Contribution of extracellular matrix components to the stiffness of skeletal muscle contractures in patients with cerebral palsy.

Purpose: Joint contractures in children with cerebral palsy contain muscle tissue that is mechanically stiffer with higher collagen content than typically developing children. Interestingly, the correlation between collagen content and stiffness is weak. To date, no data are available on collagen types or other extracellular matrix proteins in these muscles, nor any information regarding their function. Thus, our purpose was to measure specific extracellular protein composition in cerebral palsy and typically developing human muscles along with structural aspects of extracellular matrix architecture to determine the extent to which these explain mechanical properties. Materials and Methods: Biopsies were collected from children with cerebral palsy undergoing muscle lengthening procedures and typically developing children undergoing anterior cruciate ligament reconstruction. Tissue was prepared for the determination of collagen types I, III, IV, and VI, proteoglycan, biglycan, decorin, hyaluronic acid/uronic acid and collagen crosslinking. Results: All collagen types increased in cerebral palsy along with pyridinoline crosslinks, total proteoglycan, and uronic acid. In all cases, type I or total collagen and total proteoglycan were positive predictors, while biglycan was a negative predictor of stiffness. Together these parameters accounted for a greater degree of variance within groups than across groups, demonstrating an altered relationship between extracellular matrix and stiffness with cerebral palsy. Further, stereological analysis revealed a significant increase in collagen fibrils organized in cables and an increased volume fraction of fibroblasts in CP muscle. Conclusions: These data demonstrate a novel adaptation of muscle extracellular matrix in children with cerebral palsy that includes alterations in extracellular matrix protein composition and structure related to mechanical function

Muscle gene expression patterns in human rotator cuff pathology.

BackgroundRotator cuff pathology is a common source of shoulder pain with variable etiology and pathoanatomical characteristics. Pathological processes of fatty infiltration, muscle atrophy, and fibrosis have all been invoked as causes for poor outcomes after rotator cuff tear repair. The aims of this study were to measure the expression of key genes associated with adipogenesis, myogenesis, and fibrosis in human rotator cuff muscle after injury and to compare the expression among groups of patients with varied severities of rotator cuff pathology.MethodsBiopsies of the supraspinatus muscle were obtained arthroscopically from twenty-seven patients in the following operative groups: bursitis (n = 10), tendinopathy (n = 7), full-thickness rotator cuff tear (n = 8), and massive rotator cuff tear (n = 2). Quantitative polymerase chain reaction (qPCR) was performed to characterize gene expression pathways involved in myogenesis, adipogenesis, and fibrosis.ResultsPatients with a massive tear demonstrated downregulation of the fibrogenic, adipogenic, and myogenic genes, indicating that the muscle was not in a state of active change and may have difficulty responding to stimuli. Patients with a full-thickness tear showed upregulation of fibrotic and adipogenic genes; at the tissue level, these correspond to the pathologies most detrimental to outcomes of surgical repair. Patients with bursitis or tendinopathy still expressed myogenic genes, indicating that the muscle may be attempting to accommodate the mechanical deficiencies induced by the tendon tear.ConclusionsGene expression in human rotator cuff muscles varied according to tendon injury severity. Patients with bursitis and tendinopathy appeared to be expressing pro-myogenic genes, whereas patients with a full-thickness tear were expressing genes associated with fatty atrophy and fibrosis. In contrast, patients with a massive tear appeared to have downregulation of all gene programs except inhibition of myogenesis.Clinical relevanceThese data highlight the difficulty in treating massive tears and suggest that the timing of treatment may be important for muscle recovery. Specifically, earlier interventions to address tendon injury may allow muscles to respond more appropriately to mechanical stimuli

Antibacterial and antibiofilm efficacy of k21-E in root canal disinfection

Objectives: The aim of the current project was to study the antimicrobial efficacy of a newly developed irrigant, k21/E against E. faecalis biofilm. Methods: Root canals were instrumented and randomly divided into the following groups: irrigation with saline, 6% NaOCl (sodium hypochlorite), 6% NaOCl + 2% CHX (Chlorhexidine), 2% CHX, 0.5% k21/E (k21 - quaternary ammonium silane) and 1% k21/E. E. faecalis were grown (3-days) (1 × 107 CFU mL−1), treated, and further cultured for 11-days. Specimens were subjected to SEM, confocal and Raman analysis and macrophage vesicles characterized along with effect of lipopolysaccharide treatment. 3T3 mouse-fibroblasts were cultured for alizarin-red with Sortase-A active sites and Schrödinger docking was performed. TEM analysis of root dentin substrate with matrix metalloproteinases profilometry was also included. A cytotoxic test analysis for cell viability was measured by absorbance of human dental pulp cells after exposure to different irrigant solutions for 24 h. The test percentages have been highlighted in Table 1. Results: Among experimental groups, irrigation with 0.5% k21/E showed phase separation revealing significant bacterial reduction and lower phenylalanine 1003 cm−1 and Amide III 1245 cm−1 intensities. Damage was observed on bacterial cell membrane after use of k21/E. No difference in exosomes distribution between control and 0.5%k21/E was observed with less TNFα (*p \u3c 0.05) and preferential binding of SrtA. TEM images demonstrated integrated collagen fibers in control and 0.5%k21/E specimens and inner bacterial membrane damage after k21/E treatment. The k21 groups appeared to be biocompatible to the dental pulpal cells grown for 24 h. Significance: Current investigations highlight potential advantages of 0.5% k21/E as irrigation solution for root canal disinfection

Antibacterial and antibiofilm efficacy of k21-E in root canal disinfection

Objectives: The aim of the current project was to study the antimicrobial efficacy of a newly developed irrigant, k21/E against E. faecalis biofilm. Methods: Root canals were instrumented and randomly divided into the following groups: irrigation with saline, 6% NaOCl (sodium hypochlorite), 6% NaOCl + 2% CHX (Chlorhexidine), 2% CHX, 0.5% k21/E (k21 - quaternary ammonium silane) and 1% k21/E. E. faecalis were grown (3-days) (1 × 107 CFU mL−1), treated, and further cultured for 11-days. Specimens were subjected to SEM, confocal and Raman analysis and macrophage vesicles characterized along with effect of lipopolysaccharide treatment. 3T3 mouse-fibroblasts were cultured for alizarin-red with Sortase-A active sites and Schrödinger docking was performed. TEM analysis of root dentin substrate with matrix metalloproteinases profilometry was also included. A cytotoxic test analysis for cell viability was measured by absorbance of human dental pulp cells after exposure to different irrigant solutions for 24 h. The test percentages have been highlighted in Table 1. Results: Among experimental groups, irrigation with 0.5% k21/E showed phase separation revealing significant bacterial reduction and lower phenylalanine 1003 cm−1 and Amide III 1245 cm−1 intensities. Damage was observed on bacterial cell membrane after use of k21/E. No difference in exosomes distribution between control and 0.5%k21/E was observed with less TNFα (*p \u3c 0.05) and preferential binding of SrtA. TEM images demonstrated integrated collagen fibers in control and 0.5%k21/E specimens and inner bacterial membrane damage after k21/E treatment. The k21 groups appeared to be biocompatible to the dental pulpal cells grown for 24 h. Significance: Current investigations highlight potential advantages of 0.5% k21/E as irrigation solution for root canal disinfection

Synthesis and evaluation of nicotine alpha(4)beta(2) receptor radioligand, 5-(3 '-F-18-fluoropropyl)-3(2-(S)-pyrrolidinylmethoxy)pyridine, in rodents and PET in nonhuman primate

Nicotine alpha(4)beta(2) receptor subtypes are implicated in the study of Alzheimer's disease, schizophrenia, substance abuse, lung cancer, and other disorders. We report the development and evaluation of a putative antagonist, 5-(3'-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine (nifrolidine) as a PET agent for nicotine alpha(4)beta(2) receptors. Methods: In vitro binding affinity of nifrolidine was measured in rat brain slices labeled with I-125-iodoepibatidine or I-125-bungaratoxin. Selectivity of binding was measured in the presence of cytisine. F-18 radiolabeling was performed by reacting the tosylate precursor with F-18-fluoride followed by cleprotection. In vitro autoradiographic studies in rat brain slices with 5-(3'-F-18-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine (F-18-nifrolidine) were read on a phosphor imager. Rats were injected with F-18-nifrolidine (3.7 MBq each), and brain regions were counted at various times (2-120 min). Blocking studies were performed by subcutaneous injection of nicotine (10 mg/kg). A PET study of F-18-nifrolidine (approximately 148 MBq) was performed on an anesthetized rhesus monkey using a high-resolution scanner. Results: In vitro binding affinity of nifrolidine exhibited an inhibition constant of 2.89 nmol/L for the alpha(4)beta(2) sites. Radiosynthesis and high-performance liquid chromatography purifications yielded the product in approximately 20%-40% decay-corrected radiochemical yield to provide 18F-nifrolidine specific activities of approximately 111-185 GBq/mumol. In vitro autoradiography in rat brain slices revealed selective binding of F-18-nifrolidine to the anteroventral thalamic nucleus, ventral posteriomedial thalamus, dorsolateral geniculate, and, to a lesser extent, cortex and striata, which are known to contain alpha(4)beta(2) sites. This specific binding was completely abolished by 300 mumol/L nicotine. Ex vivo rat brain distribution studies indicated selective binding in the thalamus with a maximal thalamus-to-cerebellum ratio of approximately 3. The PET study revealed selective maximal uptake (0.01 % injected dose/mL) in regions of the thalamus (anteroventral and anteromedial mus, ventrolateral thalamus) and extrathalamic regions such as cingulate gyrus, lateral geniculate, temporal cortex, and frontal cortex. Conclusion: Binding of F-18-nifrolidine to alpha(4)beta(2) receptor-rich regions in rats and monkeys indicates promise as a PET agent. Additionally, the thalamus-to-cerebellum ratio approached a plateau of 1.7 in 120 min, indicating relatively faster kinetics compared with previously reported imaging agents

Heterogeneous muscle gene expression patterns in patients with massive rotator cuff tears

<div><p>Detrimental changes in the composition and function of rotator cuff (RC) muscles are hallmarks of RC disease progression. Previous studies have demonstrated both atrophic and degenerative muscle loss in advanced RC disease. However, the relationship between gene expression and RC muscle pathology remains poorly defined, in large part due to a lack of studies correlating gene expression to tissue composition. Therefore, the purpose of this study was to determine how tissue composition relates to gene expression in muscle biopsies from patients undergoing reverse shoulder arthroplasty (RSA). Gene expression related to myogenesis, atrophy and cell death, adipogenesis and metabolism, inflammation, and fibrosis was measured in 40 RC muscle biopsies, including 31 biopsies from reverse shoulder arthroplasty (RSA) cases that had available histology data and 9 control biopsies from patients with intact RC tendons. After normalization to reference genes, linear regression was used to identify relationships between gene expression and tissue composition. Hierarchical clustering and principal component analysis (PCA) identified unique clusters, and fold-change analysis was used to determine significant differences in expression between clusters. We found that gene expression profiles were largely dependent on muscle presence, with muscle fraction being the only histological parameter that was significantly correlated to gene expression by linear regression. Similarly, samples with histologically-confirmed muscle distinctly segregated from samples without muscle. However, two sub-groups within the muscle-containing RSA biopsies suggest distinct phases of disease, with one group expressing markers of both atrophy and regeneration, and another group not significantly different from either control biopsies or biopsies lacking muscle. In conclusion, this study provides context for the interpretation of gene expression in heterogeneous and degenerating muscle, and provides further evidence for distinct stages of RC disease in humans.</p></div

Fold change in expression between pooled RSA biopsies and controls.

<p>As a single pool, RSA biopsies are not significantly different from controls, though expression of pro-myogenic genes trended down while atrophic, adipogenic, and fibrotic genes trended up.</p