Bioactive impact of manuka honey and bone char incorporated into gelatin and chitosan cryogels in a rat calvarial fracture model.

Bone tissue engineered scaffolds are designed to mimic the natural environment for regeneration when typical healing is inhibited. Autografts are the current gold standard for treatment but are limited by available bone and supplementary surgical sites that broaden complications and comorbidities. Cryogels are an ideal scaffold in bone regeneration due to their mechanical integrity and marcoporous structure that elicits angiogenesis and subsequently new bone tissue formation. To aid in bioactivity and osteoinductivity, manuka honey (MH) and bone char (BC) were added to gelatin and chitosan cryogels (CG). Manuka honey has powerful antimicrobial properties to aid against graft infection, and bone char is composed of 90% hydroxyapatite, a well-studied bioactive material. These additives are natural, abundant, easy to use, and cost effective. CG cryogels incorporated with either BC or MH, and plain CG cryogels were implanted into rat calvarial fracture models for cortical bone regeneration analysis. We found indication of bioactivity with both bone char and manuka honey through the presence of woven bone structure in histology stains and micro computed tomography (microCT) data. Overall, plain CG cryogels supported greater bone regeneration capabilities than the BC or MH incorporated cryogels due to a lack of advanced organized tissue formation and collagen deposition after 8 weeks of implantation; however, future work should explore varying additive concentrations and delivery methods to further assess additive potential.

Development and validation of a method for simultaneous analysis of hair underivatized amino acids and damage biomarkers, using liquid chromatography-tandem mass spectrometry.

The forensic and medical fields are seeing growing interest in the amino acid and damage biomarker composition of hair, in order to identify adulteration of drug hair testing and for diagnostic purposes. Therefore, there is an increased demand for quick and accurate analytical methods. This study presents the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of hair amino acids and four damage biomarkers, which also implements an isotopic dilution strategy to improve recovery and precision of the acid hydrolysis-sensitive analytes. The applied strategy enabled a recovery of the hydrolysis-sensitive amino acids between 83 and 120% (vs. 33-77%, without isotopic dilution) for two different protein standards, and a precision with a relative standard deviation (RSD) between 1.3 and 7.5% (vs. 9.0-29.4%, without isotopic dilution). All 21 analytes could be measured without interferences by matrix and sample components, thus demonstrating satisfactory selectivity of the method. For spiked samples of hair hydrolyzate, recovery was between 88 and 120%, whereas precision and intermediate precision were below 10.1%. The high sensitivity of the method made it possible to reduce sample preparation to a 10000-fold dilution of the raw hydrolyzate. The wide linear range displayed by the method allowed the simultaneous quantification of minor (0.3 µmol/g of hair) and major (up to 1000 µmol/g of hair) components of the biological fiber. This method was successfully applied to the analysis of real hair samples submitted to six different treatments. Statistical data analysis by means of t-test and principal component analysis (PCA) showed a clear discrimination of the treated from the untreated hair samples and of the different treatments. Since these hair treatments can interfere with hair drug testing, the method possesses the ability of identifying hair samples with potential for attempted drug test evasion. In addition, lanthionine emerged as a new biomarker for heat damaged hair.

Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice.

[Denervation of the patella: influence on mid-term results after total knee arthroplasty]. / Retropatellare Denervierung : Einfluss auf die mittelfristigen Ergebnisse von Knietotalendoprothesenimplantationen.

BACKGROUND: The purpose of the study was to determine possible differences in the mid-term results of total knee arthroplasty in patients treated with and without denervation of the patella. PATIENTS AND METHODS: This study included 80 total knee replacements in 71 patients who were treated with total knee replacement, either with (n = 40) or without (n = 40) simultaneous denervation of the patella out of a total population with 122 knee replacements in 100 patients. Comparability of both groups was achieved by applying matching criteria. All patients were reviewed by isokinetic tests, physical and radiological examination. The mean follow-up time was 2.2 years. RESULTS: The mean hospital for special surgery (HSS) score revealed no statistically significant differences between both groups (with denervation 77.9 ± 11.1 and without denervation 77.8 ± 11.0, p = 0.976). The isokinetic torque measurements with low angle velocity (60°/s) indicated slightly higher values during extension (60.2 ± 32.2 Nm versus 55.8 ± 25.2 Nm, p = 0.497) and flexion (52.4 ± 28.3 Nm versus 46.1 ± 22.3 Nm, p = 0.272) movements of the affected knee joint. However, the differences did not reach statistical significance. At high angle velocity (180°/s) no differences could be found between both groups. No cases of postoperative necrosis of the patella were observed. Anterior knee pain after denervation was reported in 6 cases (15 %) compared to 10 cases (25 %) in patients who were treated without denervation (p = 0.402). CONCLUSION: No statistically significant differences could be found between patients with and without denervation of the patella for total knee arthroplasty.

A systemic approach to cancer treatment: tumor cell reprogramming focused on endocrine-related cancers.

The term "cancer cell reprogramming" is used to define any kind of intervention aimed at transforming cancer cells into terminally differentiated cells. Using this approach, new technologies have been applied with different methods for a more systemic approach to cancer treatment. This review reports on advances of these technologies, including our personal contributions, mainly carried out on endocrine-related cancers. Some of the interventions, aimed at reverting cancer cells into a normal phenotype, are based on the evidence that tumor development is suppressed by the embryonic microenvironment. On the basis of this rationale, experiments have been conducted using stem cell differentiation stage factors (SCDSFs) taken at different stages of development of Zebrafish embryos, oocyte extracts, or naïve human umbilical cord matrix derived stem cells (UMDSCs). SCDSFs induce significant growth inhibition on different tumor cell lines in vitro, likely because of increases in cell cycle regulatory molecules, such as p53 and pRb. Treatment with these factors activates apoptosis and differentiation related to caspase-3. This is achieved via p73 apoptotic-dependent pathway activation with a concurrent normalization of the E-cadherin and beta-catenin ratio. Extracts from prophase amphibian oocytes could reprogram relevant epigenetic alterations in MCF-7 and HCC1954 breast cancer cell lines, while un-engineered (naïve) human UMDSCs attenuated growth of MDA-231 human breast carcinoma cells. A product prepared for human treatments, containing SCDSFs at very low doses, yielded favorable results in breast cancer and in intermediate-advanced hepatocellular carcinoma. Other reprogramming interventions used in the models of breast, prostate and ovarian cancer cell lines are described. Finally, current and future perspectives of this novel technology are discussed and a new hallmark of cancer is suggested: the loss of differentiation of cancer cells.

Effect of brace design on patients with ACL-ruptures.

Different designs of functional knee braces for ACL-injury rehabilitation exist. In addition to the mechanical stabilization provided by rigid shell braces, sleeve braces also address proprioceptive mechanisms, but little is known if this leads to benefits for ACL-deficient subjects. Therefore the aim of this study was to investigate the effect of 2 different functional brace designs (shell and sleeve brace) on functional achievements in ACL-deficient patients. 28 subjects with ACL-ruptured knees performed tests for knee joint laxity, joint position sense, static and dynamic balance and isometric and dynamic lower limb extension strength in non-braced, sleeve braced and shell braced condition. The results showed a significant decrease in knee joint laxity for sleeve (33%; p<0.001) and rigid shell bracing (14%, p=0.039). The sleeve brace revealed a significant increase in dynamic balance after perturbation (20%; p=0.024) and a significant increase in dynamic lower limb peak rate of force development (17%; p=0.015) compared to the non-braced condition. The effects might be caused by the flexible area of support and the incorporated mechanisms to address proprioceptive aspects. Braces might not be needed in simple daily life tasks, but could provide beneficial support in more dynamic settings when patients return to sporting activities after an ACL-injury.

Infection, stem cells and cancer signals.

The association of cancer with preceding parasitic infections has been observed for over 200 years. Some such cancers arise from infection of tissue stem cells by viruses with insertion of viral oncogenes into the host DNA (mouse polyoma virus, mouse mammary tumor virus). In other cases the virus does not insert its DNA into the host cells, but rather commandeers the metabolism of the infected cells, so that the cells continue to proliferate and do not differentiate (human papilloma virus and cervical cancer). Cytoplasmic Epstein Barr virus infection is associated with a specific gene translocation (Ig/c-myc) that activates proliferation of affected cells (Burkitt lymphoma). In chronic osteomyelitis an inflammatory reaction to the infection appears to act through production of inflammatory cytokines and oxygen radical formation to induce epithelial cancers. Infection with Helicobacter pylori leads to epigenetic changes in methylation and infection by a parasite. Clonorchis sinensis also acts as a promoter of cancer of the bile ducts of the liver (cholaniocarcinoma). The common thread among these diverse pathways is that the infections act to alter tissue stem cell signaling with continued proliferation of tumor transit amplifying cells.

Preliminary investigation of airgap electrospun silk-fibroin-based structures for ligament analogue engineering.

The process of electrospinning has proven to be highly beneficial for use in a number of tissue-engineering applications due to its ease of use, flexibility and tailorable properties. There have been many publications on the creation of aligned fibrous structures created through various forms of electrospinning, most involving the use of a metal target rotating at high speeds. This work focuses on the use of a variation known as airgap electrospinning, which does not use a metal collecting target but rather a pair of grounded electrodes equidistant from the charged polymer solution to create highly aligned 3D structures. This study involved a preliminary investigation and comparison of traditionally and airgap electrospun silk-fibroin-based ligament constructs. Structures were characterized with SEM and alignment FFT, and underwent porosity, permeability, and mechanical anisotropy evaluation. Preliminary cell culture with human dermal fibroblasts was performed to determine the degree of cellular orientation and penetration. Results showed airgap electrospun structures to be anisotropic with significantly increased porosity and cellular penetration compared to their traditionally electrospun counterparts.

Electrospinning-aligned and random polydioxanone-polycaprolactone-silk fibroin-blended scaffolds: geometry for a vascular matrix.

Extracellular matrices are arranged with a specific geometry based on tissue type and mechanical stimulus. For blood vessels in the body, preferential alignment of fibers is in the direction of repetitive force. Electrospinning is a controllable process which can result in fiber alignment and randomization depending on the parameters utilized. In this study, arterial grafts composed of polycaprolactone (PCL), polydioxanone (PDO) and silk fibroin in blends of 100:0 and 50:50 for both PCL:silk and PDO:silk were investigated to determine if fibers could be controllably aligned using a mandrel rotational speed ranging from 500 to 8000 revolutions per minute (RPM). Results revealed that large- and small-diameter mandrels produced different degrees of fiber alignment based on a fast Fourier transform of scanning electron microscope images. Uniaxial tensile testing further demonstrated scaffold anisotropy through changes in peak stress, modulus and strain at break at mandrel rotational speeds of 500 and 8000 RPM, causing peak stress and modulus for PCL to increase 5- and 4.5-fold, respectively, as rotational speed increased. Additional mechanical testing was performed on grafts using dynamic compliance, burst strength and longitudinal strength displaying that grafts electrospun at higher rotational rates produced stiffer conduits which had lower compliance and higher burst strength compared to the lower mandrel rotational rate. Scaffold properties were found to depend on several parameters in the electrospinning process: mandrel rotational rate, polymer type, and mandrel size. Vascular scaffold design under anisotropic conditions provided interesting insights and warrants further investigation.

Angiogenic potential of human macrophages on electrospun bioresorbable vascular grafts.

The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of key angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor and acidic fibroblast growth factor. Transforming growth factor beta-1 (TGF-beta1) secretion was relatively low and there was negligible production of basic fibroblast growth factor. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis.

Physical mapping of distinct 7q22 deletions in uterine leiomyoma and analysis of a recently annotated 7q22 candidate gene.

Uterine leiomyoma (UL) is a benign, smooth muscle tumor of the uterus affecting a significant proportion of women of reproductive age. Deletions involving chromosome 7q22 are common in UL and vary in length. Previously reported 7q22 deletion intervals were physically mapped using information from the recently completed human genome sequence. Four distinct deletion intervals, which included a microdeletion reported by our laboratory, were identified. This microdeletion contains two known genes, ORC5L and LHFPL3. The single deleted marker in the microdeletion was mapped within the LHFPL3 locus. The ORC5L gene has been studied in UL. Conversely, LHFPL3 has been annotated only recently, and has therefore not been studied in UL. The predicted LHFPL3 protein sequence contained a polyalanine domain, and a signature sequence for the PMP22 Claudin protein family. Members of this family are transmembrane proteins with roles in differentiation, proliferation, and extracellular matrix formation, and have been implicated in other tumors. Differences in LHFPL3 expression were observed in both human and Eker rat UL. Our results provide evidence for four distinct 7q22 deletion intervals, each with multiple candidate genes, including the recently identified LHFPL3 gene.

Nitric oxide and gene regulation in plants.

There is increasing evidence that nitric oxide (NO), which was first identified as a unique diffusible molecular messenger in animals, plays an important role in diverse physiological processes in plants. Recent progress that has deepened our understanding of NO signalling functions in plants, with special emphasis on defence signalling, is discussed here. Several studies, based on plants with altered NO-levels, have recently provided genetic evidence for the importance of NO in gene induction. For a general overview of which gene expression levels are altered by NO, two studies, involving large-scale transcriptional analyses of Arabidopsis thaliana using custom-made or commercial DNA-microarrays, were performed. Furthermore, a comprehensive transcript profiling by cDNA-amplification fragment length polymorphism (AFLP) revealed a number of Arabidopsis thaliana genes that are involved in signal transduction, disease resistance and stress response, photosynthesis, cellular transport, and basic metabolism. In addition, NO affects the expression of numerous genes in other plant species such as tobacco or soybean. The NO-dependent intracellular signalling pathway(s) that lead to the activation or suppression of these genes have not yet been defined. Several lines of evidence point to an interrelationship between NO and salicylic acid (SA) in plant defence. Recent evidence suggests that NO also plays a role in the wounding/jasmonic acid (JA) signalling pathway. NO donors affect both wounding-induced H2O2 synthesis and wounding- or JA-induced expression of defence genes. One of the major challenges ahead is to determine how the correct specific response is evoked, despite shared use of the NO signal and, in some cases, its downstream second messengers.

Electrospun polydioxanone-elastin blends: potential for bioresorbable vascular grafts.

An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed and fabricated with mechanical properties to more closely match those of native arterial tissue, while remaining conducive to tissue regeneration. PDO was chosen to provide mechanical integrity to the prosthetic, while elastin provides elasticity and bioactivity (to promote regeneration in vitro/in situ). It is the elastic nature of elastin that dominates the low-strain mechanical response of the vessel to blood flow and prevents pulsatile energy from being dissipated as heat. Uniaxial tensile and suture retention tests were performed on the electrospun grafts to demonstrate the similarities of the mechanical properties between the grafts and native vessel. Dynamic compliance measurements produced values that ranged from 1.2 to 5.6%/100 mmHg for a set of three different mean arterial pressures. Results showed the 50:50 ratio to closely mimic the compliance of native femoral artery, while grafts that contained less elastin exceeded the suture retention strength of native vessel. Preliminary cell culture studies showed the elastin-containing grafts to be bioactive as cells migrated through their full thickness within 7 days, but failed to migrate into pure PDO scaffolds. Electrospinning of the PDO and elastin-blended composite into a conduit for use as a small diameter vascular graft has extreme potential and warrants further investigation as it thus far compares favorably to native vessel.

[Changes in muscle torque in patients after total knee arthroplasty]. / Veränderungen der muskulären Leistungsfähigkeit bei Trägern von Kniegelenkstotalendoprothesen.

AIM: The purpose of this study was to compare muscle torque during isokinetic knee movements of patients after total knee arthroplasty (TKA) with a similar healthy group. METHOD: 43 patients were examined 3 years after primary total knee arthroplasty with an isokinetic dynamometer (Cybex 340). Torque measurements were evaluated for extension and flexion with 4 different velocities. The results were compared with a group of 43 people without knee disease. Bouth groups consist of 29 women and 14 men. The mean age was also comparable (66.4 vs. 65.8 years). RESULTS: A mean peak torque of 65.9 Nm was measured during extension with low velocity (60 degrees /s) in the group of patients with TKA -- 84.3 % of the control group (p = 0.079). At the highest velocity the patients produced 30.2 Nm. This was 72.8 % of the control group (difference statistically significant, p = 0.006). No differences were found in the Extension/Flexion ratio between the two groups. CONCLUSION: The deficites of knee strength in patients after TKA are more distinct in high velocities of motion.

Monoaminergic drugs and directly observable signs of LAAM withdrawal in rhesus monkeys.

Monoaminergic ligands modified a naltrexone discriminative stimulus in rhesus monkeys dependent on 2 mg/kg per day of the mu opioid L-alpha-acetylmethadol (LAAM). This study examined a role for monoamines in the directly observable and physiologic manifestations of LAAM withdrawal induced by naltrexone in the same monkeys. The effects of saline, clonidine (0.032 mg/kg), haloperidol (0.032 mg/kg), cocaine (1.0 mg/kg), amphetamine (1.0 mg/kg) and imipramine (10.0 mg/kg) were examined in LAAM-dependent monkeys that subsequently received saline or naltrexone (0.0001-1.0 mg/kg). Naltrexone dose-dependently increased respiration, abdominal rigidity and salivation. Clonidine attenuated each of these withdrawal signs, whereas haloperidol increased some (i.e. respiration) and decreased others (i.e. salivation). When administered alone, cocaine and amphetamine increased respiration and also increased the respiratory stimulant effects of naltrexone; cocaine and amphetamine did not attenuate any measure of withdrawal. With the exception of a decrease in naltrexone-induced salivation, imipramine was without effect. These results are strikingly different from results in these same LAAM-dependent monkeys showing that cocaine and amphetamine, but not clonidine, markedly attenuated a naltrexone discriminative stimulus. That monoaminergic ligands differentially alter the directly observable and discriminative stimulus effects of naltrexone in LAAM-dependent monkeys supports the view that monoamines differentially mediate the physical manifestations (norepinephrine) and subjective experience (dopamine) of opioid withdrawal.

[Risk factors associated with heterotopic ossifications in primary total hip arthroplasty]. / Risikofaktoren für heterotope Ossifikationen in der primären Hüftgelenkstotalendoprothetik.

AIM: The purpose of this study was to determine the important predisposing factors associated with heterotopic ossifications (HO) after total hip arthroplasty. METHOD: 589 patients were examined 6 months after primary total hip arthroplasty with regard to periarticular ossifications. Several predetermining factors were evaluated using the hospitalization records and preoperative X-ray examination. RESULTS: A significantly increased frequency of heterotopic ossifications was found for male gender, patients with very high body mass index (BMI), low preoperative range of motion (ROM), long duration of operation and large preexistent osteophytes (p < 0.05). Only one out of the one hundred patients with an BMI < 22.6 developed severe HO (Brooker III). Out of the one hundred patients with the best preoperative ROM (> or = 140 degrees ) only one case developed severe ossifications (Brooker III). There was no correlation with the use of acrylic bone cement or the patient's age. The frequency of HO was significantly reduced both as well by nonsteroidal antiinflammatory drugs as from postoperative radiation prophylaxis. CONCLUSION: In patients undergoing total hip arthroplasty with low preoperative ROM in the hip joint, large osteophytes and a very high BMI an efficient prophylaxis against HO is of great importance.

[Radio-ulnar and radio-scaphoid-lunate arthrodesis in chronic polyarthritis. Clinical and radiologic follow-up of 32 cases]. / Radiolunäre und radioskapholunäre Arthrodese bei chronischer Polyarthritis. Klinische und radiologische Nachuntersuchung an 32 Fällen.

INTRODUCTION: In 75% of all cases of rheumatoid arthritis the wrist is affected and in 12% is the region of initial manifestation of this chronic inflammatory joint disease. To prevent destruction of the wrist through carpal dislocation, radiolunate and radioscapholunate arthrodeses have increased in importance. METHODS: During a 6.5-year period, 28 radiolunate and 4 radioscapholunate arthrodeses were performed in 30 patients. The indication for operation was progressive carpal translation and increasing subluxation of the wrist in which existent radiological damage had not reached more than grade III according to the classification of Larsen and co-workers. RESULTS: The results obtained during the follow-up study (median: 17.3 months) showed in most treated patients after partial arthrodesis of the wrist no or fewer complaints concerning swelling and pain and an acceptable remaining range of motion of the wrist in everyday life. In patients with preoperatively existing ulnar deviation less than 15 degrees progressive carpal collapse and dislocation could be mostly prevented. CONCLUSION: All told radiolunate and radioscapholunate arthrodeses successfully stabilized the wrist in patients suffering from rheumatoid arthritis.

Superior efficacy in suppression of heterotopic bone formation using fractionated irradiation of 5 x 2 Gy compared to a single dose of 7 Gy. An experimental study in rats.

Postoperative irradiation of the operative field is an established method to prevent heterotopic ossification in total hip arthroplasty. In this study two theoretical dose-equivalent regimens of radiation therapy were compared. Allogenic bone matrix was implanted in both thighs of 50 adult male Wistar rats to induce heterotopic ossification. Immediately after operation the implants of 40 animals were irradiated using a single-dose of 7 Gy or 5 fractions of 2 Gy each. Ten rats served as a controlgroup and did not undergo irradiation. Radiation therapy with 5 x 2 Gy led to a highly significantly better suppression of heterotopic ossification than irradiation with 1 x 7 Gy (p < 0.001; paired-t-test). Single-dose irradiation reduced the mean calcium contents to 138.87 +/- 22.84 micrograms Ca2+/mg implanted bone matrix; fractionated irradiation obtained a reduction to 63.35 +/- 21.16 micrograms Ca2+/mg implanted bone matrix. In thigh implants not exposed to irradiation the mean calcium content was 191.50 +/- 11.46 micrograms Ca2+/mg implanted bone matrix. Radiographically better suppression of bone formation could be documented after irradiation with 5 x 2 Gy compared to 1 x 7 Gy and non-irradiated implants. The histological aspect of the explanted specimens showed quantitatively more new bone formation in the non-irradiated controls than in both irradiation groups. In view of experimentally demonstrated better effects, as well as the reduced side effects, fractionated irradiation appears preferable.