.

Zinc as therapeutic agent in skin and wound care has been known for centuries, but its role is controversial and comprehensive investigations in nutrient-deficient environments are lacking. We aimed to provide a broad analysis of different zinc derivatives on proliferation, apoptosis and antimicrobial properties in a simulated nutrient-deficient environment in vitro . Human fibroblasts (CRL2522) and keratinocytes (HaCaT) were treated with a broad concentration range (10 - 0.0001 µg/mL) of zinc-sulfate (ZnSO 4 ), -gluconate (ZnGluc) and -histidine (ZnHis) for 1-6 days under nutrient-deficient media conditions. Cell proliferation was investigated by XTT assay. Targeted analyzes in proliferation ( E2F1 , PCNA ) and apoptosis ( TP53 ) associated genes were performed via qRT-PCR and apoptosis was determined via FACS (annexin V/7-AAD staining). Antimicrobial efficacy was investigated using a quantitative suspension method against S. aureus , P. aeruginosa , E. coli , and C. albicans . The results indicated that 0.1 to 0.001 µg/mL Zn increased cell proliferation in both cell lines. Fibroblasts were more susceptible with significant proliferation peaks on days 2 & 6, and days 1 & 4 for keratinocytes. No relevant changes in gene expression were detected for E2F1 and PCNA nor for TP53 . Annexin-V/7-AAD-staining of fibroblasts revealed a small, yet insignificant reduction of apoptosis induction for ZnGluc and ZnSO 4 . ZnGluc and ZnSO 4 (0.1%) achieved high microbial reductions (4-5 log 10 reductions) against tested pathogens. ZnGluc and ZnSO 4 showed relevant pro-proliferative and antimicrobial, as well as tendential anti-apoptotic features in a simulated nutrient-deficient microenvironment in vitro . This further validates a potential benefit of local zinc treatment in deficient wound microenvironments.

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.

Lifestyle diseases such as diabetes and arteriosclerosis are rising in the increasingly aging society, and the number of patients with daily intake of glucose-lowering medication has also increased. Interestingly, knowledge about oral antidiabetics with regard to wound healing is scarce. Therefore, the aim of this study was to identify possible (side) effects of the most frequently prescribed oral antidiabetics on skin cells and wound healing. Four oral antidiabetics of different substance classes (i.e., metformin, glibenclamide, sitagliptin, repaglinide) were investigated with regard to the promotion of cell metabolism and migration of human skin fibroblasts and keratinocytes by XTT and scratch assays. In addition, histological and immunohistochemical analyses were performed in a 3D wound model to address the impact of the antidiabetics on regeneration processes, such as cell migration, fibroblast activity, epidermal thickness, and cell apoptosis. In comparison to systemic application, metformin displayed the most adverse effects in vitro in nearly all analyses, interestingly at serum equivalent concentrations. In contrast, sitagliptin and glibenclamide had a slight but insignificant effect on fibroblasts compared with keratinocytes. Repaglinide tended to have a negative influence on keratinocyte metabolism. Interestingly, antidiabetics generally induced a significantly enhanced rate of apoptosis in fibroblasts, with the exception of repaglinide.Antidiabetics influenced key players in wound healing, namely, keratinocytes and fibroblasts. Particularly, metformin impaired human skin cells. These findings should be kept in mind in further studies because of their putative relevance in patients suffering from chronic wounds that do not respond to various wound therapies.

Background: Sodium hypochlorite (NaClO, SHC)/hypochlorous acid (HClO, HCA) wound irrigation solutions have experienced a renaissance in the prevention and treatment of low-level wound infections. They are attributed with lower cytotoxicity and have therefore gained increasing attention in daily clinical practice., Objectives: To determine the cytotoxicity and antimicrobial efficacy of six NaClO/HClO wound irrigation solutions., Methods: For cytotoxicity evaluation (based on DIN EN 10993-5), human keratinocytes (HaCaT) and human skin fibroblasts (BJ) were used. Staphylococcus aureus and Pseudomonas aeruginosa were used for antimicrobial efficacy evaluation (based on DIN EN 13727). Solutions were evaluated after 1, 5 and 15 min of exposure. Additionally, physicochemical properties (pH and oxidation-reduction potential values) were investigated., Results: Efficacy and cytotoxicity varied significantly between solutions. Generally, increasing antimicrobial activity was associated with decreasing cell viability. Furthermore, a concentration- and time-dependent impact on pathogens and cells was observed: cytotoxic and antimicrobial activity increased with rising NaClO/HClO solution concentrations and extended exposure times. Based on these in vitro evaluations, the following ranking (lowest to highest microbicidal effect and cytotoxic impact) was found: Microdacyn60® (SHC/HCA-M) < Granudacyn® (SHC/HCA-G) < Veriforte™ (SHC/HCA-V) < KerraSol™ (SHC-K) < Lavanox® (SHC-L) ≪ ActiMaris®forte (SHC/SM-A)., Conclusions: The presented results indicate that microbicidal effects are almost always associated with certain negative side effects on cell proliferation. Efficacy and biocompatibility of NaClO/HClO solutions depend on their specific formulation and physicochemical properties. The investigations also underline the necessity for exact product- and application-specific efficacy profiles.

Background: The number of patients who has a daily intake of antihypertensive drugs is rising, due to an also rising prevalence of lifestyle diseases. Interestingly, knowledge about effects of these drugs in terms of wound healing is low., Objective: Based on a few differing studies, the idea arose that antihypertensives may have side effects on wound healing., Methods: Five antihypertensive drugs from different substance classes (metoprolol, amlodipine, ramipril, hydrochlorothiazide, candesartan) were investigated, in terms of possible impacts on cell metabolism and migration of human skin fibroblasts and keratinocytes. Additionally, histological and immunohistochemical analyses were performed in a 3-dimensional (3D) wound model addressing the influence on regeneration processes, such as cell migration, metabolic activity, apoptosis and epidermal thickness., Results: Hydrochlorothiazide and ramipril exerted inhibiting effects in nearly all analyses, interestingly, in serum equivalent doses. In contrast, candesartan and amlodipine induced slight positive effects in 2D as well as in 3D models. The previously described positive effects of β-blockers could only partially be confirmed by metoprolol. Antihypertensive drugs affected fibroblasts more than keratinocytes - whether positively or negatively., Conclusion: Antihypertensive drugs have an influence on keratinocytes and fibroblasts; they are not neutral. Candesartan has the most positive effects on skin cells. For angiotensin-converting enzyme inhibitors and thiazide diuretics, wound healing in a 3D model is delayed. β-Receptor blockers seem to improve wound healing to a small extent just like calcium channel blockers. These results should be evaluated in a clinical trial to verify their clinical relevance., (© 2019 S. Karger AG, Basel.)

Objective: Vitamins are essential for human health. In terms of local application for wound healing, vitamins' positive effect remains unclear. However, because of the regular appearance of nutritional deficiency in chronic wound patients, a favorable impact of locally applied vitamins can be hypothesized., Methods: Vitamins B3, B5, B6, B7, B9, B10, B12, and C individually as well as different combinations of B vitamins were investigated regarding their ability to promote proliferation and migration of human skin fibroblasts and keratinocytes. Proliferation assays with and without bacterial challenge, immunocytochemical staining, and scratch assay were used to determine the most effective combination(s)., Main Results: Some vitamin combinations showed a positive impact on proliferation, especially for keratinocytes after 72 hours. In terms of wound closure, the combinations B9 and B12; B3, B5, B6, and B10; and B3, B5, and B7 improved closure rates by 25% to 30%. The improved closure rates are also reflected by immunocytochemically detected upregulation of the migration marker CXCR4 for several combinations., Conclusions: Certain combinations of B vitamins demonstrate a positive influence on human keratinocytes and fibroblasts. Vitamins especially promoted fibroblast migration, and a statistically significant induction of keratinocyte proliferation was observed. Therefore, local vitamin application could benefit the physiologic wound healing process.

Treating infected acute and/or chronic wounds still represents a major challenge in medical care. Various interactions of antiseptic dressings with wound environments regarding antimicrobial efficacy remain unclear. Therefore, this work aimed to investigate the influence of human acute wound fluid (AWF) on the antimicrobial performance of different antiseptic foam dressings in vitro against typical bacterial wound pathogens. Eight antiseptic polyurethane foam dressings containing either a silver formulation or a polyhexamethylene-biguanide (PHMB) were assessed regarding their antimicrobial potency against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using a modified time-kill assay based on ISO EN 20743. The antiseptic efficacy was evaluated standardly as well as under the influence of human AWF after 2, 4, 6, and 24 hours. The specific chemical formulation and concentration of the antiseptic substance (ionic or nanocrystalline silver, silver sulfadiazine, PHMB 0.1%/0.5%) embedded within the dressings seemed to play a key role. For certain dressings (two nanocrystalline and one ionic silver dressing), the antimicrobial efficacy was significantly reduced under the influence of AWF compared to unchallenged test series. Unchallenged the efficacy of PHMB was comparable to silver against P. aeruginosa and even significantly superior against S. aureus and E. coli. Challenged with AWF the reduction rates for silver adjusted or even exceeded (P. aeruginosa) those of PHMB. Within a challenging wound environment, especially some silver formulations demonstrated a reduced bacterial reduction. Regarding the presented in vitro results, the biomolecular interactions of antiseptic wound dressings with wound fluid should be part of more extensive investigations, considering varying factors such as bacterial species and wound (micro)environment to develop targeted therapeutic regimes for the individual., (© 2018 by the Wound Healing Society.)

In this study, polyaminopropyl biguanide (PAPB) was compared to the molecularly closely related polyhexamethylene biguanide (PHMB) with respect to chemical relationship, antiseptic efficacy and cytotoxicity in vitro. Cytotoxicity for human keratinocytes (HaCaTs) and murine fibroblasts (L929) was determined according to ISO EN 10993-5 for both substances. Antimicrobial efficacy tests were performed via determination of the MBC, quantitative suspension method for substances and investigation of two PAPB- or PHMB-containing dressings against Staphyloccoccus aureus, Escherichia coli and Pseudomonas aeruginosa, according to international standards. Prior mass spectrometry was performed for chemical differentiation of the investigated substances. PHMB showed high toxicity even in low concentrations for both tested cell lines and a high antimicrobial efficacy against S. aureus and E. coli. In the case of PAPB, no or only low cytotoxicity was detected after 72 h, whilst comparable antibacterial features are lacking, as PAPB showed no relevant antimicrobial effects. Even though chemically closely related, PAPB proved to be ineffective in bacterial eradication, whilst PHMB showed a high efficacy. The discovery and establishment of safe and effective alternative antiseptics are important issues for the treatment of infected wounds. In particular, rising bacterial resistances to established agents, as well as ongoing discussions of potential toxic or carcinogenic effects emphasize this necessity. Nevertheless, the presented results highlight that even small changes in the chemical structure of related agents such as PHMB and PAPB can dramatically affect their efficacy and, therefore, need to be carefully distinguished and assessed side by side.

Background: Due to demographical changes the number of elderly patients depending on oral anticoagulation is expected to rise. Prolonged bleeding times in case of traumatic injuries represent the drawback of these medications, not only in major trauma, but also in superficial wounds. Therefore, dressings capable of accelerating coagulation onset and shortening bleeding times are desirable for these patients., Methods: The hemostatic potential and physical properties of different types of superficial wound dressings (standard wound pad, two alginates, chitosan, collagen (Lyostypt(®)), oxidized cellulose, and QuikClot(®)) were assessed in vitro. For this purpose the clotting times of blood under the influence of the named hemostatics from healthy volunteers were compared with Marcumar(®) or ASS(®) treated patients. For that, a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity was used., Results: The fastest coagulation onset was observed for oxidized cellulose (Ø 2.47 min), Lantor alginate-L (Ø 2.50 min) and QuikClot(®) (Ø 3.01 min). Chitosan (Ø 5.32 min) and the collagen Lyostypt(®) (Ø 7.59 min) induced clotting comparatively late. Regarding physical parameters, QuikClot(®) showed the lowest absorption capacity and speed while chitosan and both alginates achieved the highest. While oxidized cellulose displayed the best clotting times, unfortunately it also revealed low absorption capacity., Conclusions: All tested specimens seem to induce clotting independently from the administered type of oral anticoagulant, providing the possibility to neglect the disadvantage in clotting times arising from anticoagulation on a local basis. QuikClot(®), oxidized cellulose and unexpectedly alginate-L were superior to chitosan and Lyostypt(®). Due to its additional well-known positive effect on wound healing alginate-L should be considered for further investigations.

The antimicrobial activity of cetylpyridinium chloride (CPC) and miramistin (MST) solutions at different concentrations (5×10(-5) to 0.4%) and a dressing, containing 0.15% CPC, were tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli after 30 (solutions) and 60 min (fleece) incubation, respectively. Furthermore, the cytotoxic effects of CPC and MST were examined in human keratinocyte (HaCaT) and murine fibroblast (L929) cell lines. A dose of 3×10(-3)% CPC or MST was sufficient to entirely eradicate S. aureus after 30 min incubation. To achieve the same effect, higher concentrations were required against E. coli (0.025% CPC; 0.0125% MST) and P. aeruginosa (0.5% CPC; 0.05% MST). The CPC-fleece showed a high antiseptic effect against all three bacterial strains, although it did not completely eliminate P. aeruginosa. Both substances showed a high cytotoxic impact at higher tested concentrations (CPC >3×10(-3)%; MST >8×10(-4)%). CPC showed high antimicrobial potency at low concentrations against S. aureus, accompanied by low cytotoxic (side) effects at these concentrations, whilst the required minimal concentration to eradicate E. coli and P. aeruginosa was shown to be cytotoxic for keratinocytes and fibroblasts. The necessary antibacterial amounts of MST were lower, but also cytotoxic in direct contact with typical human wound cells. With regard to demographic changes and increasing bacterial resistance, new effective antiseptics, such as CPC and MST, incorporated in wound dressings without releasing an active substance could help to improve the treatment and healing rates of chronic wounds., (© 2015 The Authors.)

Introduction: Different transfusion ratio concepts of packed red blood cells (pRBCs), fresh frozen plasma (FFP) and platelets (PLTs) have been implemented in trauma care, but the optimal ratios are still discussed. In this study the hemostatic potential of two predefined ratios was assessed by using an in vitro thrombelastometric approach. Furthermore, age effects of reconstituted blood were analyzed., Methods: Whole blood (WB) of voluntary donors was separated into pRBCs, FFP and PLTs and reconstituted into the ratios 1:1:1 and 3:1:1 at day 1, 4, 14, and 24. Standard blood count, electrolytes and coagulation proteins were quantified. The functional coagulation in ratio- and age-specific groups was evaluated using rotational thromboelastometry (ROTEM)., Results: Several coagulation factors reduced significantly in the 3:1:1 ratio and were consistent with increased INR, decelerated clot formation times and A10 (amplitude 10 minutes after clotting time (CT)), flattened α-angle during the EXTEM and diminished MCF for distinct time points during the INTEM, FIBTEM and APTEM assays. With rising age of pRBCs the pH, sodium and potassium reached non-physiological levels., Conclusion: Under standardized in vitro conditions the higher amount of pRBCs in the 3:1:1 ratio diluted coagulation factors significantly on the expense of its functional coagulation capacity as revealed by ROTEM results. Thus, the coagulation functionality of the 1:1:1 ratio predominated.

Background: Traumatic brain injury (TBI) is one of the leading causes of death and permanent disability world-wide. The predominant cause of death after TBI is brain edema which can be quantified by non-invasive diffusion-weighted magnetic resonance imaging (DWI)., Purpose: To provide a better understanding of the early onset, time course, spatial development, and type of brain edema after TBI and to correlate MRI data and the cerebral energy state reflected by the metabolite adenosine triphosphate (ATP)., Material and Methods: The spontaneous development of lateral fluid percussion-induced TBI was investigated in the acute (6 h), subacute (48 h), and chronic (7 days) phase in rats by MRI of quantitative T2 and apparent diffusion coefficient (ADC) mapping as well as perfusion was combined with ATP-specific bioluminescence imaging and histology., Results: An induced TBI led to moderate to mild brain damages, reflected by transient, pronounced development of vasogenic edema and perfusion reduction. Heterogeneous ADC patterns indicated a parallel, but mixed expression of vasogenic and cytotoxic edema. Cortical ATP levels were reduced in the acute and subacute phase by 13% and 27%, respectively, but were completely normalized at 7 days after injury., Conclusion: The partial ATP reduction was interpreted to be partially caused by a loss of neurons in parallel with transient dilution of the regional ATP concentration by pronounced vasogenic edema. The normalization of energy metabolism after 7 days was likely due to infiltrating glia and not to recovery. The MRI combined with metabolite measurement further improves the understanding and evaluation of brain damages after TBI.

One of the putative pathophysiological mechanisms of chronic wounds is a disturbed homing of stem cells. In this project, the stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor (CXCR) 4 and SDF-1/CXCR7 pathway were focused in human adipose-derived stem cells (ASCs). ASCs were incubated with acute (AWF) or chronic wound fluid (CWF) to analyze their effects by quantitative real-time polymerase chain reaction (SDF-1, CXCR4, CXCR7, TIMP3), enzyme-linked immunosorbent assay (SDF-1 in WFs and supernatant), and transwell migration assay with/without antagonization. Whereas SDF-1 amounted 73.5 pg/mL in AWF, it could not be detected in CWF. Incubation with AWF led to a significant enhancement (129.7 pg/mL vs. 95.5 pg/mL), whereas CWF resulted in a significant reduction (30 pg/mL vs. 95.5 pg/mL) of SDF-1 in ASC supernatant. The SDF-1 receptor CXCR7 was detected on ASCs. AWF but not CWF significantly induced ASC migration, which was inhibited by CXCR4 and CXCR7 antagonists. Expressions of SDF-1, CXCR4, and CXCR7 were significantly stimulated by AWF while TIMP3 expression was reduced. In conclusion, an uncontrolled inflammation in the chronic wound environment, indicated by a reduced SDF-1 expression, resulted in a decreased ASC migration. A disturbed SDF-1/CXCR4 as well as SDF-1/CXCR7 pathway seems to play an important role in the impaired healing of chronic wounds., (© 2015 by the Wound Healing Society.)

Current osteoporosis therapies aim to delay bone destruction and have additional anabolic effects. While they have demonstrated some positive effects on bone healing, more progress is needed in this area. This study used the well-known osteoporotic agents estrogen (E) and raloxifene (R) in conjunction with biomechanical whole body vibration (WBV) at a frequency of 70 Hz twice daily for six weeks to stimulate bone healing. Eighty-four 3-month old female Sprague-Dawley rats (12 per group) were bilaterally ovariectomized to develop osteopenia within eight weeks. Osteotomy of the metaphyseal tibiae was performed and fracture healing was then studied using mechanical tests, histomorphometry, computed tomography (μCT), and gene analysis. We found that E and R improved the structure of osteopenic bones as did WBV alone, although significant levels for WBV were seldom reached. Combination treatments significantly enhanced stiffness (R+WBV; p<0.05), endosteal bone (R+WBV; p<0.01), and trabecular density (E+WBV; p<0.05, R+WBV; p<0.05). In addition, the expression of osteoclast-specific Trap was significantly reduced after treatment with E, R, or their combination with WBV (p<0.01). The effects were additive and not inhibitory, leading us to conclude that the combined applications of WBV with E or R may improve the healing of osteopenic bones. The therapies studied are all currently approved for human use, suggesting ready applicability to clinical practice. To better understand the effects of WBV on osteopenic bones, the ideal vibration regime will require further study., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Numerous whole-body vibration (WBV) devices of various forces are available on the market, although their influence on the musculoskeletal system is not yet understood. The effect of different WBVs on bone healing and muscle function was evaluated in rats ovariectomized at 3 months of age. 2 months after ovariectomy, bilateral metaphyseal tibia osteotomy and T-plate osteosynthesis were performed. Rats were divided into groups: intact, OVX, and OVX exposed to vertical WBVs of 35, 50, 70, or 90 Hz (experiment 1) or horizontal WBVs of 30, 50, 70, or 90 Hz (experiment 2) 5 days after osteotomy (0.5 mm, 15 min/day for 30 days). The tibia and gastrocnemius and soleus muscles were collected. Vertical vibrations (>35 Hz) improved cortical and callus densities, enlarged callus area and width, suppressed the tartrate-resistant acid phosphatase gene, enhanced citrate synthase activity, accelerated osteotomy bridging (35 and 50 Hz), upregulated the osteocalcin (Oc) gene (70 Hz), and increased relative muscle weight (50 Hz). Horizontal vibrations reduced cortical width (<90 Hz) and callus density (30 Hz), enhanced alkaline phosphatase (Alp) gene expression (50 Hz), decreased the size of oxidative fibers (35 and 70 Hz), and increased capillary density (70, 90 Hz). Biomechanical data; serum Oc, Alp, and creatine kinase activities; body weight; and food intake did not change after WBVs. Vertical WBVs of 35 and 50 Hz produced more favorable results than the higher frequencies. Horizontal WBV showed no positive or negative effects. Further studies are needed to elucidate the effects of WBV on different physiological systems, and precautions must be taken when implementing WBV in the treatment of patients.

The study investigated the influence of 4-methylbenzylidene camphor (4-MBC), daidzein, and estradiol-17β-benzoate (E(2)) on either intact or osteotomized cancellous bone in ovariectomized (Ovx) rats. Three-month old Ovx rats were fed with soy-free (SF) diet over 8 weeks; thereafter, bilateral transverse metaphyseal osteotomy of tibia was performed and rats were divided into groups: rats fed with SF diet and SF diet supplemented with 4-MBC (200 mg), daidzein (50 mg), or E(2) (0.4 mg) per kilogram body weight. After 5 or 10 weeks, computed tomographical, biomechanical, histological, and ashing analyses were performed in lumbar spine and tibia of 12 rats from each group. 4-MBC and E(2) improved bone parameters in lumbar spine and tibia, were not favorable for osteotomy healing, and decreased serum osteocalcin level. However, daidzein improved bone parameters to a lesser extent and facilitated osteotomy healing. For lumbar spine, the bone mineral density was 338±9, 346±5, 361±6, and 360±5 mg/cm(3) in SF, daidzein, 4-MBC, and E(2), respectively, after 10 weeks. For tibia, the yield load was 98±5, 114±3, 90±2, and 52±4 N in SF, daidzein, 4-MBC, and E(2), respectively, after 10 weeks. Serum daidzein was 54±6 ng/ml in daidzein group and equol was not detected. Alp and Igf1 genes were down-regulated in callus after daidzein and E(2) compared with 4-MBC (week 5). The response of bone tissue and serum markers of bone metabolism could be ordered: daidzein<4-MBC

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Influence of human parathyroid hormone (hPTH 1-34) on muscle and bone healing was studied in either orchiectomized (Orx at 8 months of age) or sham-operated male rats. Eleven-month-old Sprague-Dawley rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into four groups (n=12): 1) sham-vehicle, 2) sham group-PTH everyday, 3) Orx-vehicle, 4) Orx-PTH everyday, and 5) Orx-PTH every other day. PTH dosage was 40  μg/kg body weight. After 5 weeks, fiber cross-sectional area, capillary density, and enzyme activity (lactate dehydrogenase, citrate synthase, and complex I) were measured in soleus (MS), gastrocnemius (MG), and longissimus (ML) muscles; tibiae were analyzed by computed tomographical, histological, and gene expression analyses. The effect of PTH in all rats was increased serum osteocalcin, cortical and callus densities and callus area. In sham rats capillary density was increased in limb muscles (MS: 1.3-1.7, MG: 1.2-1.4 capillaries/fiber), and rate of osseous bridging of osteotomy was enhanced (67-100%). In Orx rats serum creatine kinase was decreased (6670-2847 U/l), and bone genes (Igf-1, osteoprotegerin, and receptor activator of nuclear factor kB ligand) were up-regulated. Cross-sectional area, enzyme activity, food intake, weight of body, visceral organs, adipose tissue, MG, and MS were not affected by PTH. PTH had a favorable effect on muscle capillary density and improved bone healing being more effective in sham rats and having no adverse systemic effect. The effect was less if PTH was applied every other day. The findings may show up trends for therapeutic treatment of male patients.

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.

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.

Three experiments were conducted to investigate the effect of intermittent administration of parathyroid hormone (PTH) (1-34) applied at different regimes on fracture healing and muscle in healthy and ovariectomized (Ovx at 3 months of age) rats. Five-month old rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into groups (12 rats each). In Exp 1, Ovx rats were either treated with PTH (7x/w, 1-35d), with oral estradiol-17beta-benzoate (0.4 mg/kg BW, 1-35d) or untreated. In Exp. 2, there were 3 groups: healthy untreated or treated with PTH (5x/w, 1-35d or 7-35d). In Exp. 3, there were 7 groups: healthy, Ovx, "healthy PTH 5x/w 7-35d", "Ovx PTH 5x/w 7-35d, 14-35d or 14-28d", "Ovx PTH every other day 7-35d". Single dosage of PTH was 40 microg/kg BW. After 35 days of healing one tibia was analyzed by computed tomographical, biomechanical, histological analyses. The other tibia was used in analyses of Alp, Oc, Trap 1, Igf-1, Rankl, Opg genes (Exp.2, 3). Serum Oc and Alp were measured. Body, uterus weight was recorded. M. gastrocnemius was analyzed for weight (Exp. 2), fiber size and mitochondrial respiratory activity (MRA) (Exp.3). Estrogen enhanced uterus weight, prevented body increase, however, did not improve bone healing in Ovx rats (Exp. 1). PTH administration from days 1 and 7 improved bone parameters in all rats regardless of the application frequency (7, 5x/w or every other day) (Exp. 1, stiffness Ovx: 118+13 N/mm, Ovx PTH: 250+/-20 N/mm) being more effective in healthy rats (Exp. 3, stiffness improvement Healthy: 59 to 174 N/mm, Ovx: 52 to 98 N/mm). Serum Oc level was elevated in PTH treated rats. Application from day 14 proved to be less effective (Exp. 3). PTH had no effect (P>0.05) on body, uterus and muscle weight, muscle fiber size, MRA and expression of bone markers. PTH promoted bone healing in Ovx and healthy rats, when it is applied during early stage of healing without having any adverse systemic effect. In perspective, PTH may represent a treatment for enhancement of fracture healing. The findings need to be confirmed by follow-up studies on other animals., (Copyright 2010 Elsevier Inc. All rights reserved.)

This study investigated the effect of vibration on bone healing and muscle in intact and ovariectomized rats. Thirty ovariectomized (at 3 months of age) and 30 intact 5-month old female Sprague-Dawley rats underwent bilateral metaphyseal osteotomy of tibia. Five days later, half of the ovariectomized and of the intact rats were exposed to whole-body vertical vibration (90 Hz, 0.5 mm, 4 x g acceleration) for 15 min twice a day during 30 days. The other animals did not undergo vibration. After decapitation of rats, one tibia was used for computed tomographic, biomechanical, and histological analyses; the other was used for gene expression analyses of alkaline phosphatase (Alp), osteocalcin (Oc), tartrate-resistant acid phosphatase 1, and insulinlike growth factor 1. Serum Alp and Oc were measured. Mitochondrial activity, fiber area and distribution, and capillary densities were analyzed in M. gastrocnemius and M. longissimus. We found that vibration had no effect on body weight and food intake, but it improved cortical and callus densities (97 vs. 99%, 72 vs. 81%), trabecular structure (9 vs. 14 trabecular nodes), blood supply (1.7 vs. 2.1 capillaries/fiber), and oxidative metabolism (17 vs. 23 pmol O(2)/s/mg) in ovariectomized rats. Vibration generally increased muscle fiber size. Tibia biomechanical properties were diminished after vibration. Oc gene expression was higher in vibrated rats. Serum Alp was increased in ovariectomized rats. In ovariectomized rats, vibration resulted in an earlier bridging; in intact rats, callus bridging occurred later after vibration. The chosen vibration regimen (90 Hz, 0.5 mm, 4 x g acceleration, 15 min twice a day) was effective in improving musculoskeletal tissues in ovariectomized rats but was not optimal for fracture healing.

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.

Osteoporosis and its accompanying, predominantly metaphyseal, fractures are a major health problem. Black cohosh (Cimicifuga racemosa) and estrogen positively influence osteoporotic bone. Both substances may improve fracture healing in early osteoporosis as well. In 48 twelve-week-old ovariectomized or, respectively, sham-operated (SHAM) rats, a standardized metaphyseal tibia osteotomy with bridging T-plate fixation was performed. During the healing process of 35 days, rats received soy-free (SHAM, osteopenic C), estrogen- (E) or Cimicifuga racemosa- (CR) supplemented diets. After sacrifice, the callus formation was analyzed with regard to biomechanical quality, morphology, quantity, time course of new bone built and gene expression. CR induced a high rate of metaphyseal callus formation. The biomechanical properties and the amount of new callus formation indicated that fracture healing was still in progress. Therefore, gene expression of osteoblasts was comparatively high. Body weight and the trabecular structure were influenced little by CR. Estrogen improved the biomechanical properties of the callus. Resistance to microfracturing was significantly enhanced in the E group and even superior to SHAM. Remodeling of the callus formation had already begun. The trabecular network and the typical endosteal fracture healing were especially improved. Osteoporotic metaphyseal fracture healing was improved by estrogen more than by Cimicifuga racemosa. The process of fracture healing occurred nearly physiologically. The generation of callus formation was supported by Cimicifuga racemosa as well, but the five-week duration of application was too short for Cimicifuga racemosa to show its complete potential. Already-initiated Cimicifuga racemosa therapy for menopausal symptoms could be continued during fracture healing without hesitation., (Georg Thieme Verlag KG Stuttgart-New York.)

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.

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.

The isoflavonoids found in soy have attracted great interest as dietary phytoestrogens that might be effective for postmenopausal hormone replacement therapy. Special attention has been devoted to the hormonal effects of various isoflavonoids, like genistein (GEN) and daidzein's (DAID) potent metabolite, equol (EQ). Here we aimed to investigate the short-term effects of genistein and equol on the proximal femur of ovariectomized (OVX) rats. Forty-eight, 3-month-old female Sprague-Dawley rats were ovarectomized; after eight weeks the bilateral osteotomy and osteosynthesis (OS) of their tibiae was performed and the rats were randomly divided into the following four groups: OVX control group (C), treated with estradiol-17beta (E2) -benzoate (E; daily intake 0.086 mg/d per animal), genistein (GEN; daily intake 12.7 mg/d per animal) and equol (EQ; daily intake 4.65 mg/d per animal). At 5 weeks postoperatively (OS), the breaking test was performed on the trochanteric region of femur. Additionally, histomorphometric assessment, and trabecular and cortical bone microstructure analyses were performed. The relative gain of body weight (BW) in the EQ (24 %) group was significantly (p < 0.05) lower than in the C (33 %) and GEN (30 %) groups. After treatment for 5 weeks, the maximal load (F(max)) and yield load (yL) were higher (p < 0.05 for the weight-adapted results) in the E (188.4 N resp. 113.1 N) and EQ (177.3 N resp. 112 N) groups as compared to C (162.8 N resp. 109.1 N) and GEN (165.7 N resp. 108.8 N). In the histomorphometric tests the E- (trabecular area (Tb.Ar) = 74.93 %, trabecular nodes/mm(2) (N.Nd/mm(2)) = 48.65) and EQ-treated (Tb.Ar = 63.13 %, N.Nd/mm(2) = 43.72) animals showed significant improvement with regard to Tb.Ar and trabecular connectivity (N.Nd./mm(2)) in comparison to C (Tb.Ar = 46.84, N.Nd/mm(2) = 31.86) and GEN (Tb.Ar = 48.22 %, N.Nd/mm(2) = 34.15). There were no differences in relative cortical width (Ct.Wi) among the four groups. The treatment with EQ resulted in improved biomechanical and histomorphometric properties as compared to the treatment with GEN. Thus, of the studied substances, EQ seems to be a possible alternative to hormone replacement therapy, but further studies are needed., (Georg Thieme Verlag KG Stuttgart-New York.)

Osteoporosis is accompanied by predominantly metaphyseal fractures with a delayed and qualitatively reduced healing process. This study addressed the question of whether fracture healing in the context of osteoporosis prophylaxis is improved with estrogen (E) or alendronate (ALN). Thirty-six ovariectomized and 12 sham-operated 12-week-old rats received soy-free (osteoporotic C, sham), E-, or ALN- supplemented diets. After 10 weeks, a metaphyseal tibia osteotomy and standardized T-plate fixation were performed. After a 5-week healing process, the fracture callus was evaluated qualitatively by biomechanical bending test and quantitatively in microradiographic sections. The time course of callus formation was examined using fluorochrome-labeled histological sections. Administration of E improved the biomechanical properties of callus (stiffness [N/mm]: sham: 110.2 + or - 76.07, C: 41.28 + or - 33.70, E: 85.72 + or - 47.24, ALN: 72.07 + or - 34.68). The resistance to microfracturing seen in E-treated animals was significantly enhanced and even superior to sham (yield load [N] sham: 27.44 + or - 9.72, C: 21.04 + or - 12.47, E: 42.85 + or - 13.74(Delta), ALN: 25.28 + or - 6.4(.)) (* P < 0.05 vs. sham group, (Delta) P < 0.05 vs. C group, (*) P < 0.05 vs. E group). Trabecular bone in particular was improved, indicating the presence of physiological endosteal bridging (Tr.Dn [%] sham: 10.53 + or - 18.9, C: 1.01 + or - 0.14, E: 24.13 + or - 34.09(Delta), ALN: 3.99 + or - 8.3(.)). ALN did not help bone healing, as shown by mechanical tests. Compared to the C group, statistically, ALN did not show worse properties. The induction of callus formation under ALN treatment was slightly delayed (Tt.Cl [mm(2)] sham: 3.68 + or - 0.66, C: 3.44 + or - 0.42, E: 3.69 + or - 0.58, ALN: 3.06 + or - 0.56). Osteoporotic metaphyseal fracture healing was qualitatively and quantitatively improved by E prophylaxis. The process of fracture healing occurred nearly physiologically (shamlike). Notably, ALN hardly improved metaphyseal callus properties when assessed as osteoporosis prophylaxis, but to a lesser extent than E.

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.