Collagen Structure Research Articles

Quantitative intravital imaging for real-time monitoring of pancreatic tumor cell hypoxia and stroma in an orthotopic mouse model.

Pancreatic cancer is a lethal disease with few successful treatment options. Recent evidence demonstrates that tumor hypoxia promotes pancreatic tumor invasion, metastasis, and therapy resistance. However, little is known about the complex relationship between hypoxia and the pancreatic tumor microenvironment (TME). In this study, we developed a novel intravital fluorescence microscopy platform with an orthotopic mouse model of pancreatic cancer to study tumor cell hypoxia within the TME in vivo, at cellular resolution, over time. Using a fluorescent BxPC3-DsRed tumor cell line with a hypoxia-response element (HRE)/green fluorescent protein (GFP) reporter, we showed that HRE/GFP is a reliable biomarker of pancreatic tumor hypoxia, responding dynamically and reversibly to changing oxygen concentrations within the TME. We also characterized the spatial relationships between tumor hypoxia, microvasculature, and tumor-associated collagen structures using in vivo second harmonic generation microscopy. This quantitative multimodal imaging platform enables the unprecedented study of hypoxia within the pancreatic TME in vivo.

Spectral Features Differentiate Aging-Induced Changes in Parchment-A Combined Approach of UV/VIS, µ-ATR/FTIR and µ-Raman Spectroscopy with Multivariate Data Analysis.

From the moment of production, artworks are constantly exposed to changing environmental factors potentially inducing degradation. Therefore, detailed knowledge of natural degradation phenomena is essential for proper damage assessment and preservation. With special focus on written cultural heritage, we present a study on the degradation of sheep parchment employing accelerated aging with light (295-3000 nm) for one month, 30/50/80% relative humidity (RH) and 50 ppm sulfur dioxide with 30/50/80%RH for one week. UV/VIS spectroscopy detected changes in the sample surface appearance, showing browning after light-aging and increased brightness after SO2-aging. Band deconvolution of ATR/FTIR and Raman spectra and factor analysis of mixed data (FAMD) revealed characteristic changes of the main parchment components. Spectral features for degradation-induced structural changes of collagen and lipids turned out to be different for the employed aging parameters. All aging conditions induced denaturation (of different degrees) indicated by changes in the secondary structure of collagen. Light treatment resulted in the most pronounced changes for collagen fibrils in addition to backbone cleavage and side chain oxidations. Additional increased disorder for lipids was observed. Despite shorter exposure times, SO2-aging led to a weakening of protein structures induced by transitions of stabilizing disulfide bonds and side chain oxidations.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

Size effect of fish gelatin nanoparticles on the mechanical properties of collagen film based on its hierarchical structure

Fish gelatin nanoparticles (FGNPs) of different sizes (∼155–410 nm) were successfully obtained by adjusting the ratio of gelatin and glutaraldehyde (Gel/Glu ratio), and their possible generation mechanisms were revealed by Fourier transform infrared (FTIR) analysis and transmission electron microscopy images. The FGNPs were then used to fabricate collagen composite films. Compared to a pure collagen film, the tensile strength, elongation at break, and Young's modulus of composite films increased by 37.1%, 34.4%, and 79.2%, respectively, while the water vapor permeability decreased by 51.5%. Meanwhile, scanning electron microscopy images, FTIR, and X-ray diffraction analysis suggested that small-sized and uniformly distributed FGNPs maintained the original crystal structure and well-arranged super helical structure of collagen, while large-sized and cross-linked FGNPs destabilized the collagen structure. A Gel/Glu ratio of 12:4 afforded FGNPs with a size of ∼155 nm, which was the best collagen composite film-forming size. Moreover, the FGNPs could bind to collagen through weak but countless non-covalent bonds (hydrogen bonds and ionic bonds), thereby improving the mechanical properties of the films. Overall, the FGNPs improved the performances characteristic of collagen films by influencing their structure, including hydrogen bonding interaction, crystalline structure, and superhelix structure, which would advance the understanding of collagen-based films.

Mechanochemistry of Collagen.

The collagen molecular family is the result of nearly one billion years of biological evolution. It is a unique family of proteins, the majority of which provide general mechanical support to biological tissues. Fibril forming collagens are the most abundant collagens in vertebrate animals and are generally found in positions that resist tensile loading. In animals, cells produce fibril-forming collagen molecules that self-assemble into larger structures known as collagen fibrils. Collagen fibrils are the fundamental, continuous, load-bearing elements in connective tissues, but are often further aggregated into larger load-bearing structures, fascicles in tendon, lamellae in cornea and in intervertebral disk. We know that failure to form fibrillar collagen is embryonic lethal, and excessive collagen formation/growth (fibrosis) or uncontrolled enzymatic remodeling (type II collagen: osteoarthritis) is pathological. Collagen is thus critical to vertebrate viability and instrumental in maintaining efficient mechanical structures. However, despite decades of research, our understanding of collagen matrix formation is not complete, and we know still less about the detailed mechanisms that drive collagen remodeling, growth, and pathology. In this perspective, we examine the known role of mechanical force on the formation and development of collagenous structure. We then discuss a mechanochemical mechanism that has the potential to unify our understanding of collagenous tissue assembly dynamics, which preferentially deposits and grows collagen fibrils directly in the path of mechanical force, where the energetics should be dissuasive and where collagen fibrils are most required. We term this mechanism: Mechanochemical force-structure causality. STATEMENT OF SIGNIFICANCE: : Our mechanochemical-force structure causality postulate suggests that collagen molecules are components of mechanochemically-sensitive and dynamically-responsive fibrils. Collagen molecules assemble preferentially in the path of applied strain, can be grown in place by mechanical extension, and are retained in the path of force through strain-stabilization. The mechanisms that drive this behavior operate at the level of the molecules themselves and are encoded into the structure of the biomaterial. The concept might change our understanding of structure formation, enhance our ability to treat injuries, and accelerate the development of therapeutics to prevent pathologies such as fibrosis. We suggest that collagen is a mechanochemically responsive dynamic element designed to provide a substantial "material assist" in the construction of adaptive carriers of mechanical signals.

Quantitative nanohistology of aging dermal collagen.

The skin is the largest organ in the body and is essential for protecting us from environmental stressors such as UV radiation, pollution, and pathogens. As we age, our skin undergoes complex changes that can affect its function, appearance, and health. These changes result from intrinsic (chronological) and extrinsic (environmental) factors that can cause damage to the skin's cells and extracellular matrix. As higher-resolution microscopical techniques, such as Atomic Force Microscopy (AFM), are being deployed to support histology, it is possible to explore the biophysical properties of the dermal scaffold's constituents, such as the collagen network. In this study, we demonstrate the use of our AFM-based quantitative nanohistology, performed directly on unfixed cryosections of 30 donors (female, Caucasian), to differentiate between dermal collagen from different age groups and anatomical sites. The initial 420 (10 × 10μm2) Atomic Force Microscopy images were segmented into 42,000 (1 × 1μm2) images before being classified according to four pre-defined empirical collagen structural biomarkers to quantify the structural heterogeneity of the dermal collagen. These markers include interfibrillar gap formation, undefined collagen structure, and registered or unregistered dense collagen fibrillar network with evident D-banding. The structural analysis was also complemented by extensive nanoindentation (∼1,000 curves) performed on individual fibrils from each section, yielding 30,000 indentation curves for this study. Principal Component Analysis was used to reduce the complexity of high-dimensional datasets. The % prevalence of the empirical collagen structural biomarkers between the papillary and reticular dermis for each section proves determinant in differentiating between the donors as a function of their age or the anatomical site (cheek or breast). A case of abnormal biological aging validated our markers and nanohistology approach. This case also highlighted the difference between chronological and biological aging regarding dermal collagen phenotyping. However, quantifying the impact of chronic and pathological conditions on the structure and function of collagen at the sub-micron level remains challenging and lengthy. By employing tools such as the Atomic Force Microscope as presented here, it is possible to start evaluating the complexity of the dermal matrix at the nanoscale and start identifying relevant collagen morphology which could be used toward histopathology standards.

COMPARISON IN CORNEAL COLLAGEN STRUCTURE BETWEEN 20% PLATELET RICH PLASMA AND 20% AUTOLOGOUS SERUM THERAPY POST NATRIUM HIDROKSIDA EXPOSURE

Objective to compare therapy response between 20% PRP and AS 20% on cornea post sodium hydroxide exposure on collagen structure. Methods this is a true experimental study with post test only design on 18 New Zealand white rabbits. Sample were divided randomly to two groups, each with 9 rabbits. The right eye of each rabbit were exposed to alkali injury with 1N NaOH under general anesthesia. First group was given 20% PRP eyedrops and 20% AS for the second group. And the end of the 7 day periode, all rabbits were euthanized and enucleated to obtain histopthological data. Collagen density, collagen thickness and keratosit cell were evaluated. The result will be analyzed, ratio scale data will be tested with independent T- test, and ordinal scaled data will be tested with Mann-Whitney test. Result The collagen density in the 20% PRP group shows 88.9% grade 2 and 44.4 % grade 2 for the 20% AS group. The collagen thickness in the 20% PRP group is 55.41 and 67.62 for the 20% AS group. The keratosit cell count in the 20% PRP group are 54.56 and 45.47 in the 20% AS group. There is significant difference in the collagen density between 20% PRP and 20% AS treatment (p= 0.066). There is significant difference in the collagen thickness between 20% PRP and 20% AS treatment (p= 0,224). There is significant difference in the keratosit cell count between 20% PRP and 20% AS treatment (p= 0,227). Conclusion From the statistik analysis shows that there is no significant difference in corneal collagen structure between 20% PRP and 20% AS therapy post natrium hidroksida exposure, from our study there is no significant better corneal healing parameters in post alkali chemical ocular injury with 20% PRP eyedrops compare to 20% AS eyedrops.

Properties of Bovine Collagen as Influenced by High-Pressure Processing.

The physical properties and structure of collagen treated with high-pressure technologies have not yet been investigated in detail. The main goal of this work was to determine whether this modern gentle technology significantly changes the properties of collagen. High pressure in the range of 0-400 MPa was used, and the rheological, mechanical, thermal, and structural properties of collagen were measured. The rheological properties measured in the area of linear viscoelasticity do not statistically significantly change due to the influence of pressure or the duration of pressure exposure. In addition, the mechanical properties measured by compression between two plates are not statistically significantly influenced by pressure value or pressure hold time. The thermal properties Ton and ∆H measured by differential calorimetry depend on pressure value and pressure hold time. Results from amino acids and FTIR analyses show that exposure of collagenous gels to high pressure (400 MPa), regardless of applied time (5 and 10 min), caused only minor changes in the primary and secondary structure and preserved collagenous polymeric integrity. SEM analysis did not show changes in collagen fibril ordering orientation over longer distances after applying 400 MPa of pressure for 10 min.

Effect of Ultrasonic Amplitude on the Yield and Properties of Barramundi (Lates calcarifer) Skin Collagen

Barramundi skin, a by-product of the fish processing industry, has shown potential as an alternative collagen source. However, the commonly used acid extraction method to produce collagen rendered a low yield requires a lengthy time and is not environmentally friendly. As a result, the adoption of greener technology, such as ultrasound, to improve the conventional extraction process is emerging. This study aimed to investigate the effect of different ultrasonication amplitudes on collagen recovery from barramundi skin. The resulting collagens were evaluated for their protein, hydroxyproline and moisture content, colour, molecular weight distribution, and FTIR spectra. Ultrasound-assisted extraction (UAE) was performed at 40 (UAE40), 60 (UAE60) and 80 (UAE80) % amplitude for 20 min. For comparison, acetic acid extraction was also carried out to produce acid-soluble collagen (ASC). UAE increased the yield (p<0.05) of collagen from barramundi skin, with UAE80 exhibiting a 7-fold increment compared to ASC. Increasing the ultrasonic amplitude increased the yield considerably but decreased the hydroxyproline content, indicating a reduction in collagen quality. Furthermore, the protein content and SDS-PAGE profile of the extracted collagens revealed that UAE promoted protein degradation. FTIR spectra indicated that despite slightly varying wavenumbers, no detrimental effect on the triple helical structure was seen following UAE with the presence of amides A, B, I, II, and III. Also, the α1, α2 and ß-chains were found in all samples, although the band intensity reduced as the amplitude increased. In conclusion, given the right conditions, UAE could improve the extraction yield without influencing the collagen structure.

Multimodal microscopic imaging with deep learning for highly effective diagnosis of breast cancer

Accurate and rapid diagnosis of breast cancer is particularly important since it becomes the primary malignant tumor threatening women's health. Although pathological diagnosis with H&E staining section is the gold standard in tumor diagnosis, it is hard to meet quick intraoperative inspection because of its complex procedures and hysteresis. This study adopts multimodal microscopic imaging technology (bright-field imaging, auto-fluorescence imaging and orthogonal polarization imaging) combined with deep learning to achieve rapid intelligent diagnosis of breast cancer by getting the rich information of tissue morphology, content and structure of collagen in tissue slices. By using both fusion classification models of multimodal images at pixel level and decision level, the AUC score and accuracy is 0.9366 and 89.01% for the pixel fusion classification, as well as 0.9421 and 87.53% for the decision fusion classification, respectively. The results suggest that the multimodal microscopic imaging technique proposed in this study has unique advantages in accuracy, speed and feasibility in clinic for breast cancer diagnosis, and has strong clinical potential and application prospects when combined with deep learning.

Cancer Cells Promote Immune Regulatory Function of Macrophages by Upregulating Scavenger Receptor MARCO Expression.

Expression of macrophage receptor with collagenous structure (MARCO) by tumor-associated macrophages is associated with poor prognosis of multiple types of cancer. In this article, we report that cancer cells (e.g., breast cancer and glioblastoma cell lines) can upregulate surface MARCO expression on human macrophages not only via IL-6-induced STAT3 activation but also via sphingosine-1-phosphate receptor (S1PR)-mediated IL-6 and IL-10 expression followed by STAT3 activation. We further found that MARCO ligation induces activation of the MEK/ERK/p90RSK/CREB signaling cascade, leading to IL-10 expression followed by STAT3-dependent PD-L1 upregulation. Such MARCO-induced macrophage polarization is accompanied by increased expression of PPARG, IRF4, IDO1, CCL17, and CCL22. Ligation of surface MARCO can thus result in decreased T cell responses mainly by reduction of their proliferation. Taken together, cancer cell-induced MARCO expression and its intrinsic regulatory function within macrophages are, to our knowledge, new aspects of cancer immune evasion mechanisms that need to be further studied in the future.

Purification of recombinant bacterial collagens containing structural perturbations.

Streptococcus pyogenes-derived recombinant bacterial collagen-like proteins (CLPs) are emerging as a potential biomaterial for biomedical research and applications. Bacterial CLPs form stable triple helices and lack specific interactions with human cell surface receptors, thus enabling the design of novel biomaterials with specific functional attributes. Bacterial collagens have been instrumental in understanding collagen structure and function in normal and pathological conditions. These proteins can be readily produced in E. coli, purified using affinity chromatography, and subsequently isolated after cleavage of the affinity tag. Trypsin is a widely used protease during this purification step since the triple helix structure is resistant to trypsin digestion. However, the introduction of Gly→X mutations or natural interruptions within CLPs can perturb the triple helix structure, making them susceptible to trypsin digestion. Consequently, removing the affinity tag and isolating collagen-like (CL) domains containing mutations is impossible without degradation of the product. We present an alternative method to isolate CL domains containing Gly→X mutations utilizing a TEV protease cleavage site. Protein expression and purification conditions were optimized for designed protein constructs to achieve high yield and purity. Enzymatic digestion assays demonstrated that CL domains from wild-type CLPs could be isolated by digestion with either trypsin or TEV protease. In contrast, CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease cleaved the His6-tag, enabling the isolation of mutant CL domains. The developed method can be adapted to CLPs containing various new biological sequences to develop multifunctional biomaterials for tissue engineering applications.

517 Long Term Scarring Assessment of Burn Wounds Treated with Bioengineered Allogeneic Cellularized Construct

Abstract Introduction Bioengineered allogeneic cellularized construct (BACC) has recently been proven to be effective for treatment of burn wounds and now has FDA approval. There is a paucity of data describing long term (≥1 year) scar characteristics following treatment with BACC, including its potential to modulate the formation of hypertrophic scar (HTS). HTS causes significant physical and psychosocial implications which can result in diminished quality of life. This study aims to characterize post-burn scars resulting from BACC-treated wounds compared to within subject matched autografted wounds using clinical and histologic metrics. Methods Adult patients (n=5) were previously enrolled in a Phase III clinical trial for treatment of their deep partial thickness acute burn wounds. Subjects had clinical scar examinations including imaging, Scar Assessment Scales (POSAS), and non-invasive skin measurements to quantify skin elasticity, erythema, and melanin. Punch biopsies were collected and fixed in formalin from the autograft-treated site (A), the BACC-treated site (B), the donor-site (D), and an area of normal skin (NS). The formalin-fixed biopsies were then embedded in paraffin, sectioned, and stained with H&E, Picrosirius Red, and Herovici. Results The use of BACC produced no difference in POSAS score and achieved elasticity measurements comparable to NS (Table 1). The erythema measurement demonstrated differences between scar sites and each scar site was different from NS. The donorsite also had some variation from NS. The measured melanin levels were different between each scar site, and both were different from NS. The donorsite also had detectable differences in melanin from NS. Measurements of epidermal thickness demonstrated differences between scar sites with BACC being similar to NS. Rete ridge ratio were different between scar sites and between autograft vs. NS. Papillary dermis cellularity had minor differences between scar sites and both scar sites were different from NS. Reticular dermis cellularity was equivalent between autograft and BACC sites and both were different from NS. Papillary and reticular alignment were not different between scar sites. Reticular orientation was not different between autograft and BACC but was different in BACC vs. NS. Papillary dermal thickness was different between autograft and BACC. Conclusions The HTSs resulting from wound closure achieved using BACC are similar to autografting in metrics related to clinical POSAS scores, extracellular matrix, and collagen structure. Scars following autografting or the use of BACC to achieve wound closure differ in metrics related to pigmentation, epidermal structure, and papillary dermal thickness and cellularity. Applicability of Research to Practice These findings may suggest that the use of BACC is a more optimal treatment for certain burn depths in certain patients because some HTS outcomes are equivalent, and it eliminates the need for a donor site.

Developmental Aspects of Cardiac Adaptation to Increased Workload

The heart is capable of extensive adaptive growth in response to the demands of the body. When the heart is confronted with an increased workload over a prolonged period, it tends to cope with the situation by increasing its muscle mass. The adaptive growth response of the cardiac muscle changes significantly during phylogenetic and ontogenetic development. Cold-blooded animals maintain the ability for cardiomyocyte proliferation even in adults. On the other hand, the extent of proliferation during ontogenetic development in warm-blooded species shows significant temporal limitations: whereas fetal and neonatal cardiac myocytes express proliferative potential (hyperplasia), after birth proliferation declines and the heart grows almost exclusively by hypertrophy. It is, therefore, understandable that the regulation of the cardiac growth response to the increased workload also differs significantly during development. The pressure overload (aortic constriction) induced in animals before the switch from hyperplastic to hypertrophic growth leads to a specific type of left ventricular hypertrophy which, in contrast with the same stimulus applied in adulthood, is characterized by hyperplasia of cardiomyocytes, capillary angiogenesis and biogenesis of collagenous structures, proportional to the growth of myocytes. These studies suggest that timing may be of crucial importance in neonatal cardiac interventions in humans: early definitive repairs of selected congenital heart disease may be more beneficial for the long-term results of surgical treatment.

Morphological identification of the squirrel (Callosciurus notatus) tongue through Scanning Electron Microscopy (SEM) and histochemistry

Abstract. Megawati EI, Pradipta SID, Damia U, Kustiati U, Wihadmadyatami H, Kusindarta DL. 2023. Morphological identification of the squirrel (Callosciurus notatus) tongue through Scanning Electron Microscopy (SEM) and histochemistry. Biodiversitas 24: 2302-2314. Squirrel (Callosciurus notatus) plays an important role in the agricultural ecosystem.Their urine and feces kill coconut tree pests and help preserve coconut farming productivity. This research focuses on the morphological identification of the tongue and lingual papillae distribution of the Callosciurus notatus’stongue. The tongue morphology and lingual papillae distribution of Callosciurus notatus were investigated through two different methods: using scanning electron microscopy (SEM) and histology. This paper provides macroscopic and histological tongue morphology and lingual papillae distribution of Callosciurus notatus. Macroscopically, the tongue is divided into the apex, corpus, and radix. The dorsal surface of the apex the groove and lateral prominence were occur. SEM and light microscopy observation showed that the apex bears three types of papillae: leaf-like filiform, bifid filiform, and fungiform. The corpus includes wide leaf-like filiform papillae, saw-like filiform papillae, bifid filiform papillae, and fungiform papillae. On the radix, there are conical like filiform papillae, fungiform papillae, conical and vallatae papillae. The lateral part of radix showed Weber’s glands that react strongly positively by Alcian Blue (AB) pH 2.5 and periodic acid Schiff (PAS) staining; Weber’s glands consist of neutral and acidic mucin. Von Ebner’s glands were at the textus muscularis striatus syncytialis near the vallatae papillae, which react weakly positively to PAS stain and did not react to AB pH 2.5 stain; von Ebner’s glands consist of neutral mucin. Masson Trichromestain revealed collagen structure on lamina propria mucosae and textus muscularis striatus syncytialis of apex; moderately on lamina propria mucosae and lesser on textus muscularis striatus syncytialis of the corpus and radix.

Long-Chain Acyl Carnitines Aggravate Polystyrene Nanoplastics-Induced Atherosclerosis by Upregulating MARCO.

Exposure to micro- and nanoplastics (MNPs) is common because of their omnipresence in environment. Recent studies have revealed that MNPs may cause atherosclerosis, but the underlying mechanism remains unclear. To address this bottleneck, ApoE-/- mice are exposed to 2.5-250mg kg-1 polystyrene nanoplastics (PS-NPs, 50nm) by oral gavage with a high-fat diet for 19 weeks. It is found that PS-NPs in blood and aorta of mouse exacerbate the artery stiffness and promote atherosclerotic plaque formation. PS-NPs activate phagocytosis of M1-macrophage in the aorta, manifesting as upregulation of macrophage receptor with collagenous structure (MARCO). Moreover, PS-NPs disrupt lipid metabolism and increase long-chain acyl carnitines (LCACs). LCAC accumulation is attributed to the PS-NP-inhibited hepatic carnitine palmitoyltransferase 2. PS-NPs, as well as LCACs alone, aggravate lipid accumulation via upregulating MARCO in the oxidized low-density lipoprotein-activated foam cells. Finally, synergistic effects of PS-NPs and LCACs on increasing total cholesterol in foam cells are found. Overall, this study indicates that LCACs aggravate PS-NP-induced atherosclerosis by upregulating MARCO. This study offers new insight into the mechanisms underlying MNP-induced cardiovascular toxicity, and highlights the combined effects of MNPs with endogenous metabolites on the cardiovascular system, which warrant further study.

Comparison of the Characteristics of Three Acellular Dermal Matrices Subjected to Distinct Processing Methods Using Five Types of Histochemical Staining.

Acellular dermal matrix (ADM) is treated using various devitalization and aseptic processing methods. The processing effects on ADM were evaluated by histochemical tests. From January 2014 to December 2016, 18 patients [average age, 43.0 (range, 30-54) years] who underwent breast reconstruction with an ADM and tissue expander were prospectively enrolled. During the permanent implant replacement, a biopsy of the ADM was performed. We used three different human-derived products, namely, Alloderm®, Allomend®, and Megaderm®. Hematoxylin and eosin, CD68, CD3, CD31, and smooth muscle actin were used to evaluate the collagen structure, inflammation, angiogenesis, and myofibroblast infiltration. Each ADM was semi-quantitatively analyzed. Significant differences in collagen degradation, acute inflammation, and myofibroblast infiltration were observed among the ADMs. Collagen degeneration (p<0.001) and myofibroblast infiltration (smooth muscle actin-positive, p=0.018; CD31-negative, p=0.765) were the most severe in Megaderm®. Acute inflammation, represented by CD68, was most severe in Alloderm® (p=0.024). Both radiation and freeze-drying treatment physically damaged the collagen structure. Collagen degeneration was most severe in Megaderm®, followed by Allomend® and Alloderm®. Since Alloderm® is treated using chemicals, an assessment of the chemical irritation is warranted. The biopsy results were inconclusive. Therefore, to better interpret processing, more large-scale, serial, histochemical studies of each ADM are needed. This journal requires that authors 38 assign a level of evidence to each article. For a full 39 description of these Evidence-Based Medicine ratings, 40 please refer to the Table of Contents or the online 41 Instructions to Authors www.springer.com/00266 .

A Novel and Efficient Collagen Extraction Method Assisted by Microwave Irradiation

A new and efficient microwave heating method was developed to extract intact Type I collagen from bovine limed splits. Orthogonal experiment was conducted to optimize the parameters of extraction, and the extraction yield was measured by ultraviolet spectra (UV). The hierarchical structures of the obtained collagen were determined by amino acid analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV, Fourier transform infrared spectra (FT-IR), circular dichroism spectra (CD), fluorescence spectra (FL) and ultra-sensitive differential scanning calorimetry (US-DSC). The results indicated the optimal conditions for this microwave assisted extraction were 37°C for 7 h by using solid to liquid ratio of 1:45. The resultant yield of collagen was 14.35% under the optimal conditions, which is 1.5 times higher than that obtained by the control water bath-heating method. Amino acid analysis, UV and SDS-PAGE revealed that the primary structure of collagen extracted with microwave assistance was type I collagen. Although the extraction yield was promoted drastically with microwave assisting, the secondary structure of the collagen involving triple helix still maintained according to FT-IR, CD, FL and VP-DSC results. In brief, microwave irradiation could be a new routine for collagen extraction with advantages of fast and effective. Moreover, this study might offer a potential choice for utilizing hide or skin limed split wastes.

Immune-Modifying Microparticles for Treatment of Acute Graft-Versus-Host-Disease

Abstract Background: Acute Graft versus host disease (aGVHD) is one of the most common complication of allogeneic hematopoietic stem cell transplant. We have previously shown that infused immune-modifying particles (IMPs) can reduce clinical symptoms and improve survival in inflammatory conditions. IMPs are taken up by inflammatory monocytes (θIM) via the macrophage receptor with collagenous structure (MARCO) and then undergo splenic sequestration. Given these findings, IMPs may be an effective method of reducing inflammation in aGVHD. Methods: We tested the efficacy of IMP in an established aGVHD murine model. We also explored the changes in immune cell subsets and serum cytokines. To assess impact of IMP on graft-versus-tumor (GVT), we infused host mice with A20 lymphoma cells. Results: We demonstrated that treatment with IMPs effectively rescues mice in a lethal aGVHD model. Treatment with IMPs also significantly improved clinical and target organ histopathological scores. IMP treatment resulted in increased splenic and intestinal CD4 +CD25 +Foxp3 +Tregs. IMP treated mice had lower numbers of colonic CD11b +Ly6c hiθIM and a lower peak of serum inflammatory cytokines. Maintenance of GVT effect was demonstrated by comparable survival rates in host mice with co-infusion of A20 lymphoma cells. Conclusion: Systemic IMP was effective at rescuing mice in this lethal aGVHD model. This demonstrates that targeting θIMs with IMPs could be an effective strategy to treat aGVHD. Further investigation of the mechanisms by which IMPs ameliorate aGVHD is warranted, as is investigation of the use of IMPs to treat aGVHD in the clinical setting. Supported by Nanoparticle Tolerance Research Fund

ALKAPTONURIA DIAGNOSED IN A 72 YEAR OLD FEMALE AFTER TOTAL KNEE REPLACEMENT

Alkaptonuria is a rare autosomal recessive disorder of metabolism caused by deficiency of homogentisic acid oxidase and resulting in accumulation of homogentisic acid in collagenous structures. It is characterized by homogentisic aciduria, bluish-black discoloration of connective tissues (ochronosis) and arthropathy of large joints. Less common manifestations include cardiovascular abnormalities, renal, urethral and prostate calculi. Bone fractures are unusual in ochronosis. In this report, we describe a woman, 72 years of age, with a history of severe arthropathy requiring total joint replacement in both of her hips and left Knee. During the Left Total Knee Replacement, an intra operative observation of a bluish-black discoloration of the knee joint and the surrounding soft tissue raised the diagnosis of Alkaptonuria. We review the etiology, pathogenesis, clinical presentation, diagnosis and treatment of alkaptonuric ochronosis. Early detection is important for prevention and treatment of multiple systems.