Specific osteogenesis imperfecta-related Gly substitutions in type I collagen induce distinct structural, mechanical, and dynamic characteristics.

The stiffnesses, ß-structures, hydrogen bonds, and vibrational modes of wild-type collagen triple helices are compared with osteogenesis imperfecta-related mutants using integrative structural and dynamic analysis via molecular dynamics simulations and Markov state models. Differences in these characteristics are strongly related to the unwound structural states in the mutated regions that are specific to each mutation.

Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin-eosin and immunostaining.

Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine.

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs.

In vivo confocal Raman spectroscopy for intrinsic aging and photoaging assessment.

BACKGROUND: In vivo confocal Raman spectroscopy is a non-invasive method to assess either the epidermis or the dermis composition. Few studies have focused on dermis collagen alterations through intrinsic aging and photoaging. OBJECTIVE: This study evaluated the in vivo Raman spectra from the dermis of a photoexposed site versus a non-photoexposed region in different age groups, and evaluated the correlation between peak intensities and age, photoaging score and the amount of collagen assessed with histology and high frequency ultrasound (HFUS). METHODS: Fifteen volunteers aged 28-82 years were divided into three groups according to forearm photoaging degree. In vivo Raman spectra from the dermis were collected on the dorsal forearm (chronically photoexposed skin) and on the proximal medial arm (non-photoexposed skin). Cross-sectional images of the skin were obtained using a 20MHz ultrasound unit exactly on the same sites, which were further submitted to punch biopsies for histologic study (collagen I immunohistochemistry, picrosirius red staining and Verhoeff). Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) were taken in the spectral region of 796cm-1-996cm-1 to determine its potential to discriminate between different groups. The Spearman rank correlation coefficient of individual peak intensities and ratios with age, clinical score and the amount of collagen assessed by ultrasound and histology were calculated. RESULTS: PCA of pairs of groups and OPLS-DA could discriminate the intrinsically from the photoaged skin and the young group from the elderly one, with important contribution of the 938cm-1 and 855cm-1 peaks intensities. The intensity of the peaks in 855cm-1 and/or 938cm-1 presented moderate correlation with age (rho=0.579, p=0.049) and moderate to high inverse correlation with HFUS echogenicity (rho=-0.710, p=0.010) and collagen I immunohistochemistry (rho=-0.833, p=0.005) in the non-photoexposed region. The I1275/I1450 intensities ratio presented moderate to high correlation coefficients with age (rho=-0.730, p=0.007), photoaging score (rho=-0.594, p=0.042), HFUS echogenicity (rho=0.760, p<0.001) and histology (collagen I immunohistochemistry (rho=0.643, p=0.024), picrosirius (rho=0.773, p=0.005) and Verhoeff (rho=-0.727, p=0.011)) in the photoexposed site. CONCLUSION: The wavenumber region between 798 and 994cm-1 is useful for the analysis of dermal collagen alterations through the intrinsic aging process, while photoaging is better assessed by the I1275/I1450 intensities ratio. This is the first skin aging study to show a correlation between Raman peaks and the amount of collagen assessed by HFUS and histology.

Tissue integrity, costs and time associated with different agents for histological bone preparation.

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin-fixed and paraffin-embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3-µm-thick sections were obtained and stained with hematoxylin-eosin to calculate the percentage of osteocyte-occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi-quantitative analysis to verify the adjacent muscle-to-bone integrity and preservation of collagen fibres. The highest percentage of osteocyte-occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8-5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution.

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization.

The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

Dermal type I collagen assessment by digital image analysis.

Type I collagen is the main dermal component, and its evaluation is relevant to quantitative studies in dermatopathology. However, visual gradation (0 to 4+) has low precision and high subjectivity levels. This study aimed to develop and validate a digital morphometric analysis technique to estimate type I collagen levels in the papillary dermis. Four evaluators visually quantified (0 to 4+) the density of type I collagen in 63 images of forearm skin biopsies marked by immunohistochemistry and two evaluators analyzed the same images using digital morphometric techniques (RGB split colors (I) and color deconvolution (II)). Automated type I collagen density estimation in the papillary dermis (two techniques) were correlated with visual evaluations (Spearman's rho coefficients of 0.48 and 0.62 (p<0.01)). With regard to the inter-observer repeatability, the four evaluators who used visual classification had an intraclass correlation coefficient (for absolute agreement) of 0.53, while the other two evaluators who used digital analysis (algorithm II) had an intraclass correlation coefficient of 0.97.

Validation of a food frequency questionnaire for assessing macronutrient and calcium intake in adult Pakistani population.

OBJECTIVE: To develop and validate a food frequency table (FFQ) for use in urban Pakistani population. STUDY DESIGN: A validation study. PLACE AND DURATION OF STUDY: The Aga Khan University, Karachi, from June to November 2008. METHODOLOGY: Healthy adult females, aged ³ 18 years who consented to be included in the study were inducted, while males, unhealthy females, aged below 18 years or who did not consent were excluded. The FFQ was administered once while 4, 24 hours recalls spread over a period of one year were administered as the reference method. Daily intakes for energy, protein, fat, and calcium intake were estimated for both the tools. Crude and energy adjusted correlations for nutrient intakes were computed for the FFQ and mean of 4, 24 hours recalls and serum N-telopeptide of type-I collagen (NTx). RESULTS: The correlation coefficients for the FFQ with mean of 4, 24 hours recall ranged from 0.21 for protein to 0.36 for calcium, while the correlation for nutrient estimates from the FFQ with NTx ranged from -0.07 for calcium to 0.01 for energy. CONCLUSION: Highly significant correlations were found for nutrient intakes estimated from the FFQ vs. those estimated from the mean of 4, 24 hours recalls but no correlations was found between nutrient estimates from the FFQ and serum NTx levels. FFQ was concluded to be a valid tool for assessing dietary intake of adult females in Pakistan.

[Use of oral fluid metabolic rates for the assessment of reparative osteogenesis after bone augmentation procedures].

The use of osteoplastic materials allows extending indications for dental implants placement by considerable alveolar bone atrophy. The aim of the study was to reveal bone tissue metabolites which can be used as early bone destruction markers after bone augmentation procedure before any radiological signs occur. For this purpose the content of osteocalcinum, C-telopeptides, alkaline phosphatase and lactate dehydrogenase was examined in oral fluid. The increase of osteocalcinum and C-telopeptides and decrease activity of alkaline phosphatase and lactate dehydrogenase in comparison with control represented the pattern specific for bone destruction.

Quantitative histopathological assessment of retardation of islets of langerhans degeneration in rosiglitazone-dosed obese ZDF rats using combined insulin and collagens (I and III) immunohistochemistry with automated image analysis and statistical modeling.

Islets of Langerhans represent a heterogeneous population in insulin resistant and diabetic animals and humans as histological appearances and function vary substantially. Mathematical representation that reflects this morphological diversity will assist in assessment of degeneration and regeneration, enabling comparisons between species, strains, and experimental investigations. Our investigative approach used a model of islet degeneration in diabetic male obese Zucker Diabetic Fatty (ZDF) rats and evaluated its prevention using rosiglitazone treatment. Immunohistochemical staining (insulin and collagens I/III) with automated image analysis reliably measured numbers, area, clustering, and staining intensity of ß-cells and degree of islet fibrosis. Finite mixture mathematical modeling for the joint probability distribution of seven islet parameters to represent islet numerical data variation provided an automatic procedure for islet category allocations as normal or abnormal. Allocations for obese ZDF controls and rosiglitazone-treated animals were significantly different, with no significant difference between the latter and lean ZDF controls, indicative of differences within islet populations of individual animals, between lean and obese rat strains and following drug treatment. Islet morphology showed clear association with mathematical characterization. Information on islet morphology obtained by histopathological assessment of single pancreatic tissue sections was confirmed by this method showing drug-induced retardation of islet of Langerhans degeneration.

Influence of aging on the quality of the skin of white women: the role of collagen.

PURPOSE: Evaluate the influence of aging on the quality of the skin of white women, analyzing the dermal collagen. METHODS: Pre-auricular flaps were collected for histological and morphometric analysis of 218 white women who underwent spontaneous facial aesthetic plastic surgery. Picrosirius ultrared stain was used for analysis and quantification of collagen in five age groups (<40 years, 40 to 49 years, 50 to 59 years, 60 to 69 years and 70 to 79 years) . RESULTS: Histological analysis showed changes suggestive of skin aging (fragmentation and disorganization of collagen fibers), especially in patients over 60 years. There were no significant changes in the relationship of age with the thickness of the dermis and epidermis, but there was with the percentage of the collagen I, III and total (p<0.001), which decreased with increasing aging. CONCLUSION: There is reduction in collagen with increasing age, and an increase in its degradation, leading to fragmentation of the fibers.

Influence of aging on the quality of the skin of white women: the role of collagen / Influência do envelhecimento na qualidade da pele de mulheres brancas: o papel do colágeno

PURPOSE: Evaluate the influence of aging on the quality of the skin of white women, analyzing the dermal collagen. METHODS: Pre-auricular flaps were collected for histological and morphometric analysis of 218 white women who underwent spontaneous facial aesthetic plastic surgery. Picrosirius ultrared stain was used for analysis and quantification of collagen in five age groups (<40 years, 40 to 49 years, 50 to 59 years, 60 to 69 years and 70 to 79 years) . RESULTS: Histological analysis showed changes suggestive of skin aging (fragmentation and disorganization of collagen fibers), especially in patients over 60 years. There were no significant changes in the relationship of age with the thickness of the dermis and epidermis, but there was with the percentage of the collagen I, III and total (p<0.001), which decreased with increasing aging. CONCLUSION: There is reduction in collagen with increasing age, and an increase in its degradation, leading to fragmentation of the fibers.

OBJETIVO: Avaliar a influência do envelhecimento na qualidade da pele de mulheres brancas analisando o colágeno dérmico. MÉTODOS: Realizou-se análise histológica e morfométrica de 218 retalhos pré-auriculares de mulheres brancas que se submeteram espontaneamente à cirurgia estética facial. Foi usada a coloração de Picrosirius Ultrared para analisar e quantificar os colágenos I, III e total em cinco grupos etários (<40 anos, 40 a 49 anos, 50 a 59 anos, 60 a 69 anos e 70 a 79 anos). RESULTADOS: A análise histológica mostrou alterações sugestivas de envelhecimento cutâneo (fragmentação e desorganização das fibras de colágeno), especialmente em pacientes acima de 60 anos. Não houve diferenças significantes entre a idade e a espessura da derme e da epiderme, mas houve diferenças significantes entre as percentagens de colágeno I, III e total (p<0,001) com o aumento da idade. CONCLUSÃO: Existe redução do colágeno com o aumento da idade e um aumento na sua degradação, levando à fragmentação das fibras.

A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

[Is bone biopsy necessary for the diagnosis of metabolic bone diseases? Non- invasive assessment of bone turn over markers could define the cause of metabolic bone diseases].

Recent advances of the measurement of bone turn over markers contribute to non-invasive assessment of bone-metabolic disorders. We can detect the cause of the metabolic disorders with bone turn over markers and hormonal profiles more easily than before. Today, we can diagnose and treat metabolic bone diseases without invasive procedure such as bone biopsy.

Assessment of osteoclast number and function: application in the development of new and improved treatment modalities for bone diseases.

Numerous experimental and clinical observations suggest that overall changes in bone resorption during menopause or treatment with hormone replacement therapy (HRT) are combined effects of changes in osteoclast number and function. Moreover, due to a coupling between osteoclastic bone resorption and osteoblastic bone formation, pronounced alteration of osteoclast number will eventually lead to alteration of osteoblastic bone formation. Fragments of type I collagen, such as the C- and N-terminal telopeptides of collagen type I (CTX and NTX, respectively), are generated during bone resorption and hence can be used as surrogate markers of osteoclast function. Circulating levels of different enzymes in the serum, such as TRAP 5b and cathepsin K are proportional to the number of osteoclasts, and hence can be used as surrogate markers of osteoclast number. Since antiresorptive effects can be obtained in different ways, we felt it was timely to discuss the different scenarios, highlight differences specific to different pharmacological interventions with different mechanisms of action, and discuss how these bone markers can assist us in a deeper analysis of the pharmacodynamics and safety profile of existing and upcoming drug candidates.

Assessment of morphological and immunohistological alterations in long-term keloid skin explants.

One of the major impediments in keloid research is the lack of a keloid animal model that can mimic human keloid. This imposes investigative constraints on studying cellular interactions and biochemical processes that normally occur in vivo. Our main objective is to establish an in vitro model for maintaining long-term viable keloid dermal explants as a tool for investigating the pathogenesis of keloid scar formation. Explants of adult keloid scars were cultured in vitro by embedding them in enriched collagen gel matrix and maintaining them for up to 6 weeks, whereupon changes in tissue morphology and cellular differentiation were examined. The effects of medium enrichment, air versus liquid submersion, and different substrates on the explants were examined. Our results indicated that keloid explants embedded in a collagen gel matrix were morphologically better preserved than explants placed on a plastic substrate. Explants with epidermis at the air-liquid interface had better morphology than collagen-submerged explants, and there were no differences between serum-free and serum-supplemented explant cultures. Immunohistochemical and apoptotic analyses were performed to assess cellular viability and differentiation. In situ hybridization confirmed that keloid fibroblasts had sustained collagen type I gene expression throughout the 6 weeks in culture, thus validating the integrity of a long-term keloid culture system. In conclusion, the collagen-embedded skin explant system demonstrates that keloid tissues could be maintained for up to 6 weeks for long-term in vitro studies.